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PubMed2001

Purification of anthrax edema factor from Escherichia coli and identification of residues required for binding to anthrax protective antigen.

The structural gene for anthrax edema factor (EF) was expressed in Escherichia coli under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography. From 1 liter of culture, 2.5 mg of biologically active EF was easily purified. This is the first report of purification of anthrax EF from E. coli. EF purified from E. coli was biologically and functionally as active as its Bacillus anthracis counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen. The adenylate cyclase activity of the mutants remained unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells.

Adenylyl Cyclases↗

PubMed2004

Differentiating inhalational anthrax from other influenza-like illnesses in the setting of a national or regional anthrax outbreak.

The workup of a febrile patient who presents with an influenza-like illness in the setting of a national or regional anthrax outbreak presents a unique challenge to the physicians who initially evaluate this patient, and the diagnostic challenge can be even more profound during the influenza season. Based on information gathered from the recent inhalational anthrax cases, we have developed an algorithm to be used by physicians in the emergency department to evaluate patients with influenza-like illnesses during a national or regional anthrax outbreak. The algorithm necessitates stays no longer than 24 hours in the emergency department or hospital.

Algorithms↗

PubMed2004

Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax.

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

Animals↗

PubMed1989

Evaluation of serologic tests for diagnosis of anthrax after an outbreak of cutaneous anthrax in Paraguay.

An outbreak of at least 21 cases of cutaneous anthrax occurred in rural Paraguay. A case-control study revealed that disease was associated with touching the raw meat of an ill cow (odds ration = 16.5, P = .02). Serum drawn from 12 cases and 16 colony and 2 noncolony controls 6 w after the outbreak were analyzed by electrophoretic-immunotransblots (EITB) to detect serum antibodies to the protective antigen (PA) and lethal factor components of anthrax toxin. Serum was also tested by enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies to poly-D-glutamic acid capsule. Of 12 cases, 11 had a positive PA screen, for a sensitivity of 91.7% (76.1%-100%, 95% confidence interval [CI]) whereas none of the 18 controls was positive for a specificity of 100% (84.8%, one-sided binomial 95% CI). Only 6 (50%) of 12 cases (21.7%-78.3%, 95% CI) had positive lethal factor titers; all controls were negative. At a cutoff of greater than or equal to 1:32 for antibodies to capsule, 11 (91.7%) of 12 (76.1%-100%, 95% CI) were positive; 16 (88.9%) of 18 controls (74.5%-100%, 95% CI) were negative. These data suggest that the EITB for detection of antibody to PA, and ELISA for detection of anticapsule antibodies are both sensitive for the retrospective diagnosis of anthrax. Both tests were specific, but EITB may be more so than ELISA.

Animals↗

PubMed2004

Risk factors for human anthrax among contacts of anthrax-infected livestock in Kazakhstan.

A retrospective cohort analysis was conducted in Kazakhstan to define modifiable risk factors during seven outbreaks of human anthrax. Fifty-three cases and 255 non-ill persons with an epidemiologic link to an infected animal were enrolled. Cases were 58% male and had a median age of 35 years (range = 5-71). Nearly all cases had cutaneous disease (96%). Two patients (4%) were diagnosed with gastrointestinal disease. Although all cases had some contact with an infected animal other than consumption, in multivariable analysis the act of butchering an animal (relative risk [RR] = 3.6, 95% confidence interval [CI] = 1.5-9.6) and the presence of visible cuts on the hands were associated with anthrax (RR = 3.0, 95% CI = 0.9-9.6). Contact with infected livestock, in particular butchering, is associated with developing anthrax. The risk may be exacerbated by the presence of cuts on the hands at the time of contact with the animal or animal products.

Adolescent↗

PubMed1986

[Hereditary resistance to anthrax and sensitivity to the anthrax toxin in mice].

The degree of the hereditary susceptibility of mice to anthrax caused by noncapsular and capsule-forming Bacillus anthracis strains has been found to be directly related to the sensitivity of the animals to the edematogenic and immunosuppressing action of anthrax toxin. The genetic analysis indicates that resistance to anthrax is probably controlled by a dominant gene, not linked with histocompatibility complex H-2 and, probably, unrelated to the presence of hemolytic activity in mouse sera, determined by component C5 of the complement.

Animals↗

PubMed1994

[Delayed hypersensitivity after anthrax vaccination. I--Study of guinea pigs vaccinated against anthrax].

