Anthrax is caused by B. anthracis, a Gram-positive, spore-forming, rod-shaped bacterium that primarily infects herbivores such as cattle and deer.1,3 The blood of an animal that dies of anthrax can contain upto 109 vegetative bacteria per millilitre. As the carcass decays, the bacteria form highly infectious endospores, which contaminate the local environment and can remain viable for long time periods. When spores are introduced into the body of a non-immune animal, they germinate, causing infection and death, thus completing the infectious cycle. Anthrax in humans, is rare and occurs mainly in individuals who come into close contact with farm animals or contaminated animal products such as wool or hides. Animals acquire B. anthracis infection via exposure to contaminated soil, where the organism persists in spore form. Animals also acquire infection from biting insects. Affected animals develop a bleeding diathesis as manifested by bleeding from the nose, vagina and anus. These diseased animals die suddenly after the infection. B. anthracis has gained recent notoriety as a potential biological weapon because it can be easily dispersed by aerosols to produce inhalational disease. Apart from cases associated with its use as biological agent, human cases of anthrax result from exposure to infected animals or contaminated animal products, including: hides, raw meat, hair and bone meal.
B. anthracis owes its lethality to two major virulence factors: an anti-phagocytic poly/-Dglutamic acid capsule and a toxin.4–6 The toxin, discovered in the 1950s, consists of three proteins: protective antigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and oedema factor (EF; 89 kDa). Individually, none of the three is toxic, but a mixture of PA and LF (called lethal toxin; LeTx) can cause lethal shock in experimental animals, and a mixture of PA and EF (called edema toxin; EdTx) induces oedema at the site of injection. The term binary toxin has been applied to such bacterial toxins when two discrete proteins must be combined to elicit toxicity. Genes encoding the three anthrax toxin proteins reside on a large plasmid (pXO1),7 and those for capsule synthesis, on a different plasmid (pXO2).4 Strains of B. anthracis carrying either plasmid alone are essentially avirulent, implying synergy between the two types of virulence factors. The importance of the toxin in infection and pathogenesis is further substantiated by the fact that immunization against it is protective.8 The PA is the most immunogenic component of the toxin, hence its name.
Anthrax as Bioterrorism Weapon
In a simplistic way, it is possible to grow bacteria, let them form spores and just spray a spore solution over an enemy. Alternatively, the spore solution could be dried and used as a powder. It has always been a potential fear that terrorists may use anthrax either in spray form or powder form.
Wet dispersal
When a solution is sprayed, the particle or droplet size tends to be quite large. These do not stay in 2the air very long. For an attack in a small area, the large droplets are also harmful enough. But for a big area to be covered by a military weapon, the aerosolized particles should stay in the air for a long time and the droplet size down to less than 10 µ. In these circumstances, dried spores in powder form would work better.
Dry dispersal
After the liquid culture is grown up and the bacteria sporulate, the spores are harvested by filtration or centrifugation. Thereafter, the spores and remaining cells form a very sticky paste, which forms a “brick” on drying down. When the “brick” is ground into a fine (one-micrometer) powder, the spores develop a surface charge which keeps them them to stick together It is also possible to freeze-dry the spore solution but again the surface charge will cause clumping. The basic approach is to coat the spores with a fine silica or alumina clay to neutralize the surface charge. Consequently, the spores do not stick to each other or any surface and result in a better dispersal.
Clinical Aspects of Anthrax Infection
Anthrax infection can occur in three forms: cutaneous (skin), inhalational, and gastrointestinal.
Cutaneous anthrax
Most (about 95%) anthrax infections occur when the bacterium enters a cut or abrasion on the skin, such as when handling contaminated wool, hides, leather or hair products (especially goat hair) of infected animals. Skin infection begins as a raised itchy bump that resembles an insect bite but within one to two days develops into a vesicle and then a painless ulcer, usually 1 to 3 cm in diameter, with a characteristic black necrotic (dying) area in the center. Lymph glands in the adjacent area may swell. About 20% of untreated cases of cutaneous anthrax will result in death. Deaths are rare with appropriate antimicrobial therapy.
