The Agent

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credit: CDC / Janice CarrElectron microscope image of Bacillus anthracis spores.

Anthrax is a serious disease that came into public prominence in 2001 during the bioterrorism attack in the United States. Anthrax is caused by a bacterium called Bacillus anthracis (B. anthracis). The name anthrax comes from the Greek word for coal and refers to the black skin lesions it produces. Descriptions of a disease affecting both animals and humans that appear to be anthrax have been found as early as Biblical times, and in fact anthrax has been suggested to have been the fifth plague described in the book of Exodus.

The anthrax bacterium was first described in 1823 and was the first bacterium ever shown to be the cause of a disease - in 1876, Robert Koch obtained a pure culture of B. anthracis and demonstrated that it caused disease by injecting it into animals. B. anthracis was also the first bacterium to be used for making an attenuated vaccine by Louis Pasteur in 1881.

B. anthracis is a large, rod-shaped bacterium that forms spores. These spores can survive in a dormant state in the environment, usually in soil, for many years, even decades. Once ingested, the spores are activated, and the bacteria begin to reproduce. Reproducing bacteria produce three different proteins that combine to form two toxins known as lethal toxin and edema toxin. The toxins cause a fatal buildup of fluid around the lungs that can kill infected cells and produce disease and death in infected animals or humans.

Anthrax is primarily a disease of livestock that become infected by ingesting spores found in soil. Humans usually become infected with anthrax by handling products of infected animals such as leather or wool or by inhaling anthrax spores from infected animal products. They can also become infected by eating undercooked meat from infected animals. Anthrax is not known to be spread person-to-person. Cases of transmission of anthrax from infected animals to humans are relatively rare in the United States, with an average of about five cases per year. However, in 2001, there were 22 cases of anthrax infection that were caused by deliberate spread through the United States Postal system. Letters containing anthrax killed five people and sickened 17 others and caused a temporary disruption of mail service and the forced evacuation of several buildings including Senate offices and the Supreme Court. After a massive and difficult seven year investigation, the Federal Bureau of Investigation concluded this case after its leading and sole suspect, an Army microbiologist, committed suicide.

Three Main Forms of Human Anthrax

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credit: CDC/ James H. SteeleAnthrax lesion on the skin caused by Bacillus anthracis.

Cutaneous anthrax occurs when the bacteria from infected animal products enter a break in the skin; black lesions occur at the site of infection. This is the most common form of anthrax and can be controlled with antibiotics if it is treated before the infection spreads through the body.

Gastrointestinal anthrax can occur from the ingestion of contaminated food and can be fatal if not treated immediately. This form is not known to have occurred in the United States.

Inhalation anthrax occurs when anthrax spores are inhaled. The spores travel to the lymph nodes near the lungs and produce toxins that cause severe breathing problems and shock. This form is very difficult to treat and is often lethal. Naturally occurring inhalation anthrax is very rare, but this was the type of anthrax infection that occurred during the bioterrorism attack of 2001. This is the most dangerous form, and half of the inhalation cases of anthrax led to death during the 2001 attack.

The Problem

The bacterium that causes anthrax is considered a highly dangerous potential agent for use in bioterrorism. It is classified as a Category A agent – the highest risk type - by the Centers for Disease Control and Prevention (CDC).

The reasons that anthrax is so dangerous are that:

  • it is highly toxic – the mortality rate is nearly 100% for the inhalation form, in the absence of treatment.
  • its spores are easily disseminated through the air.
  • the spores are extremely durable.

Although several different antibiotics exist that are effective against anthrax, the early symptoms are often confused with respiratory or gastrointestinal diseases, and once the obvious symptoms occur, it is usually too late to counteract the destructive effects of the anthrax toxins. There is also the serious concern that the anthrax bacterium could become resistant to currently used antibiotics. In fact some strains are already resistant to certain classes of antibiotics.

There is currently a vaccine against the bacterium that causes anthrax, but its use is restricted to military personnel and workers with an occupational risk for anthrax exposure such as laboratory workers or individuals having direct contact with livestock. Its safety or effectiveness in children, the elderly, and people with weakened immune systems has not been determined. The major problem with the current vaccine, however, is that it is given as multiple doses over an 18-month period, so that it is unlikely to provide much protection to the general public in the event of a widespread terrorist attack.