To evaluate delayed hypersensitivity after anthrax vaccination, an Anthraxin skin test was performed in 682 guinea pigs at various times after immunization with veterinary unencapsulated active anthrax vaccine. Results were compared with those obtained in unimmunized control guinea pigs (n = 216), in guinea pigs that received a non-immunizing dose of live vaccine (n = 183) and in guinea pigs inoculated with inactivated vaccine (n = 120). Anthraxin skin tests were positive in the first postvaccination days. The incidence and intensity of positive tests peaked between two weeks and one month after vaccination and then gradually decreased during the first year. Study of resistance of guinea pigs to an inoculum at a lethal dose of a virulent strain of Bacillus anthracis showed a close correlation between positive tests and resistance. These findings demonstrate development of cell-mediated immunity after anthrax vaccination. The Anthraxin skin test should have practical applications for the production of vaccines and for evaluation of the immune status of vaccinated livestock [corrected].

Animals↗

PubMed1999

Protection against anthrax toxin by vaccination with a DNA plasmid encoding anthrax protective antigen.

A DNA vaccine encoding the immunogenic and biologically active portion of anthrax protective antigen (PA) was constructed. Spleen cells from BALB/c mice immunized intramuscularly with this vaccine were stimulated to secrete IFN gamma and IL-4 when exposed to PA in vitro. Immunized mice also mounted a humoral immune response dominated by IgG1 anti-PA antibody production, the subclass previously shown to confer protection against anthrax toxin. A 1:100 dilution of serum from these animals protected cells in vitro against cytotoxic concentrations of PA. Moreover, 7/8 mice immunized three times with the PA DNA vaccine were protected against lethal challenge with a combination of anthrax protective antigen plus lethal factor.

Animals↗

PubMed1994

[Post anthrax vaccine delayed hypersensitivity. II--delayed hypersensitivity in humans vaccinated against anthrax].

To detect cell immunity characterized by delayed postvaccination hypersensitivity to anthrax in man and assess its dynamics, vaccination using unencapsulated live anthrax vaccine was performed in 668 healthy volunteers. Vaccination was performed either by scarification (n = 172), subcutaneous injection (n = 202), or low-dose (n = 202) or high-dose (n = 83) inhalation. The anthraxin intradermal tests were performed in each patient at various times during the year following vaccination (D7, D15, D90, D180, D365). This study confirm that, regardless of the mode of administration, the vaccine induces cell-mediated immunity in man, as determined by positive anthraxine skin test. The incidence of positive tests decreases with time regardless of the mode of vaccination. After one year, the test remained positive in 34.8% of subjects vaccinated by subcutaneous injection, 37.5% vaccinated by low-dose inhalation, 34.2% vaccinated by high-dose inhalation, and 22.4% vaccinated by scarification. These findings are in agreement with those obtained in clinical epidemiological studies documenting the effectiveness of encapsulated live anthrax vaccine in man.

Administration, Inhalation↗

PubMed2006

Factors influencing laboratory workers' decisions to accept or decline anthrax vaccine adsorbed (AVA): results of a decision-making study in CDC's anthrax vaccination program.

BACKGROUND: Laboratory technicians, laboratory supervisors, decontamination/remediation workers, and environmental investigators are at increased risk for repeated occupational exposure to Bacillus anthracis. In 2002, the Advisory Committee on Immunization Practices (ACIP) recommended pre-exposure vaccination for these occupational groups. OBJECTIVES: To determine (1) the factors that influenced an individual's decision to either accept or decline Anthrax Vaccine Adsorbed (AVA), and (2) if laboratory workers' concern about AVA safety was related to their information needs and trust in the information provided. METHODS: We conducted a decision-making survey of 404 participants at 44 Laboratory Response Network laboratories located throughout the United States. All participants were enrolled between October 2002 and December 2004, and all were eligible to receive AVA according to the 2002 ACIP recommendations. Chi-square tests and multivariate logistic regression were used in the analyses. RESULTS: The response rate of eligible individuals at participating laboratories was 94% (404/430). Sixty-six percent of respondents accepted and 34% declined AVA. Laboratory workers who declined AVA were more likely to rate their risk of exposure to inhalation anthrax as low (OR = 6.9; 95%CI 1.7, 28.3), report being very concerned (OR = 4.1; 95%CI 1.8, 9.3) or concerned (OR = 2.0; 95%CI 1.3, 3.1) about the safety of the vaccine, report that they did not trust the information provided in the Vaccine Information Statement (VIS) (OR = 2.3; 95%CI 1.1, 4.5), and to be enrolled in the study during 2002 (OR = 24.7; 95%CI 6.4, 95.3) or 2003 (OR = 5.0; 95%CI 2.5, 9.8), the first 2 years of the study. Furthermore, we found a significant association between a participant's level of concern about the safety of AVA and their perception that they received enough information and/or trusted the information in the VIS. CONCLUSIONS: Low perceived necessity, concern about the safety of the vaccine, and a lack of trust in the VIS were associated with the decision of laboratory workers to decline AVA. Results of this decision-making study may be used to try to improve acceptance rates of AVA among persons considered at high risk, and may inform educational efforts for other adult vaccines.