Inhalational anthrax
Initial symptoms may resemble a common cold and the present may present with a sore throat, mild fever, muscle aches and malaise. After several days, the symptoms may progress to severe breathing problems and shock. Inhalational anthrax is usually fatal.
Gastrointestinal anthrax
The intestinal disease form of anthrax may follow the consumption of contaminated meat and is characterized by an acute inflammation of the intestinal tract. Initial signs of nausea, loss of appetite, vomiting, fever are followed by abdominal pain, vomiting of blood, and severe diarrhoea. Intestinal anthrax results in death in 25 to 60% of cases.
B. anthracis infection causes a variety of clinical symptoms such as fever, accompanied with chills and night sweats, flu, cough, chest discomfort, shortness of breath, fatigue and sore throat. Affected individuals often develop non-specific respiratory symptoms that quickly evolve to prostration and death, with overwhelming bacteraemia. The manifestations of anthrax-associated sepsis can be present in association with any form of anthrax, though they are more typically associated with pulmonary and gastrointestinal disease. Sepsis is often marked by high-grade bacteremia. Haemorrhage into body fluids, such as, cerebrospinal fluid (CSF) and pleural fluid and organs (regional lymph nodes, lung, and gastrointestinal tract) is also a characteristic feature of anthrax.
Anthrax is diagnosed by isolating B. anthracis from the blood, skin lesions, or respiratory secretions or by measuring specific antibodies in the blood of persons with suspected cases. In patients with symptoms compatible with anthrax, providers should confirm the diagnosis by obtaining the appropriate laboratory specimens based on the clinical form of anthrax that is suspected (i.e. cutaneous, inhalational, or gastrointestinal).
Prevalence and Risk Factors
Anthrax in humans, is rare. Most cases develop in people, whose occupations place them in close contact with livestock or the contaminated products of livestock such as wool, goat skin, and pelts. Direct human-to-human transmission of anthrax is extremely unlikely. In people, cutaneous anthrax accounts for about 95% of all natural infections and develops when B. anthracis enters the skin through existing cuts or abrasions. Without antibiotic treatment, the death rate from cutaneous anthrax is approximately 20%; if appropriately treated, death is rare. Intestinal anthrax results from consumption of contaminated and undercooked meat. Affected individuals may experience nausea, inappetence, vomiting, and fever, followed by abdominal pain, blood in the vomitus, and severe diarrhoea. Mortality is estimated to be 25 to 75%. Inhalational anthrax may initially present as a flu-like illness. A short period of improvement may follow, after which the patient rapidly deteriorates with high fever, respiratory distress, and shock. Case fatality rate approaches 95%, if not treated within the first 48 hours. Overall, anthrax prevalence is less in the first two decades.
Anthrax Toxin and Anthrax Vaccines
Anthrax toxin shows two distinct properties: (i) the EF/LF and PA interact only after being secreted from the bacteria; and (ii) the toxin has two A moieties (EF and LF) that enter the cell via a single B moiety (PA). The PA, EF, and LF can form toxic complexes either on the surface of receptor-bearing cells or in solution, but the cell-surface pathway is likely to be physiologically more important. The toxin, probably plays an important role early in infection, when its concentration is low. However, the soluble complexes develop late when the concentration of the toxin reaches high levels in blood.9 Also, such complexes convert to an inactive, insoluble form at neutral pH of the blood. Assembly of the three toxin proteins is initiated when PA binds to a proteinaceous cellular receptor10 and is activated by a member of the furin family of cellular proteases.11 This cleaves the molecule into two fragments: PA20 (20 kDa), corresponding to the N-terminus of the protein, and PA63 (63 kDa), corresponding to the C-terminus. The PA20 slowly dissociates from PA63 and diffuses into the surrounding medium, leaving PA63 bound to the receptor. Receptor-bound PA63 then spontaneously self-associates to form ring-shaped, heptameric oligomers.12 The EF and LF bind competitively to high-affinity sites spanning the interface of the PA63 subunits13,14 leading to formation of a series of toxic complexes containing 1 to 3 bound molecules of EF and/or LF per PA63 heptamer.
