Search

Bacillus anthracis , the causative agent of anthrax, is among the most likely bacterial pathogens to be used in a biological attack. Inhalation anthrax is a serious, life-threatening form of infection, and the mortality from acute inhaled anthrax can approach 100% if not treated early and aggressively. Food and Drug Administration-approved antibiotics indicated for post-exposure prophylaxis (PEP) or treatment of anthrax are limited. This study assessed the in vitro activity and in vivo efficacy of omadacycline and comparators against clinical isolates of B. anthracis, including a ciprofloxacin-resistant isolate. Minimum inhibitory concentrations (MICs) of omadacycline, ciprofloxacin, and doxycycline were determined against animal and human clinical isolates of B. anthracis , including the ciprofloxacin-resistant Ames strain BAC r 4-2. Mice were challenged with aerosolized BAC r 4-2 spores, and survival was monitored for 28 days post-challenge. Treatment was initiated 24 h after aerosol challenge and administered for 14 days. Omadacycline demonstrated in vitro activity against 53 B. anthracis isolates with an MIC range of ≤0.008-0.25 µg/mL, and an MIC 50 /MIC 90 of 0.015/0.03 µg/mL. Consistent with this, omadacycline demonstrated in vivo efficacy in a PEP mouse model of inhalation anthrax caused by the Ames BAC r 4-2 ciprofloxacin-resistant B. anthracis isolate. Omadacycline treatment significantly increased survival compared with the vehicle control group and the ciprofloxacin treatment group. As antibiotic resistance rates continue to rise worldwide, omadacycline may offer an alternative PEP or treatment option against inhalation anthrax, including anthrax caused by antibiotic-resistant B. anthracis ., Competing Interests: H.S.H., B.K.P., and G.D. have no conflicts to disclose. D.A., S.K.T., and A.W.S. are employees of Paratek Pharmaceuticals, Inc.

Inhalation anthrax is the most severe form of Bacillus anthracis infection, often progressing to fatal conditions if left untreated. While recommended antibiotics can effectively treat anthrax when promptly administered, strains engineered for antibiotic resistance could render these drugs ineffective. Telavancin, a semisynthetic lipoglycopeptide antibiotic, was evaluated in this study as a novel therapeutic against anthrax disease. Specifically, the aims were to (i) assess in vitro potency of telavancin against 17 B. anthracis isolates by minimum inhibitory concentration (MIC) testing and (ii) evaluate protective efficacy in rabbits infected with a lethal dose of aerosolized anthrax spores and treated with human-equivalent intravenous telavancin doses (30 mg/kg every 12 hours) for 5 days post-antigen detection versus a humanized dose of levofloxacin and vehicle control. Blood samples were collected at various times post-infection to assess the level of bacteremia and antibody production, and tissues were collected to determine bacterial load. The animals' body temperatures were also recorded. Telavancin demonstrated potent bactericidal activity against all strains tested (MICs 0.06-0.125 μg/mL). Further, telavancin conveyed 100% survival in this model and cleared B. anthracis from the bloodstream and organ tissues more effectively than a humanized dose of levofloxacin. Collectively, the low MICs against all strains tested and rapid bactericidal in vivo activity demonstrate that telavancin has the potential to be an effective alternative for the treatment or prophylaxis of anthrax infection., Competing Interests: I.M.-P. is an employee of Cumberland Pharmaceuticals, Inc., manufacturer of the test compound (telavancin); however, the project was funded entirely by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Human Health Services, under contract number HHSN272201700040I/75N93019F00271 (PMCID Task Order A29).

Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Background: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking., Methods: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics., Results: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached., Conclusion: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Anthrax is a notifiable disease in Turkey. In order to control the human disease, animal foci are being monitored and prevention and control activities are being implemented by the Ministry of Health in coordination with the Ministry of Agriculture and Forestry. The objective of our study was to evaluate the national surveillance data and control activities in the last decade. A total of 1174 anthrax cases and 9 deaths have been reported. Anthrax was frequent in eastern provinces and in big cities where large animal movements were significant. The incidence rate was 1.5 times higher in males than in females. The disease was more common in the 30-64 age group. The number of cases increased in the summer and autumn seasons. Human anthrax is still being reported though in decreasing numbers in Turkey. A collaborative control programme continues to be needed.

Bacillus anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA-approved vaccine to prevent anthrax in humans is anthrax vaccine adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against protective antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five-course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain. In addition, it appears to be ineffective against strains that lack anthrax toxin. Here, we investigated a vaccine formulation consisting of recombinant proteins from a surface-localized heme transport system containing near-iron transporter (NEAT) domains and its efficacy as a vaccine for anthrax disease. The cocktail of five NEAT domains was protective against a lethal challenge of inhaled bacillus spores at 3 and 28 weeks after vaccination. The reduction of the formulation to three NEATs (IsdX1, IsdX2, and Bslk) was as effective as a five-NEAT domain cocktail. The adjuvant alum, approved for use in humans, was as protective as Freund's Adjuvant, and protective vaccination correlated with increased anti-NEAT antibody reactivity and reduced bacterial levels in organs. Finally, the passive transfer of anti-NEAT antisera reduced mortality and disease severity, suggesting the protective component is comprised of antibodies. Collectively, these results provide evidence that a vaccine based upon recombinant NEAT proteins should be considered in the development of a next-generation anthrax vaccine., (Copyright © 2020 American Society for Microbiology.)