Adult↗

PubMedDate not supplied

Safety of anthrax vaccine: a review by the Anthrax Vaccine Expert Committee (AVEC) of adverse events reported to the Vaccine Adverse Event Reporting System (VAERS).

PURPOSE: To assess the safety of a licensed anthrax vaccine given to nearly 400,000 US military personnel, reports of adverse events (AEs) submitted to the Vaccine Adverse Event Reporting System (VAERS) were reviewed and evaluated medically. METHODS: The Anthrax Vaccine Expert Committee (AVEC), a civilian panel of private-sector physicians and other scientists, reviewed 602 VAERS reports using a Delphic approach (structured expert consensus) to assess the causal relationship between vaccination and the reported AEs and sought to identify unexpected patterns in the occurrence of medically important events. Reports were entered into a database and used to profile AEs with respect to person, type/location, relative frequency, severity/impact, concomitant illness or receipt of other drugs or vaccines, and vaccine lot. RESULTS: Nearly half the reports noted a local injection-site AE, with more than one-third of these involving a moderate to large degree of inflammation. Six events qualified as serious AEs (SAEs), and all were judged to be certain consequences of vaccination. Three-quarters of the reports cited a systemic AE (most common: flu-like symptoms, malaise, rash, arthralgia, headache), but only six individual medically important events were judged possibly or probably due to vaccine (aggravation of spondyloarthropathy (2), anaphylactoid reaction, arthritis (2), bronchiolitis obliterans organizing pneumonia). CONCLUSIONS: Since some cases of local inflammation involved distal paresthesia, AVEC recommends giving subcutaneous injections of AVA over the inferior deltoid instead of the triceps to avoid compression injury to the ulnar nerve. At this time, ongoing evaluation of VAERS reports does not suggest a high frequency or unusual pattern of serious or other medically important AEs.

Adolescent↗

PubMed2003

The anthrax team: a novel teaching approach to increase anthrax and bioterrorism awareness.

A team approach to educating staff regarding anthrax and bioterrorism awareness was implemented after the acts of terrorism that began September 11, 2001. "The Anthrax Team" developed algorithms on the basis of 5 different scenarios, an educational brochure, and a PowerPoint presentation. With use of the algorithms and PowerPoint material, the team conducted informal educational sessions to increase awareness and allay fears. On the basis of the success of this educational method, the teaching process will be applied to address staff on other biologic and chemical terrorism agents.

Algorithms↗

PubMed2005

An anthrax lethal factor-neutralizing monoclonal antibody protects rats before and after challenge with anthrax toxin.

Lethal factor (LF) is a component of anthrax lethal toxin (LeTx). We generated anti-LF murine monoclonal antibodies (MAbs) that show LeTx-neutralizing activity in vitro and in vivo. Anti-LF MAbs were generated by immunization with recombinant LF, and the MAbs showing LeTx-neutralizing activity in vitro were selected. Two MAbs with the highest affinities, 5B13B1 (dissociation constant [K(d)], 2.62 nM) and 3C16C3 (K(d), 8.18 nM), were shown to recognize the same or closely overlapping epitopes on domain III of LF. The 50% inhibitory concentration of 5B13B1 (0.21 microg/ml) was approximately one-third that of 3C16C3 (0.63 microg/ml) in the in vitro LeTx-neutralization assay. The 5B13B1 antibody, which had the highest neutralizing activity, provided perfect protection against LeTx challenge in an in vivo LeTx neutralization assay using Fisher 344 rats. In addition, the antibody showed pre- and postexposure prophylactic effects in the animal experiments. This is the first report that an MAb binding to domain III of LF has neutralizing activity against LeTx. The 5B13B1 antibody may be useful in prophylaxis against anthrax poisoning.

Animals↗

PubMed2004

Bichat guidelines for the clinical management of anthrax and bioterrorism-related anthrax.

