$9.2 Million Grant Enables Scripps Scientists to Design Anthrax Antitoxin Nanosponges

By Jason Socrates Bardi

A large, multi-center program project grant has been awarded to a team of scientists at The Scripps Research Institute (TSRI), Harvard Medical School, and The Salk Institute for Biological Studies to discover and develop novel anthrax antitoxins and ways of delivering them.

The overall goal of the program is to design anti-anthrax nanosponges—antitoxin particles that could be administered to someone who has been exposed to anthrax.

"They would basically bind up all the toxin and render it ineffective," says TSRI Assistant Professor Marianne Manchester, who is the principal investigator on the grant.

The "program project" grant was awarded by the National Institute of Allergy and Infectious Diseases (NIAID) and provides five years of funding for five projects led by investigators at these three institutions as well as common cores that will support the projects.

The Threat of Anthrax

Anthrax is a deadly disease that is caused by infection with the bacterium Bacillus anthracis. It is an ancient disease—both Homer and Virgil wrote about a disease that was probably anthrax.

The Greeks named the disease anthrax, which means coal, because of the characteristic black ulcers that form on the skin of people and animals infected with the bacterium. This cutaneous form of the disease was responsible for widespread outbreaks among livestock through the centuries, and Louis Pasteur famously demonstrated the first anthrax vaccine in 1881, which helped confirm the germ theory of disease.

In the 20th century, the disease and the bacterium that causes it grew to infamy because of its potential as a biological weapon. Over the years, several countries developed weaponized B. anthracis spores, which cause inhalation anthrax. B. anthracis naturally forms spores when conditions are not right for the bacterium to replicate. When it converts into a spore, it can lie dormant inside its protective, almost indestructible protein coat. When spores of anthrax are breathed in, they are taken up through the lungs by cells called macrophages. The macrophages transport ingested spores to other parts of the body, where they germinate into bacteria and begin reproducing and making toxins.

Protecting against inhalation anthrax is a major public health priority, especially after the U.S. Postal Service attacks of late 2001. There must be an effective way to treat individuals who have been exposed to spores as a last line of defense.

Exposure to anthrax can be treated with antibiotics, but the effectiveness of antibiotics diminishes over time. If the exposure is not detected quickly enough, antibiotics alone may not be able to save the patient. This is because B. anthracis produces a virulent toxin that kills cells and, in high enough doses, can kill infected people. That's the rub—even if the infection is brought under control, the bacteria may have produced enough toxin to be lethal.

Finding a way to neutralize the effect of the toxins would be a great boon to public health preparedness against anthrax exposure. That's exactly what the team on the program project grant is trying to do.

Anthrax Toxin and Where It Binds

The anthrax "toxin" is actually a system of molecules composed of three separate proteins released by the bacterium. Two are virulent proteins that interact with human cells. These are the "lethal factor," which is a metalloprotease (an enzyme that chops up other proteins), and the "edema factor," which is an adenylate cyclase (a protein that makes cAMP, an important "second messenger" molecule in the body that has a variety of systemic effects).

The third protein produced by the bacterium, called protective antigen, is important for getting lethal factor and edema factor into cells. Protective antigen binds to the surface of human cells and forms a sort of cat door that allows the lethal factor and edema factor to pass through to the interior of the cell where they can do their damage. Once inside cells, the lethal and edema factors lead to cell death.

The details of how the lethal and edema factors kill cells are still somewhat murky, but what is clear is that protective antigen, lethal factor, and edema factor work together to make Bacillus anthracis deadly.

A few years ago, Professor John Collier of the Harvard University Medical School and Professor John A.T. Young, who was then at the University of Wisconsin Medical School, discovered a human receptor of the anthrax toxin and began to elucidate the structural details whereby anthrax toxin enters human cells.

When protective antigen binds to these human receptors, it inserts itself into the membrane of a cell and self-assembles into a seven-membered heptamer, with one bound protective antigen associating with six other identical protective antigen proteins and forming a seven-spiked crown sticking out of the membrane of that human cell.

This heptamer then binds to the lethal and edema factors and acts like a pore to deliver them into the cellular membrane. Normally, human cellular membranes—bilayers of fat, protein, sugars, and other molecules—would normally be impenetrable to the lethal and edema factors. But the protective antigen heptamers enable them to pass right through the membrane and into the cell.

Collier has had a distinguished career of studying the mechanisms by which bacterial toxins cause disease. In the 1960s, he showed that diphtheria toxin works by entering human cells and inactivating an intracellular target molecule.

"As time went on, more and more bacterial toxins were found to act inside cells," says Collier.

Anthrax toxins turn out to be one of these, and in the new program project grant, Collier is attempting to understand structural details of how the protective antigen binds to human receptors on the surface of a human cell by making crystal structures of the receptor and toxins together.

Young, who is now at the Salk Institute, has recently detected another human receptor—called capillary morphogenesis gene-2—to which the protective antigen also binds. In the project he is directing, Young will be looking at the interaction between the protective antigen and the capillary morphogenesis gene-2 receptor, asking how these interactions lead to the entry of the toxin.

"The discovery of a second type of anthrax toxin receptor came as quite a surprise," says Young. "We are now attempting to better understand how interactions between protective antigen and both of its receptors contribute to the pathogenesis of anthrax disease."