The in vivo efficacy of liposomal encapsulated ciprofloxacin in two formulations, lipoquin and apulmiq, were evaluated against the causative agent of anthrax, Bacillus anthracis. Liposomal encapsulated ciprofloxacin is attractive as a therapy since it allows for once daily dosing and achieves higher concentrations of the antibiotic at the site of initial mucosal entry but lower systemic drug concentrations. The in vivo efficacy of lipoquin and apulmiq delivered by intranasal instillation was studied at different doses and schedules in both a post exposure prophylaxis (PEP) therapy model and in a delayed treatment model of murine inhalational anthrax. In the mouse model of infection, the survival curves for all treatment cohorts differed significantly from the vehicle control. Ciprofloxacin, lipoquin and apulmiq provided a high level of protection (87-90%) after 7 days of therapy when administered within 24 hours of exposure. Reducing therapy to only three days still provided protection of 60-87%, if therapy was provided within 24 hours of exposure. If treatment was initiated 48 hours after exposure the survival rate was reduced to 46-65%. These studies suggest that lipoquin and apulmiq may be attractive therapies as PEP and as part of a treatment cocktail for B. anthracis., Competing Interests: J. B. is an employee of Aradigm Corp and has been named on Aradigm patents. C.W.S., M.C. and S.J. declare no potential conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Epidemiologic data indicate a global distribution of anthrax outbreaks associated with certain ecosystems that promote survival and viability of Bacillus anthracis spores. Here, we characterized three anthrax outbreaks involving humans, livestock, and wildlife that occurred in the same locality in Kenya between 2014 and 2017. Clinical and epidemiologic data on the outbreaks were collected using active case finding and review of human, livestock, and wildlife health records. Information on temporal and spatial distribution of prior outbreaks in the area was collected using participatory epidemiology. The 2014-2017 outbreaks in Nakuru West subcounty affected 15 of 71 people who had contact with infected cattle (attack rate = 21.1%), including seven with gastrointestinal, six with cutaneous, and two with oropharyngeal forms of the disease. Two (13.3%) gastrointestinal human anthrax cases died. No human cases were associated with infected wildlife. Of the 54 cattle owned in 11 households affected, 20 died (attack rate = 37%). The 2015 outbreak resulted in death of 10.5% of the affected herbivorous wildlife at Lake Nakuru National Park, including 745 of 4,500 African buffaloes (species-specific mortality rate = 17%) and three of 18 endangered white rhinos (species-specific mortality rate = 16%). The species mortality rate ranged from 1% to 5% for the other affected wildlife species. Participatory epidemiology identified prior outbreaks between 1973 and 2011 in the same area. The frequency and severity of outbreaks in this area suggests that it is an anthrax hotspot ideal for investigating risk factors associated with long-term survival of anthrax spores and outbreak occurrence.

Anthrax infection is primarily initiated by B. anthracis endospores that on entry into the host germinate to vegetative cells and cause severe bacteremia and toxaemia employing an array of host colonisation factors and the lethal tripartite toxin. The protective efficacy of conventional protective antigen (PA) based anthrax vaccines is improved by co-administration with inactivated spores or its components. In the present study, using structural vaccinology rationale we synthesized a bivalent protein r-PB encompassing toxin (PAIV) and spore components (BclACTD) and characterized its protective efficacy against B. anthracis infection. Active immunization of mice with r-PB generated high titer circulating antibodies which facilitated the phagocytic uptake of spores, inhibited their germination to vegetative cells and completely neutralized anthrax toxins in vivo resulting in 100 % survival against anthrax toxin challenge. Proliferation of CD4+ T cell subsets with up-regulation of Th1 (IFN-γ, IL-2, and IL-12), Th2 (IL-5, IL-10) cytokines and balanced expression of IgG1:IgG2a antibody isotypes indicated the stimulation of both Th1 and Th2 subsets. The immunized mice exhibited 100 % survival upon challenge with B. anthracis spores or toxin indicating the ability of r-PB to provide comprehensive protection against anthrax. Our results thus demonstrate r-PB an efficient vaccine candidate against anthrax infection.

The recommended management of inhalational anthrax, a high-priority bioterrorist threat, includes antibiotics and antitoxins. Obiltoxaximab, a chimeric monoclonal antibody against anthrax protective antigen (PA), is licensed under the U.S. Food and Drug Administration's (FDA's) Animal Rule for the treatment of inhalational anthrax. Because of spore latency, disease reemergence after treatment cessation is a concern, and there is a need to understand the development of endogenous protective immune responses following antitoxin-containing anthrax treatment regimens. Here, acquired protective immunity was examined in New Zealand White (NZW) rabbits challenged with a targeted lethal dose of Bacillus anthracis spores and treated with antibiotics, obiltoxaximab, or a combination of both. Survivors of the primary challenge were rechallenged 9 months later and monitored for survival. Survival rates after primary and rechallenge for controls and animals treated with obiltoxaximab, levofloxacin, or a combination of both were 0, 65, 100, and 95%, and 0, 100, 95, and 89%, respectively. All surviving immune animals had circulating antibodies to PA and serum toxin-neutralizing titers prior to rechallenge. Following rechallenge, systemic bacteremia and toxemia were not detected in most animals, and the levels of circulating anti-PA IgG titers increased starting at 5 days postrechallenge. We conclude that treatment with obiltoxaximab, alone or combined with antibiotics, significantly improves the survival of rabbits that received a lethal inhalation B. anthracis spore challenge dose and does not interfere with the development of immunity. Survivors of primary challenge are protected against reexposure, have rare incidents of systemic bacteremia and toxemia, and have evidence of an anamnestic response., (Copyright © 2018 Henning et al.)

The recombinant phage endolysins AP50-31 and LysB4 were developed using genetic information from bacteriophages AP50 and B4 and were produced by microbial cultivation followed by chromatographic purification. Subsequently, appropriate formulations were developed that provided an acceptable stability of the recombinant endolysins. The bacteriolytic properties of the formulated endolysins AP50-31 and LysB4 against several bacterial strains belonging to the Bacillus genus including Bacillus anthracis (anthrax) strains were examined. AP50-31 and LysB4 displayed rapid bacteriolytic activity and broad bacteriolytic spectra within the Bacillus genus, including bacteriolytic activity against all the B. anthracis strains tested. When administered intranasally, LysB4 completely protected A/J mice from lethality after infection with the spores of B. anthracis Sterne. When examined at 3 days post-infection, bacterial counts in the major organs (lung, liver, kidney, and spleen) were significantly lower compared with those of the control group that was not treated with endolysin. In addition, histopathological examinations revealed a marked improvement of pathological features in the LysB4-treated group. The results of this study support the idea that phage endolysins are promising candidates for developing therapeutics against anthrax infection.