The Research

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credit: CDC/ Judy SchmidtWoman receiving an intramuscular vaccine injection

In the Department of Molecular Virology and Microbiology at Baylor College of Medicine, research on anthrax is being performed in several areas – vaccine evaluation and development, antibiotic resistance, the identification and analysis of all of the proteins that make up the anthrax bacterium (an approach called proteomics), and an investigation into the mechanisms by which B. anthracis acquires the essential nutrient iron and overcomes a normal host defense against infection.

There is currently only one vaccine licensed in the United States against anthrax, anthrax vaccine adsorbed (AVA, also known as BioThrax). Its use is limited to healthy adults with a high risk of exposure. The recommended vaccine schedule is six doses administered over the course of 18 months. AVA is delivered subcutaneously (under the skin) and is commonly associated with reactions at the site of injection. Due to the number of injections required, the length of time needed to acquire immunity, and frequent side effects, a new vaccine – or at the very least an improved AVA vaccination schedule – is needed.

The anthrax bacterium produces several toxins that are responsible for many of the disease manifestations. Antibodies to one part of the toxins - the protective antigen (PA) have been found to protect against infection and disease. Therefore, most vaccines for protection against anthrax, including AVA, consist of or contain the PA protein.

Evaluation of new ways of delivering the licensed AVA vaccine and development of new vaccines based on PA have been a major focus of the BCM Vaccine Research Center headed by Dr. Wendy Keitel. A number of studies and clinical trials have been conducted to assess a diverse group of licensed and experimental anthrax vaccines.

In one study, MVM researchers Drs. Keitel and Hana El Sahly participated in a congressionally-mandated, CDC-sponsored phase 4 clinical trial of AVA. The goal was to develop a simpler and better-tolerated way of giving the vaccine. Healthy volunteers were the given the standard number of AVA doses under the skin or into the muscle, or reduced dose schedules into the muscle. Reactions and immune responses after vaccination were compared between the different groups or control groups receiving only salt water, and antibody and cell-mediated immune responses were measured and correlated with responses that are associated with protection in animal models.

The results of this study indicated that the initial three or four doses of AVA given in the muscle provided immunological protection comparable to four standard injections under the skin, but with the benefit of significantly reducing the incidence of reactions at the site of injection. This study demonstrated that it is possible to eliminate a dose and to change the administration route from the skin to the muscle to reduce adverse effects at the injection site without compromising the effectiveness of the vaccine. A change in the recommended anthrax vaccine schedule has since been approved by the FDA. Reducing the number of times the vaccine must be given will reduce the cost and increase the number of doses available for use. The need for subsequent booster doses is under study.

Although minimizing side effects and reducing the number of doses needed of the current vaccine is beneficial, the ultimate goal is to develop a safe, more effective vaccine that would provide immunity to anthrax within a shorter period of time. To rationally develop a new anthrax vaccine, scientists need to understand how the anthrax vaccine works in humans to elicit an antibody response and provide protection. For this purpose, Dr. Keitel and colleagues have conducted studies to analyze in detail the interactions between PA, the primary immunogenic component of the current vaccine, as well as proposed next-generation anthrax vaccines, and the antibodies produced in humans. The results of this work will guide researchers in the future design of more effective anthrax vaccines.

One candidate for a new anthrax vaccine is based on a purified recombinant PA protein. Dr. Keitel and colleagues have completed a phase I clinical trial of the purified recombinant PA protein vaccine (rPA102). The goals of this study were to evaluate the safety and immunogenicity of the vaccine, and to select dosage levels for evaluation in a larger phase II clinical trial. Healthy volunteers received different dose amounts of rPA120 administered into the muscle on three occasions over the course of 8 weeks, while some volunteers received AVA, the currently licensed vaccine. The conclusions of this study were that the rPA120 vaccine did not cause serious side effects, and that the vaccine was effective in producing an anti-PA antibody response that increased with increasing vaccine dose levels and number of doses. Neutralization activity at the highest rPA120 dosage was similar to that seen in subjects receiving AVA injections. This vaccine will require further testing in additional and larger studies.