As a consequence of concern over the severity of human infection caused by these toxins and the potential for criminal usage of this organism, vaccination seems to be the most cost-effective form of mass protection. The currently licensed vaccine approved for use in humans in the United States of America (USA) against infection with B. anthracis [Anthrax Vaccine Adsorbed (AVA) - Biothrax), prepared by adsorbing filtered culture supernatants of the V770-NP1-R strain of B. anthracis to aluminum hydroxide (alhydrogel), calls for a daunting series of three injections at short intervals followed by three injections at six-month intervals.15 In addition, administration of AVA results in reactogenicity due to contamination of the actual vaccine preparation with other bacterial products like LF and EF.16 Furthermore, the efficacy of AVA has been shown to vary in different animal models and there is no direct evidence of the efficacy of this vaccine in humans.17,18 Protective antigen is the primary immunogen of the licensed vaccines being produced currently because of its central role in pathophysiology.19 However, the traces of LF and EF associated with the previous vaccine preparations contributed to the vaccine side effects.16 Therefore, an effective expression system that can provide clean, safe, and efficacious vaccine preparation is required.
4In this context, recently an E. coli based expression system for producing recombinant PA (rPA) has been developed.20 Recombinant anthrax vaccine (adsorbed) is a sterile whitish suspension. This vaccine is manufactured by recombinant deoxyribonucleic acid (DNA) technology. The rPA is purified using different physico-chemical procedures. The purified rPA is finally adsorbed on an aluminium phosphate gel in presence of different stabilizers. Evaluation of the biological activity of this rPA revealed that it mediated all the functions as the receptor binding moiety of anthrax toxin in manner similar to native PA.20 Further, as a part of the preclinical evaluation of a vaccine candidate, there is a need to study its immunological behavior in appropriate animal models. Several animal models, including guinea pigs,17,21 rhesus monkeys22,23 and rabbits,24 have been developed to study vaccine efficacies and to evaluate PA-based vaccine formulations. While non-human primates are often deemed the most desirable animal model of inhalational anthrax, the New Zealand white (NZW) rabbit is considered to be the reliable small animal model.17,23,24
Vaccine Administration and Adverse Effects
Standard dose of rPA vaccine (0.5ml) is administered by intramuscular injection. One dose of rPA vaccine comprises of 50 µg of PA as a protein. Recombinant anthrax vaccine is administered for the active immunization against B. anthracis in individuals between 18 and 65 years of age who come in contact with animal products such as hides, hair or bones that come from the anthrax endemic areas, and those who are already infected with B. anthracis spores. This vaccine is also indicated for individuals who are at high risk of exposure to B. anthracis spores such as veterinarians, laboratory workers and whose occupation involves handling of potentially infected animals or other contaminated materials. Since the anthrax infection in general population is low, routine immunization is not recommended.
Administration
A separate 22-gauge sterile needle and syringe should be used for each patient to avoid transmission of viral hepatitis and other infectious agents. A different site should be used for each sequential injection of this vaccine and the vaccine should not be mixed with any other product in the syringe. Steps involved in administration of vaccine in a particular individual are as follows: (i) shake the bottle thoroughly to ensure that the suspension is homogeneous during withdrawal and visually inspect the product for particulate matter and discard the vial; (ii) wipe the rubber stopper with an alcohol swab and allow it to dry before inserting the needle; (iii) clean the area to be injected with an alcohol swab or other suitable antiseptic; and (iv) holding the needle at a 45° angle to the skin, inject the vaccine intramuscularly.
Care should be taken to ensure that the vaccine is not injected intravenously. After injecting, the needle is withdrawn and the injection site is and gently massaged to promote dispersal of the vaccine.
This product should be stored at 2° to 8°C. It should not be frozen and shoud not be used after the expiration date written on the package.
Adverse effects
Anthrax vaccine is associated with a series of serious adverse events that can have significant impact on multiple organ systems within the body, and result in permanent disability. Anthrax vaccine contains rPA toxin combined with an aluminum adjuvant that may work synergistically, to produce temporally related adverse reactions in susceptible vaccine recipients. Soreness, redness, itching and a lump where the shot was given, muscle aches or joint aches, headaches, fatigue chills, fever and nausea are the basic side effects related to the administration of anthrax vaccine.