As one of the virulence factors of Bacillus anthracis, lethal toxin (LT) induces various pathogenic responses including the suppression of the coagulation system. In this study, we observed that LT markedly increased the circulating soluble P-selectin (sP-sel) levels and microparticle (MP) count in wild-type but not P-selectin (P-sel, Selp -/- ) or P-sel ligand-1 (PSGL-1, Selplg -/- ) knockout mice. Because sP-sel induces a hypercoagulable state through PSGL-1 pathway to generate tissue factor-positive MPs, we hypothesized that the increase in plasma sP-sel levels can be a self-rescue response in hosts against the LT-mediated suppression of the coagulation system. In agreement with our hypothesis, our results indicated that compared with wild-type mice, Selp -/- and Selplg -/- mice were more sensitive to LT. In addition, the recombinant sP-sel treatment markedly ameliorated LT-mediated pathogenesis and reduced mortality. As a result, elicitation of circulating sP-sel is potentially a self-rescue response, which is beneficial to host recovery from an LT-induced hypocoagulation state. These results suggest that the administration of sP-sel is likely to be useful in the development of a new strategy to treat anthrax.

Anthrax, caused by the spore-forming bacterium Bacillus anthracis, poses a threat to wood bison (Bison bison athabascae) conservation. We used descriptive epidemiology to characterize a large outbreak of anthrax in the Mackenzie bison population in the Northwest Territories, Canada, in 2012 and investigated historical serologic exposure of the bison to the bacterium in nonoutbreak years. Between late June and early August 2012, 451 bison carcasses were detected; mortality peaked from 13-19 July. A substantial number of calves, yearlings, and adult females died in the 2012 outbreak, unlike in two previous anthrax outbreaks in this population that killed mostly mature males. On the basis of the difference in estimates of population size prior to the outbreak (2012) and after the outbreak (2013), it is possible that not all dead bison were found during the outbreak. We assessed serologic history of exposure to B. anthracis by using samples from the Mackenzie wood bison population collected between 1986 and 2009. Overall, 87 of 278 samples were positive (31%). Seroprevalence was lower in females (18%, 10/55) than males (36%, 72/203). The highest proportion of positive submissions (90%) was from 1994, the year following the only anthrax outbreak within the historical data set. Both adult males and females had a higher likelihood of being seropositive than the younger age categories. There was a trend toward declining antibody levels between the 1993 and 2012 outbreak years.

The fluorocycline TP-271 was evaluated in mouse and nonhuman primate (NHP) models of inhalational anthrax. BALB/c mice were exposed by nose-only aerosol to Bacillus anthracis Ames spores at a level of 18 to 88 lethal doses sufficient to kill 50% of exposed individuals (LD 50 ). When 21 days of once-daily dosing was initiated at 24 h postchallenge (the postexposure prophylaxis [PEP] study), the rates of survival for the groups treated with TP-271 at 3, 6, 12, and 18 mg/kg of body weight were 90%, 95%, 95%, and 84%, respectively. When 21 days of dosing was initiated at 48 h postchallenge (the treatment [Tx] study), the rates of survival for the groups treated with TP-271 at 6, 12, and 18 mg/kg TP-271 were 100%, 91%, and 81%, respectively. No deaths of TP-271-treated mice occurred during the 39-day posttreatment observation period. In the NHP model, cynomolgus macaques received an average dose of 197 LD 50 of B. anthracis Ames spore equivalents using a head-only inhalation exposure chamber, and once-daily treatment of 1 mg/kg TP-271 lasting for 14 or 21 days was initiated within 3 h of detection of protective antigen (PA) in the blood. No (0/8) animals in the vehicle control-treated group survived, whereas all 8 infected macaques treated for 21 days and 4 of 6 macaques in the 14-day treatment group survived to the end of the study (56 days postchallenge). All survivors developed toxin-neutralizing and anti-PA IgG antibodies, indicating an immunologic response. On the basis of the results obtained with the mouse and NHP models, TP-271 shows promise as a countermeasure for the treatment of inhalational anthrax., (Copyright © 2017 American Society for Microbiology.)

Bacillus cereus biovar anthracis (Bcbva) is a member of the B. cereus group which carries both B. anthracis virulence plasmids, causes anthrax-like disease in various wildlife species and was described in several sub-Saharan African rainforests. Long-term monitoring of carcasses in Taï National Park, Côte d'Ivoire, revealed continuous wildlife mortality due to Bcbva in a broad range of mammalian species. While non-lethal anthrax infections in wildlife have been described for B. anthracis, nothing is known about the odds of survival following an anthrax infection caused by Bcbva. To address this gap, we present the results of a serological study of anthrax in five wildlife species known to succumb to Bcbva in this ecosystem. Specific antibodies were only detected in two out of 15 wild red colobus monkeys (Procolobus badius) and one out of 10 black-and-white colobus monkeys (Colobus polykomos), but in none of 16 sooty mangabeys (Cercocebus atys), 9 chimpanzees (Pan troglodytes verus) and 9 Maxwell's duikers (Cephalophus maxwellii). The combination of high mortality and low antibody detection rates indicates high virulence of this disease across these different mammalian species.

In September 2011, a total of 511 human cases of anthrax (Bacillus anthracis) infection and 5 deaths were reported in a game management area in the district of Chama, Zambia, near where 85 hippopotamuses (Hippopotamus amphibious) had recently died of suspected anthrax. The human infections generally responded to antibiotics. To clarify transmission, we conducted a cross-sectional, interviewer-administered household survey in villages where human anthrax cases and hippopotamuses deaths were reported. Among 284 respondents, 84% ate hippopotamus meat before the outbreak. Eating, carrying, and preparing meat were associated with anthrax infection. Despite the risk, 23% of respondents reported they would eat meat from hippopotamuses found dead again because of food shortage (73%), lack of meat (12%), hunger (7%), and protein shortage (5%). Chronic food insecurity can lead to consumption of unsafe foods, leaving communities susceptible to zoonotic infection. Interagency cooperation is necessary to prevent outbreaks by addressing the root cause of exposure, such as food insecurity.