Another candidate for an anthrax vaccine is composed of two segments of DNA that express PA and lethal factor (LF), two of the three components of anthrax toxins. In a VTEU-supported study, Drs. Keitel and El Sahly and colleagues, evaluated the safety of the DNA-based vaccine and tested whether it could stimulate antibody responses that are associated with protection against infection. Healthy adults were given different amounts of the DNA-based vaccine (known as VCL-AB01), or a placebo, administered in three doses over the course of two months. The vaccine was generally well tolerated by the study participants. A greater percentage of subjects developed antibodies to PA or LF among the groups receiving the higher doses of the vaccine.

The researchers further tested the DNA-based anthrax vaccine in nonhuman primates to evaluate immunogenicity and to determine if the vaccine could provide protection against challenge with a lethal dose of B. anthracis spores. Low levels of antibody were detected in the monkeys after vaccination. Importantly, 75% of the animals survived the lethal challenge. The conclusion of this study was that the vaccine provided immunity at doses that are generally well tolerated by humans.

In addition to vaccine evaluation and development, several other lines of research are ongoing within the department. One group of MVM researchers is studying antibiotic resistance of the anthrax bacterium. Treatment for anthrax infections includes use of the antibiotics ciprofloxacin, doxycycline, and penicillin G. The problem is that some strains of the anthrax bacterium are naturally resistant to penicillin, so that penicillin is ineffective in treating patients with these particular strains. Resistance to penicillin is often due to enzymes made by bacteria that are called β-lactamases (these enzymes basically break apart penicillin). One strain of B. anthracis makes two such enzymes (called Bla1 and Bla2). Dr. Timothy Palzkill and his group are studying these enzymes to learn in detail how they allow some strains of B. anthracis to resist antibiotics. Their goal is to develop compounds that can inhibit the action of the β-lactamase enzymes, so that they are no longer effective in blocking the action of antibiotics.

A further area of anthrax research utilizes an approach called functional genomics. Drs. Joseph Petrosino, George Weinstock, and Timothy Palzkill are studying the genome sequences of different B. anthracis strains and using this information to understand critical areas of B. anthracis biology. The genome sequence of multiple B. anthracis strains and other Bacillus species has been determined. Two independent virulence plasmids distinguish B. anthracis from other Bacillus species. These plasmids, required for B. anthracis virulence, encode 224 genes, including the three anthrax toxin subunits (Protective Antigen-PA, Lethal Factor-LF, and Edema Factor-EF). Because of their uniqueness to B. anthracis, these virulence plasmid genes have been targeted for further study.

Drs. Petrosino and Weinstock have cloned each of the 224 virulence plasmid genes for the purpose of expression and purification in laboratory strains of Escherichia coli. Purified proteins are being used in immunological screens with immune sera from Rhesus macaque monkeys that have been infected with B. anthracis (in collaboration with Dr. Conrad Quinn, CDC and Dr. Johnny Peterson, UTMB). The screens are designed to identify virulence plasmid-encoded proteins recognized by the macaque immune response to anthrax. Subsequent screens will test sera from human volunteers immunized with the current anthrax vaccine to determine which virulence plasmid-encoded proteins are present in the vaccine and are recognized by the human immune response. The proteins identified will be unique to anthrax and may be ideal candidates for further study in the development of a new generation of anthrax subunit vaccines and diagnostic tools.

Studies by Dr. Anthony Maresso investigate iron acquisition by B. anthracis. Iron is an essential nutrient used by almost all organisms. Bacterial pathogens must acquire iron in order to grow inside mammalian hosts. The host, however, limits the availability of free iron, thereby providing an effective defense strategy against infection. In response, bacteria have evolved clever ways to subvert host sequestration of iron. Dr. Maresso has uncovered secreted proteins produced by B. anthracis which specifically bind and transport the body’s prominent iron-carrier molecule, heme. The acquisition of mammalian heme may allow B. anthracis to attain enough iron to grow to very high densities during infection. Dr. Maresso has also uncovered a group of small molecule inhibitors which specifically target and inactivate enzymes in the pathway of iron acquisition in pathogens like B. anthracis. An understanding of the mechanisms of iron uptake in B. anthracis will allow for the development of therapeutic agents to combat infections by related Gram-positive bacteria.