The spore-forming Bacillus anthracis must be considered as one of the most serious potential biological weapons. The recent cases of anthrax caused by a deliberate release reported in 2001 in the United States point to the necessity of early recognition of this disease. Infection in humans most often involves the skin, and more rarely the lungs and the gastrointestinal tract. Inhalational anthrax is of particular interest for possible deliberate release: it is a life-threatening disease and early diagnosis and treatment can significantly decrease the mortality rate. Treatment consists of massive doses of antibiotics and supportive care. Isolation is not necessary. Antibiotics such as ciprofloxacin are recommended for post-exposure prophylaxis during 60 days.

Anthrax↗

PubMed1976

[Preparation and study of the anthrax protective antigen. II. Development of test preparations for the assessment of the quality of anthrax chemical vaccines and of the antigens obtained at various stages of its preparation].

A homogeneous anthrax protective antigen (from the ST-I strain) characterized by a high immunogenicity and stability was obtained. Hyperimmune, monospecific to this preparation antiserum was prepared. It is suggested that these preparations can be used for the assessment of the quality of the anthrax chemical vaccine and of the antigens obtained at various stages of its preparation.

Animals↗

PubMed2006

Anthrax oedema toxin induces anthrax toxin receptor expression in monocyte-derived cells.

Bacillus anthracis, the causative agent of anthrax, secretes two bipartite toxins that help the bacterium evade the immune system and contribute directly to pathogenesis. Both toxin catalytic moieties, lethal factor (LF) and oedema factor (OF), are internalized into the host-cell cytosol by a third factor, protective antigen (PA), which binds to cellular anthrax toxin receptors (ANTXRs). Oedema factor is an adenylate cyclase that impairs host defences by raising cellular cAMP levels. Here we demonstrate that oedema toxin (PA + OF) induces an increase in ANTXR expression levels in macrophages and dendritic cells resulting in an increased rate of toxin internalization. Furthermore, we show that increases in ANTXR mRNA levels depends on the ability of OF to increase cAMP levels, is mediated through protein kinase A-directed signalling and is monocyte-lineage-specific. To our knowledge, this is the first report of a bacterial toxin inducing host target cells to increase toxin receptor expression.

Adenylyl Cyclases↗

PubMed2006

Immunization against anthrax using Bacillus subtilis spores expressing the anthrax protective antigen.

Protective immunity to anthrax can be achieved by antibodies raised against the secreted protective antigen (PA) and this forms the basis of the current acellular vaccines for human use. Bacillus subtilis spores have previously been used for delivery of heterologous antigens by the oral and nasal routes and their intrinsic heat-stability make them attractive vaccine vehicles. In this study we have expressed PA, or segments of PA, in B. subtilis using two strategies. First, display on the spore coat, and second, in the germinated spore (or vegetative cell). Using parenteral delivery we show that recombinant spores can be used to confer protective immunity in a murine model using an in vitro toxin neutralization assay and a challenge experiment with the latter showing protection to 100 median lethal dose of B. anthracis spores. PA must be secreted from the live bacterium or alternatively displayed on the spore surface to confer protective immunity. Intracellular expression of PA failed to confer protective immunity. The highest levels of protective immunity were achieved when PA was displayed on the spore surface as well as in the germinating spore.

Animals↗

PubMed1951

Studies on immunity in anthrax. I. Variation in the serum T-agglutinin during anthrax infection in the rabbit.

An abrupt decrease in the titer of serum T-agglutinin frequently occurs during the acute phase of anthrax infection in the rabbit. In partially immunized animals which survive the infection the titer returns to normal during convalescence, but has not been observed to rise significantly above the normal level. The presence of a substance capable of inhibiting T-agglutination may be demonstrated in the sera of many of those animals in which a marked decrease in T-agglutinin has occurred. The polysaccharide and polypeptide antigens of B. anthracis do not possess this activity. Sera containing the T-inhibitory substance usually produce a slow hemolysis of T-erythrocytes and also of normal human type O erythrocytes; hemolysis of erythrocytes of other species is irregular. This reactivity of the sera withstands inactivation at 56 degrees C. but is inhibited by citrate. T-inhibitory and hemolytic activities can frequently be increased by incubation of the acute-phase serum at 37 degrees C. The ability of normal serum to neutralize the T-inhibitory and hemolytic activities of acute-phase serum is not significantly increased after recovery from the infection. The meaning of the results is discussed.

Animals↗