Background: B. anthracis anti-toxin agents are approved and included in the Strategic National Stockpile based primarily on animal infection trials. However, in the only anthrax outbreak an approved anti-toxin agent was administered in, survival did not differ comparing recipients and non-recipients, although recipients appeared sicker., Objective: Employ a systematic review and meta-analysis to investigate preclinical studies supporting anthrax anti-toxin agents., Data Source: PubMed, EMBASE, and Scopus., Study Eligibility: Compared survival with an anti-toxin agent versus control in B. anthracis challenged, antibiotic treated animals., Study Methods: Examine model and study design and the effect of anti-toxin agents on relative risk of death(95%CI) (RR)., Results: From 9 studies, 29 experiments were analyzed which included 4 species (748 animals) and 5 agents; LFI, AIG, AVP-21D9, Raxibacumab, and ETI-204. Only five experiments were blinded and no experiment included the cardiopulmonary support sick B. anthracis patients receive. Only one agent in a single un-blinded experiment reduced RR significantly [0.45(0.22,0.940]. However, in six studies testing an agent in more than one experiment in the same species, agents had consistent survival effects across experiments [I2 = 0, p≥0.55 in five and I2 = 42%, p = 0.16 in one]. Within each species, agents had effects on the side of benefit; in one study testing AVP-21D9 in mice [0.11(0.01,1.82)] or guinea pigs [0.70(0.48,1.03)]; across eight rabbit studies testing LFI, Raxibacumab, AIG or ETI-204 [0.62(0.45,0.87); I2 = 17.4%, p = 0.29]; and across three monkey studies testing Raxibacumab, AIG or ETI-204 [0.66(0.34,1.27); I2 = 25.3%, p = 0.26]. Across all agents and species, agents decreased RR [0.64(0.52,0.79); I2 = 5.3%, p = 0.39]., Limitations: Incidence of selective reporting not identifiable., Conclusions: Although overall significant, individually anti-toxin agents had weak beneficial effects. Lack of study blinding and relevant clinical therapies further weakened studies. Although difficult, preclinical studies with more robust designs and results are warranted to justify the resources necessary to maintain anti-toxin agents in national stockpiles.

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation.

Health officials lack field-implementable tools for forecasting the effects that a large-scale release of Bacillus anthracis spores would have on public health and hospitals. We created a modeling tool (combining inhalational anthrax caseload projections based on initial case reports, effects of variable postexposure prophylaxis campaigns, and healthcare facility surge capacity requirements) to project hospitalizations and casualties from a newly detected inhalation anthrax event, and we examined the consequences of intervention choices. With only 3 days of case counts, the model can predict final attack sizes for simulated Sverdlovsk-like events (1979 USSR) with sufficient accuracy for decision making and confirms the value of early postexposure prophylaxis initiation. According to a baseline scenario, hospital treatment volume peaks 15 days after exposure, deaths peak earlier (day 5), and recovery peaks later (day 23). This tool gives public health, hospital, and emergency planners scenario-specific information for developing quantitative response plans for this threat.

We studied anthrax immune globulin intravenous (AIG-IV) use from a 2009-2010 outbreak of Bacillus anthracis soft tissue infection in injection drug users in Scotland, UK, and we compared findings from 15 AIG-IV recipients with findings from 28 nonrecipients. Death rates did not differ significantly between recipients and nonrecipients (33% vs. 21%). However, whereas only 8 (27%) of 30 patients at low risk for death (admission sequential organ failure assessment score of 0-5) received AIG-IV, 7 (54%) of the 13 patients at high risk for death (sequential organ failure assessment score of 6-11) received treatment. AIG-IV recipients had surgery more often and, among survivors, had longer hospital stays than did nonrecipients. AIG-IV recipients were sicker than nonrecipients. This difference and the small number of higher risk patients confound assessment of AIG-IV effectiveness in this outbreak.

Anthrax vaccine adsorbed (AVA, BioThrax) was recently approved by the Food and Drug Administration (FDA) for a post-exposure prophylaxis (PEP) indication in adults 18-65years of age. The schedule is three doses administered subcutaneous (SC) at 2-week intervals (0, 2, and 4weeks), in conjunction with a 60-day course of antimicrobials. The Public Health Emergency Medical Countermeasures Enterprise (PHEMCE) developed an animal model to support assessment of a shortened antimicrobial PEP duration following Bacillus anthracis exposure. A nonhuman primate (NHP) study was completed to evaluate the efficacy of a two dose anthrax vaccine absorbed (AVA) schedule (0, 2weeks) aerosol challenged with high levels of B. anthracis spores at week4- the time point at which humans would receive the third vaccination of the approved PEP schedule. Here we use logistic regression models to combine the survival data from the NHP study along with serum anthrax lethal toxin neutralizing activity (TNA) and anti-PA IgG measured by enzyme linked immunosorbent assay (ELISA) data to perform a cross-species analysis to estimate survival probabilities in vaccinated human populations at this time interval (week4 of the PEP schedule). The bridging analysis demonstrated that high levels of NHP protection also yield high predicted probability of human survival just 2weeks after the second dose of vaccine with the full or half antigen dose regimen. The absolute difference in probability of human survival between the full and half antigen dose was estimated to be at most approximately 20%, indicating that more investigation of the half-antigen dose for vaccine dose sparing strategies may be warranted., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The Centers for Disease Control and Prevention recommend adjunctive antitoxins when systemic anthrax is suspected. Obiltoxaximab, a monoclonal antibody against protective antigen (PA), is approved for treatment of inhalational anthrax in combination with antibiotics and for prophylaxis when alternative therapies are not available. The impact of toxin neutralization with obiltoxaximab during pre- and postexposure prophylaxis was explored, and efficacy results that supported the prophylaxis indication are presented here. New Zealand White rabbits and cynomolgus macaques received obiltoxaximab as a single intramuscular or intravenous dose of 2 to 16 mg/kg of body weight at various times relative to Bacillus anthracis aerosol spore challenge. The primary endpoint was survival, and effect of treatment timing was explored. In rabbits, obiltoxaximab administration 9 h postchallenge singly or combined with a 5-day levofloxacin regimen protected 89% to 100% of animals compared to 33% with levofloxacin monotherapy. In cynomolgus macaques, a single intramuscular dose of 16 mg/kg obiltoxaximab led to 100% survival when given 1 to 3 days preexposure and 83% to 100% survival when given 18 to 24 h postexposure and prior to systemic bacteremia onset. Obiltoxaximab administration after bacteremia onset resulted in lower (25% to 50%) survival rates reflective of treatment setting. Prophylactic administration of obiltoxaximab before spore challenge or to spore-challenged animals before systemic bacterial dissemination is efficacious in promoting survival, ameliorating toxemia, and inhibiting bacterial spread to the periphery., (Copyright © 2016 Yamamoto et al.)