Precautions to avoid adverse effects
Before administration of anthrax vaccine, the immunization history of the patient should be reviewed for possible vaccine sensitivities and/or previous vaccination related adverse events in order to determine the existence of any contraindications to immunization. Pregnant women should not be vaccinated against anthrax unless the potential benefits of vaccination clearly outweigh the potential risks to the foetus. However, administration of non-live vaccines during breastfeeding is not medically contraindicated. Clinicians administering anthrax vaccine should be equipped and prepared to manage anaphylactic shock in case this complication develops.
Potential Benefits of Anthrax Vaccination
The rPA vaccine might potentially prevent the development of all milder forms of anthrax in vaccinated individuals. It could also potentially prevent cutaneous and gastrointestinal anthrax in vaccinated individuals and thereby decrease the risk of anthrax infection in non-vaccinated persons also. The efficacy of the vaccine for protection against cutaneous and inhalational forms of anthrax is estimated to be 92.5%. Anthrax vaccine, in due course, will be most beneficial for those who work directly with the organism in the laboratory, those who handle potentially infected animal products and military personnel deployed in areas with high risk of exposure to the organism. Stockpiling a supply of the vaccine can be helpful in the event of a bioterrorist attack.
Evaluation Aspects of an Efficacious and Novel Vaccine
Preclinical evaluation of immune responses is critical to the development of any novel vaccine approach. However, the relatively rare occurrence of human inhalational anthrax cases makes it impractical to perform human protection studies with these vaccines. For this reason, the Food and Drug Administration in the United States has decided that any new vaccine against anthrax can be licensed using protection data from two relevant animal models, supported by well-defined correlates of protection,25 also known as the “animal rule.” The best animal model for comparative purposes with human inhalational anthrax is the Rhesus macaque.22,23 Rabbits have been developed as a surrogate animal model in addition to the non-human primate model.24 The pathology of rabbit anthrax aerosol model is remarkably similar to the pathology observed in humans and non-human primates exposed by the aerosol route, with the exception that rabbits are more susceptible to infection and die more rapidly. But the antibody titre of non-human primates inoculated with anthrax vaccine preparations has been reported to decrease over time.26 Thus, comparative immunological studies focused on generation of immune responses in rabbits, rhesus monkeys and humans are very few. The hurdles for carrying out such studies are: (i) the time scale of the onset of disease; (ii) kinetics of protective immune response generation; and (iii) disease progression, in different models. Complete evaluation of the effectiveness of a vaccine involves demonstration of protection against live challenge. Research is needed on the proportion of anthrax caused in developing countries like India. More evidence, including the efficacy of vaccine in high risk populations such as human immunodeficiency virus (HIV) infected persons, is required. More importantly, data on cost effectiveness of the rPA vaccine according to developing countries is required, to safeguard the future vaccination against anthrax.
Acknowledgements
We would like to express our gratitude towards Department of Biotechnology (DBT), Govt. of India and Panacea Biotec Ltd. for their constant support.
Conflict of Interest: None
Key Points
- B. anthracis is a potential biological terrorism agent due to its easy dispersal in air
- Inhalational anthrax is the most fatal form of anthrax
- Protective antigen is the most immunogenic component of anthrax toxin
- Anthrax in humans is rare but inhalational anthrax results in 95% fatality if not treated within 48 hours
- The rPA vaccine is recommended for healthy individuals between 18 to 65 years of age who come in contact with infected animal products or directly with the B. anthracis in laboratory
- A standard dose (0.5ml) is administered by intramuscular injection
- Three dose schedule is recommended at 0, 1, 6 months followed by boosters at 12 and 18 months. Yearly booster afterwards is recommended for long term immunity
- The rPA vaccine might potentially prevent the development of all milder forms of anthrax
- Most importantly cost effectiveness of vaccine needs to be thoroughly examined
- Stockpiling a supply of the vaccine can be helpful in the event of bioterrorist attack.
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