Collier and Young are both using this structural information to come up with ways of inhibiting the interaction of the protective antigen with the human receptors. In particular, they are asking what portions of these human receptors are necessary for the entry of the toxins and if these same parts could be used to attach to the toxins in solution.

This is the first step in designing anti-toxins—soluble peptides or other small molecules that could act like molecular decoys to grab the toxins out of the bloodstream.

Nanosponges

The decoy molecules are only half the story. The other half of is to find a good vehicle to deliver them, and that's what Manchester and several other investigators at TSRI are working on as their portion of the program project grant.

Peptides are more potent if they are displayed polyvalently, which is a way of increasing the efficacy of a drug by presenting multiple copies of it.

In fact, a few years ago, Collier identified a peptide that was not able to inhibit the binding of protective antigen to the human receptors on its own but could do so when it was made in a multimeric form where one particle displayed a score of these peptides.

"This gave us the idea that multimerization strategies could be useful," says Manchester.

Using the targets that Collier and Young produce, the scientists at TSRI are adapting technology that has been developed at the institute to display these molecular decoys in multiple numbers on the surface of nanoparticles—to make something that they call antitoxin nanosponges.

Manchester is looking at displaying the molecular decoys on the surface of particles of Cowpea mosaic virus (CPMV). CPMV withers and stunts the leaves and pods of the Vigna unguiculata plant—an important crop and source of protein in many parts of the world. Like most plant viruses, CPMV is delivered by insects into plant cells, and like most plant viruses, CPMV has little need for its viral envelope to facilitate entry into cells. All these envelopes are, basically, are rigid, stable containers—shells.

The shell of a CPMV particle is some 30 nanometers in diameter and is formed by 60 identical copies of a pair of viral proteins surrounding a single strand of viral RNA. These 60 pairs constitute 60 equivalent sites for displaying antitoxins.

Similarly, TSRI Associate Professor Anette Schneemann is working with Flock house virus particles, which infect insects, and is studying whether these will serve as an effective platform for presenting these peptides.

The Flock house virus is about 35 nanometers in diameter and is formed by 180 copies of a coat protein surrounding two strands of RNA. The 180 copies each have four potential sites for displaying an antitoxin, which means that one Flock house virus particle could potentially display 720 copies

The structures of both of these viruses were solved a few years ago by TSRI Professor John Johnson, who is also involved with the grant. This means that scientists know at the atomic level what these viruses looks like, and they can apply this knowledge to making designer viral particles.

By changing the genetic makeup of the virus to modify the capsid proteins, they can display peptides of interest on the surface of the virus without altering the virus's basic structure.

In this case, Manchester and Schneeman are taking the substances that are produced by Collier and Young and displaying them on the surface of their virions. Multiple copies of these peptides can be displayed in different locations on the particle surface, and this, they hope, will allow them to prevent the toxin from binding to the receptor and the toxin from entering the cells.

By Comparison

Collier and Young are also developing soluble forms of the antitoxins that can be attached to the particle through a chemical linker, a complementary approach to Manchester's and Schneemann's that explores attaching the antitoxins to the viral particles chemically .

TSRI Associate Professor M.G. Finn and Assistant Professor Vijay Reddy, who lead this project, will compare the viral particles to other types of scaffolds—like those made from organic polymer or tiny flecks of gold.

"We want to be able to compare a virus to a different type of nanoparticle to see which is more effective and why," says Finn.

One of the advantages of the particles is that they have the ability to traffic throughout the body and to stay in the bloodstream for a long time. These particles are also more stable in the gut than peptides would be, which makes them potentially bioavailable—an important advantage to any potential therapeutic.

The chemical or biological approaches might also be used to create a vaccine in the traditional sense by using antigen molecules from B. anthracis to stimulate an immune response. This response would have the added benefit of blocking an infection from a later exposure.

NIAID is a component of the National Institutes of Health, an agency of the Department of Health and Human Services. NIAID supports basic and applied research to prevent, diagnose, and treat infectious and immune-mediated illnesses, including HIV/AIDS and other sexually transmitted diseases, illness from potential agents of bioterrorism, tuberculosis, malaria, autoimmune disorders, asthma, and allergies. For more information, see: http://www.niaid.nih.gov.

Harvard Medical School is one of the world's preeminent institutions in medical education and research. The breadth and depth of its scientific and clinical disciplines are unsurpassed. The School has nearly 8,000 faculty and 17 affiliated facilities.

The Salk Institute for Biological Studies, located in La Jolla, Calif., is an independent nonprofit organization dedicated to fundamental discoveries in the life sciences, the improvement of human health and conditions, and the training of future generations of researchers. Jonas Salk, M.D., founded the institute in 1960 with a gift of land from the City of San Diego and the financial support of the March of Dimes Birth Defects Foundation.

The Scripps Research Institute is one of the largest, private, non-profit scientific research organizations in the world. It stands at the forefront of basic biomedical science, a vital segment of medical research that seeks to comprehend the most fundamental processes of life. TSRI is recognized for its research in molecular and cellular biology, chemistry, immunology, the neurosciences, and molecular medicine. TSRI abides by all local, state, and federal guidelines concerning environmental health and safety and biological materials.

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TSRI Assistant Professor Marianne Manchester is the principal investigator on a new, multi-center grant. Photo by Jason Socrates Bardi.