Inhalational anthrax has high mortality even with antibiotic treatment, and antitoxins are now recommended as an adjunct to standard antimicrobial regimens. The efficacy of obiltoxaximab, a monoclonal antibody against anthrax protective antigen (PA), was examined in multiple studies conducted in two animal models of inhalational anthrax. A single intravenous bolus of 1 to 32 mg/kg of body weight obiltoxaximab or placebo was administered to New Zealand White rabbits (two studies) and cynomolgus macaques (4 studies) at disease onset (significant body temperature increase or detection of serum PA) following lethal challenge with aerosolized Bacillus anthracis spores. The primary endpoint was survival. The relationship between efficacy and disease severity, defined by pretreatment bacteremia and toxemia levels, was explored. In rabbits, single doses of 1 to 16 mg/kg obiltoxaximab led to 17 to 93% survival. In two studies, survival following 16 mg/kg obiltoxaximab was 93% and 62% compared to 0% and 0% for placebo (P = 0.0010 and P = 0.0013, respectively). Across four macaque studies, survival was 6.3% to 78.6% following 4 to 32 mg/kg obiltoxaximab. In two macaque studies, 16 mg/kg obiltoxaximab reduced toxemia and led to survival rates of 31%, 35%, and 47% versus 0%, 0%, and 6.3% with placebo (P = 0.0085, P = 0.0053, P = 0.0068). Pretreatment bacteremia and toxemia levels inversely correlated with survival. Overall, obiltoxaximab monotherapy neutralized PA and increased survival across the range of disease severity, indicating clinical benefit of toxin neutralization with obiltoxaximab in both early and late stages of inhalational anthrax., (Copyright © 2016 Yamamoto et al.)

Objective: The aim of this study was to describe and define an outbreak of human anthrax in two villages in the northern savannah region of Togo., Patients and Method: In December 2009, localised groups of deaths occurred among villagers and their livestock, confirmed to be due to anthrax at the district hospital of Dapaong in Northern Togo. The National Disease Control department undertook an investigation to describe the epidemiological, clinical and bacteriological characteristics of this outbreak., Results: Thirty-four individuals presented with clinical manifestations of anthrax. All patients were known to have consumed meat from cattle who had died of unknown causes or had been killed as a result of unknown illness. All patients presented with muco-cutaneous lesions; some had gastro-intestinal, neurological or meningeal symptoms, or septicaemia. One patient was co-infected with Plasmodium falciparum. Six deaths (17.6%) were reported at the beginning of the epidemic; 28 patients were successfully treated with a 10-day course of intravenous Penicillin or oral Amoxicillin. The two factors that contributed to the ultimate resolution of the anthrax outbreak were the increase of community awareness toward health promotion and vaccination of all farm animals., Conclusion: Although six deaths occurred among families' members who were infected, new human anthrax cases were prevented by rapid treatment of victims as well as aggressive public health interventions. However the risk of re-emergence of infection and exposure still exists as there are no existing epidemiological mapping and no identification of infected zones; and furthermore, no functional anthrax surveillance system exists in the affected region., (© The Author(s) 2015.)

Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis. Live attenuated vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔhtrA strain secretes functional anthrax toxins but is 10-10(4)-fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits). In spite of this attenuation, double or even single immunization with SterneΔhtrA spores elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization.

Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization., (Copyright © 2015. Published by Elsevier Ltd.)

Whereas DNA provides the information to design life and proteins provide the materials to construct it, the metabolome can be viewed as the physiology that powers it. As such, metabolomics, the field charged with the study of the dynamic small-molecule fluctuations that occur in response to changing biology, is now being used to study the basis of disease. Here, we describe a comprehensive metabolomic analysis of a systemic bacterial infection using Bacillus anthracis, the etiological agent of anthrax disease, as the model pathogen. An organ and blood analysis identified approximately 400 metabolites, including several key classes of lipids involved in inflammation, as being suppressed by B. anthracis. Metabolite changes were detected as early as 1 day postinfection, well before the onset of disease or the spread of bacteria to organs, which testifies to the sensitivity of this methodology. Functional studies using pharmacologic inhibition of host phospholipases support the idea of a role of these key enzymes and lipid mediators in host survival during anthrax disease. Finally, the results are integrated to provide a comprehensive picture of how B. anthracis alters host physiology. Collectively, the results of this study provide a blueprint for using metabolomics as a platform to identify and study novel host-pathogen interactions that shape the outcome of an infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Respiratory anthrax is a fatal disease in the absence of early treatment with antibiotics. Rabbits are highly susceptible to infection with Bacillus anthracis spores by intranasal instillation, succumbing within 2 to 4 days postinfection. This study aims to test the efficiency of antibiotic therapy to treat systemic anthrax in this relevant animal model. Delaying the initiation of antibiotic administration to more than 24 h postinfection resulted in animals with systemic anthrax in various degrees of bacteremia and toxemia. As the onset of symptoms in humans was reported to start on days 1 to 7 postexposure, delaying the initiation of treatment by 24 to 48 h (time frame for mass distribution of antibiotics) may result in sick populations. We evaluated the efficacy of antibiotic administration as a function of bacteremia levels at the time of treatment initiation. Here we compare the efficacy of treatment with clarithromycin, amoxicillin-clavulanic acid (Augmentin), imipenem, vancomycin, rifampin, and linezolid to the previously reported efficacy of doxycycline and ciprofloxacin. We demonstrate that treatment with amoxicillin-clavulanic acid, imipenem, vancomycin, and linezolid were as effective as doxycycline and ciprofloxacin, curing rabbits exhibiting bacteremia levels of up to 10(5) CFU/ml. Clarithromycin and rifampin were shown to be effective only as a postexposure prophylactic treatment but failed to treat the systemic (bacteremic) phase of anthrax. Furthermore, we evaluate the contribution of combined treatment of clindamycin and ciprofloxacin, which demonstrated improvement in efficacy compared to ciprofloxacin alone., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Anthrax is a frequently fatal infection of many animal species and men. The causative agent Bacillus anthracis propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissection. Mice were challenged into foot pads with spores of toxinogenic, unencapsulated Sterne strain. The spores quickly migrated to the regional popliteal lymph nodes and spread to the bloodstream as early as 3 h post challenge. All mice died before 72 h post challenge from the systemic disease accompanied by a widespread LN tissue damage by bacteria, including the hemorrhagic necrotizing lymphadenitis, infiltration of CD11b+ and CD3+ cells, and massive proliferation of bacteria in lymph nodes. A macrophage scavenger receptor CD68/macrosialin was upregulated and found in association with vegetative bacteria likely as a marker of their prior interaction with macrophages. The major signaling findings among the 65 tested proteins included the reduced MAPK signaling, upregulation of STAT transcriptional factors, and altered abundance of a number of pro- and anti-apoptotic proteins with signaling properties opposing each other. Downregulation of ERK1/2 was associated with the response of CD11b+ macrophages/dendritic cells, while upregulation of the pro-apoptotic Puma indicated a targeting of CD3+ T-cells. A robust upregulation of the anti-apoptotic survivin was unexpected because generally it is not observed in adult tissues. Taken together with the activation of STATs it may reflect a new pathogenic mechanism aimed to delay the onset of apoptosis. Our data emphasize a notion that the net biological outcome of disease is determined by a cumulative impact of factors representing the microbial insult and the protective capacity of the host.

This retrospective, descriptive case-series reviews the clinical presentations and significant laboratory findings of patients diagnosed with and treated for injectional anthrax (IA) since December 2009 at Monklands Hospital in Central Scotland and represents the largest series of IA cases to be described from a single location. Twenty-one patients who fulfilled National Anthrax Control Team standardized case definitions of confirmed, probable or possible IA are reported. All cases survived and none required limb amputation in contrast to an overall mortality of 28% being experienced for this condition in Scotland. We document the spectrum of presentations of soft tissue infection ranging from mild cases which were managed predominantly with oral antibiotics to severe cases with significant oedema, organ failure and coagulopathy. We describe the surgical management, intensive care management and antibiotic management including the first description of daptomycin being used to treat human anthrax. It is noted that some people who had injected heroin infected with Bacillus anthracis did not develop evidence of IA. Also highlighted are biochemical and haematological parameters which proved useful in identifying deteriorating patients who required greater levels of support and surgical debridement.

Humoral and cell-mediated immune correlates of protection (COP) for inhalation anthrax in a rhesus macaque (Macaca mulatta) model were determined. The immunological and survival data were from 114 vaccinated and 23 control animals exposed to Bacillus anthracis spores at 12, 30, or 52 months after the first vaccination. The vaccinated animals received a 3-dose intramuscular priming series (3-i.m.) of anthrax vaccine adsorbed (AVA) (BioThrax) at 0, 1, and 6 months. The immune responses were modulated by administering a range of vaccine dilutions. Together with the vaccine dilution dose and interval between the first vaccination and challenge, each of 80 immune response variables to anthrax toxin protective antigen (PA) at every available study time point was analyzed as a potential COP by logistic regression penalized by least absolute shrinkage and selection operator (LASSO) or elastic net. The anti-PA IgG level at the last available time point before challenge (last) and lymphocyte stimulation index (SI) at months 2 and 6 were identified consistently as a COP. Anti-PA IgG levels and lethal toxin neutralization activity (TNA) at months 6 and 7 (peak) and the frequency of gamma interferon (IFN-γ)-secreting cells at month 6 also had statistically significant positive correlations with survival. The ratio of interleukin 4 (IL-4) mRNA to IFN-γ mRNA at month 6 also had a statistically significant negative correlation with survival. TNA had lower accuracy as a COP than did anti-PA IgG response. Following the 3-i.m. priming with AVA, the anti-PA IgG responses at the time of exposure or at month 7 were practicable and accurate metrics for correlating vaccine-induced immunity with protection against inhalation anthrax., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: We have evaluated the therapeutic efficacy of AIGIV when given in combination with levofloxacin and the effective window of treatment to assess the added benefit provided by AIGIV over standard antibiotic treatment alone in a New Zealand white rabbit model of inhalational anthrax., Methods: Rabbits were exposed to lethal dose of aerosolized spores of Bacillus anthracis (Ames strain) and treated intravenously with either placebo, (normal immune globulin intravenous, IGIV) or 15 U/kg of AIGIV, along with oral levofloxacin treatment at various time points (30-96 hours) after anthrax exposure., Results: The majority of treated animals (>88%) survived in both treatment groups when treatment was initiated within 60 hours of post-exposure. However, reduced survival of 55%, 33% and 25% was observed for placebo + levofloxacin group when the treatment was initiated at 72, 84 and 96 hours post-exposure, respectively. Conversely, a survival rate of 65%, 40% and 71% was observed in the AIGIV + levofloxacin treated groups at these time points., Conclusions: The combination of AIGIV with antibiotics provided an improvement in survival compared to levofloxacin treatment alone when treatment was delayed up to 96 hours post-anthrax exposure. Additionally, AIGIV treatment when given as an adjunct therapy at any of the time points tested did not interfere with the efficacy of levofloxacin.

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacillus anthracis toxins can be neutralized by antibodies against protective antigen (PA), a component of anthrax toxins. Anthrivig (human anthrax immunoglobulin), also known as AIGIV, derived from plasma of humans immunized with BioThrax (anthrax vaccine adsorbed), is under development for the treatment of toxemia following exposure to anthrax spores. The pharmacokinetics (PK) of AIGIV was assessed in naive animals and healthy human volunteers, and the efficacy of AIGIV was assessed in animals exposed via inhalation to aerosolized B. anthracis spores. In the clinical study, safety, tolerability, and PK were evaluated in three dose cohorts (3.5, 7.1, and 14.2 mg/kg of body weight of anti-PA IgG) with 30 volunteers per cohort. The elimination half-life of AIGIV in rabbits, nonhuman primates (NHPs), and humans following intravenous infusion was estimated to be approximately 4, 12, and 24 days, respectively, and dose proportionality was observed. In a time-based treatment study, AIGIV protected 89 to 100% of animals when administered 12 h postexposure; however, a lower survival rate of 39% was observed when animals were treated 24 h postexposure, underscoring the need for early intervention. In a separate set of studies, animals were treated on an individual basis upon detection of a clinical sign or biomarker of disease, namely, a significant increase in body temperature (SIBT) in rabbits and presence of PA in the serum of NHPs. In these trigger-based intervention studies, AIGIV induced up to 75% survival in rabbits depending on the dose and severity of toxemia at the time of treatment. In NHPs, up to 33% survival was observed in AIGIV-treated animals. (The clinical study has been registered at ClinicalTrials.gov under registration no. NCT00845650.).

Bacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.

Anthrax outbreaks in white-tailed deer, Odocoileus virginianus, are frequent in west Texas, USA, particularly across the Edwards Plateau. However, the outbreak severity varies among years. We summarize the outbreak history in white-tailed deer at a ranch north of Del Rio, Texas, from 2001 to 2010 and compare mortality rates to remotely sensed vegetation indices derived from Moderate Resolution Imaging Spectroradiometer satellite data. It has long been posited that the occurrence of mid- to high-latitude epizootics is associated with hot, dry summer conditions preceded by a wet spring, with cases occurring after summer rain events. Here we employed vegetation green-up indices as a proxy for such environmental conditions. Annual trajectories of vegetation indices identified a clear pattern of early green springs with dry summers in severe outbreak years. In contrast, later, less intense spring green-up with greener summers were associated with enzootic mortality years, when few cases occurred. There was a statistically significant difference in the annual timing and intensity of spring green-up from vegetation indices for epizootic and enzootic years. Years with epizootics have early, intense spring conditions, whereas enzootic years have low-intensity green-up. These results suggest that early green-up signatures may be useful in identifying epizootic climatic conditions ahead of the summer anthrax period. Such analyses are required to ultimately develop an early warning system for wildlife managers and veterinary public health officials.

Inhalation anthrax is often described as a toxin-mediated disease. However, the toxaemia model does not account for the high mortality of inhalation anthrax relative to other forms of the disease or for the pathology present in inhalation anthrax. Patients with inhalation anthrax consistently show extreme bacteraemia and, in contrast to animals challenged with toxin, signs of sepsis. Rather than toxaemia, we propose that death in inhalation anthrax results from an overwhelming bacteraemia that leads to severe sepsis. According to our model, the central role of anthrax toxin is to permit the vegetative bacteria to escape immune detection. Other forms of B. anthracis infection have lower mortality because their overt symptoms early in the course of disease cause patients to seek medical care at a time when the infection and its sequelae can still be reversed by antibiotics. Thus, the sepsis model explains key features of inhalation anthrax and may offer a more complete understanding of disease pathology for researchers as well as those involved in the care of patients., (© 2013 The Authors. Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Background and Objective: Cutaneous anthrax usually has a mortality rate of less than 1 per cent. However, since December 2009 there have been more than 13 deaths in the UK due to anthrax-contaminated heroin. We therefore wish to raise clinical awareness of this treatable disease., Case Report: We describe the case of a heroin user with an equivocal presentation of cellulitis in the neck. Within 36 hours, this led to death due to cutaneous anthrax., Conclusion: Whilst cutaneous anthrax remains rare, this case report aims to raise awareness of the fact that the symptoms and signs of this condition in intravenous drug users may not always fit the typical picture.

Nonhuman primates (NHPs) and rabbits are the animal models most commonly used to evaluate the efficacy of medical countermeasures against anthrax in support of licensure under the FDA's "Animal Rule." However, a need for an alternative animal model may arise in certain cases. The development of such an alternative model requires a thorough understanding of the course and manifestation of experimental anthrax disease induced under controlled conditions in the proposed animal species. The guinea pig, which has been used extensively for anthrax pathogenesis studies and anthrax vaccine potency testing, is a good candidate for such an alternative model. This study was aimed at determining the median lethal dose (LD50) of the Bacillus anthracis Ames strain in guinea pigs and investigating the natural history, pathophysiology, and pathology of inhalational anthrax in this animal model following nose-only aerosol exposure. The inhaled LD50 of aerosolized Ames strain spores in guinea pigs was determined to be 5.0 × 10(4) spores. Aerosol challenge of guinea pigs resulted in inhalational anthrax with death occurring between 46 and 71 h postchallenge. The first clinical signs appeared as early as 36 h postchallenge. Cardiovascular function declined starting at 20 h postexposure. Hematogenous dissemination of bacteria was observed microscopically in multiple organs and tissues as early as 24 h postchallenge. Other histopathologic findings typical of disseminated anthrax included suppurative (heterophilic) inflammation, edema, fibrin, necrosis, and/or hemorrhage in the spleen, lungs, and regional lymph nodes and lymphocyte depletion and/or lymphocytolysis in the spleen and lymph nodes. This study demonstrated that the course of inhalational anthrax disease and the resulting pathology in guinea pigs are similar to those seen in rabbits and NHPs, as well as in humans.

Background: The poly-gamma-D-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT., Methods: To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K(d) values of 0.8 microM and 2.6 microM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed., Results: Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen., General Significance: Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.

Inhaled Bacillus anthracis spores germinate and the subsequent vegetative growth results in bacteremia and toxin production. Anthrax toxin is tripartite: the lethal factor and edema factor are enzymatic moieties, while the protective antigen (PA) binds to cell receptors and the enzymatic moieties. Antibiotics can control B. anthracis bacteremia, whereas raxibacumab binds PA and blocks lethal toxin effects. This study assessed plasma PA kinetics in rabbits following an inhaled B. anthracis spore challenge. Additionally, at 84 h post-challenge, 42% of challenged rabbits that had survived were treated with either levofloxacin/placebo or levofloxacin/raxibacumab. The profiles were modeled using a modified Gompertz/second exponential growth phase model in untreated rabbits, with added monoexponential PA elimination in treated rabbits. Shorter survival times were related to a higher plateau and a faster increase in PA levels. PA elimination half-lives were 10 and 19 h for the levofloxacin/placebo and levofloxacin/raxibacumab groups, respectively, with the difference attributable to persistent circulating PA-raxibacumab complex. PA kinetics were similar between untreated and treated rabbits, with one exception: treated rabbits had a plateau phase nearly twice as long as that for untreated rabbits. Treated rabbits that succumbed to disease had higher plateau PA levels and shorter plateau duration than surviving treated rabbits.

Tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) are the two well-characterized anthrax toxin receptors, each containing a von Willebrand factor A (vWA) domain responsible for anthrax protective antigen (PA) binding. Recently, a cell-based analysis was used to implicate another vWA domain-containing protein, integrin β1 as a third anthrax toxin receptor. To explore whether proteins other than TEM8 and CMG2 function as anthrax toxin receptors in vivo, we challenged mice lacking TEM8 and/or CMG2. Specifically, we used as an effector protein the fusion protein FP59, a fusion between the PA-binding domain of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas aeruginosa exotoxin A. FP59 is at least 50-fold more potent than LF in the presence of PA, with 2 μg PA + 2 μg FP59 being sufficient to kill a mouse. While TEM8(-/-) and wild type control mice succumbed to a 5 μg PA + 5 μg FP59 challenge, CMG2(-/-) mice were completely resistant to this dose, confirming that CMG2 is the major anthrax toxin receptor in vivo. To detect whether any toxic effects are mediated by TEM8 or other putative receptors such as integrin β1, CMG2(-/-)/TEM8(-/-) mice were challenged with as many as five doses of 50 μg PA + 50 μg FP59. Strikingly, the CMG2(-/-)/TEM8(-/-) mice were completely resistant to the 5-dose challenge. These results strongly suggest that TEM8 is the only minor anthrax toxin receptor mediating direct lethality in vivo and that other proteins implicated as receptors do not play this role.

An anthrax outbreak occurred in November 2010 in five villages of Sesheke district in Western Zambia. Control measures and data collection was carried out immediately the outbreak was reported. The prevalence of the disease in cattle was estimated at 7.4% (45/609) while the average herd size of infected cattle in affected villages was estimated at 121.8 (95% CI 48.8-194.8). Individual mortality per herd varied between 1.70% (3/179) and 20.25% (6/79). The relative risk of infection of cattle in the five affected villages varied between 0.18 (95% CI 0.4-5.7) and 3.7 (95% CI 1.99-6.68). In humans, the disease only affected three people and was characterized by cutaneous carbuncles. The ratio of infected persons per number of infected carcasses varied between 1:37 and 1:49 in affected villages while the overall ratio of people at risk to the number of carcasses was 42:1 indicating that despite availability of a large number of carcasses, human contact with infected carcasses was low. The findings of this study underline the importance of timely disease control measures in reducing the risk of human infections to anthrax in the face of an outbreak., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Because of a variety of caveats, the safety and effectiveness of interventions in human subjects can only be speculated from animal studies. Careful synthesis of data from multiple animal studies is needed to begin to assess the likelihood of successful cross-species translation (Fay et al., this issue).

Because clinical trials to assess the efficacy of vaccines against anthrax are not ethical or feasible, licensure for new anthrax vaccines will likely involve the Food and Drug Administration's "Animal Rule," a set of regulations that allow approval of products based on efficacy data only in animals combined with immunogenicity and safety data in animals and humans. U.S. government-sponsored animal studies have shown anthrax vaccine efficacy in a variety of settings. We examined data from 21 of those studies to determine whether an immunological bridge based on lethal toxin neutralization activity assay (TNA) can predict survival against an inhalation anthrax challenge within and across species and genera. The 21 studies were classified into 11 different settings, each of which had the same animal species, vaccine type and formulation, vaccination schedule, time of TNA measurement, and challenge time. Logistic regression models determined the contribution of vaccine dilution dose and TNA on prediction of survival. For most settings, logistic models using only TNA explained more than 75% of the survival effect of the models with dose additionally included. Cross-species survival predictions using TNA were compared to the actual survival and shown to have good agreement (Cohen's κ ranged from 0.55 to 0.78). In one study design, cynomolgus macaque data predicted 78.6% survival in rhesus macaques (actual survival, 83.0%) and 72.6% in rabbits (actual survival, 64.6%). These data add support for the use of TNA as an immunological bridge between species to extrapolate data in animals to predict anthrax vaccine effectiveness in humans.

The efficacy of multi-agent DNA vaccines consisting of a truncated gene encoding Bacillus anthracis lethal factor (LFn) fused to either Yersinia pestis V antigen (V) or Y . pestis F1 was evaluated. A/J mice were immunized by gene gun and developed predominantly IgG1 responses that were fully protective against a lethal aerosolized B. anthracis spore challenge but required the presence of an additional DNA vaccine expressing anthrax protective antigen to boost survival against aerosolized Y. pestis., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics against Bacillus anthracis. We investigated the roles of B. anthracis capsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon (capBCADE), lethal factor (lef), edema factor (cya), or protective antigen (pagA). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability of B. anthracis to traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wild-type and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA- mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA- mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.

There has been a reduction of incidences of anthrax in the developed countries but it is still a public health problem in the developing countries where communities live in interface areas with wildlife. An outbreak of anthrax in Hippopotamus amphibious was observed in Zambia. Following the death of hippopotamuses, suspected human cases were reported. The objective of this study was to isolate and confirm Bacillus anthracis and to determine the antimicrobial susceptibility for the management of the disease. Of the specimens collected, 29.4% (95% confidence interval [CI], 11.4-56.0) were from humans, 42.1% (95% CI, 21.1-66.0) were from hippopotamuses and 20.0% (95% CI, 6.61-44.3) from the soil were found to be positive were for B. anthracis. An antimicrobial susceptibility test revealed that all the isolates were found to be sensitive to the recommended antibiotics. The disease control was achieved by case management and by explaining to the communities that they should avoid contact with animals that die from unknown causes.