Outreach Lecture Series Volume 19

Outreach Lecture Series Volume 19
Biological Weapons and Bioterrorism The History, the Danger, and What American Science is Doing About It by Dr. Brent L. Iverson Outreach Lecture Series Volume 19 Produced by and for the Outreach Lecture Series of the Environmental Science Institute. We request that the use of any of these materials include an acknowledgement of Dr. Brent L. Iverson and the Outreach Lecture Series of the Environmental Science Institute of the University of Texas at Austin. We hope you find these materials educational and enjoyable.

The University of Texas at Austin
Biological Weapons and Bioterrorism The History, the Danger, and What American Science is Doing About It Biological Weapons and Bioterrorism: The History, the Danger, and What American Science is Doing About It Dr. Brent L. Iverson The University of Texas at Austin The University of Texas at Austin Dr. Brent L. Iverson

Take Home Lessons: Biological weapons are cheap to make and easy to conceal. They have been of little military significance thus far, but of tremendous value from a propaganda perspective Points 1 and 2 make biological weapons ideal for terrorism American scientists are still playing “catch-up”, but have created several promising approaches to reduce the threat of biological weapons

What are Biological Weapons?
Biological weapons are any disease causing bacteria, virus, or natural toxin that can be used against an enemy Anthrax HIV Ebola Influenza A Definitions: bacteria: tiny, unicellular, prokaryotic organisms that reproduce by cell division and usually have cell walls; can be shaped like spheres, rods or spirals and can be found in virtually any environment. virus: submicroscopic infectious parasites of plants, animals, and bacteria, which often cause disease. Replicates only within cells of living hosts. toxin: a poisonous substance Images: Clockwise from top left: Gram stain of cerebrospinal fluid showing B. anthracis in Bioterrorism-Related Inhalational Anthrax: The First 10 Cases Reported in the United States, Emerging Infectious Diseases, Centers for Disease Control and Prevention. Scanning electron micrograph of human immunodeficiency virus (HIV), grown in cultured lymphocytes. Virions are seen as small spheres on the surface of the cells. PHIL Photomicrograph of Bacillus anthracis; anthrax. PHIL Colorized Transmission Electron Micrograph of the Ebola Virus. Hemorrhagic Fever, RNA Virus. PHIL Transmission electron micrograph of influenza A virus, early passage. PHIL Scanning electron micrograph of a preparation of Bacillus anthracis spores. Two elongated bacilli are also present among the oval-shaped spores. Original magnification x Photograph: Courtesy of John Ezzell, Ph.D., US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Md.

There is a so-called “Australia Group list” of potential bioweapons for use against humans that has 20 viruses (ex. smallpox, Ebola), 13 bacteria (ex. anthrax, plague), and 19 toxins (ex. botulinum toxin, ricin) Australia Group List

Some are infectious, some are not, some kill you quickly, some only make you feel real bad. Almost all start out with mild symptoms……… “I thought it was the flu” clip art images: Copyright © 2000 Discovery.com, Inc. There is an analogous list for crop plants and livestock

Highly contagious agents are easy to spread on a small scale, just have an infected individual walk through an airport. Photo credits: Cynthia Yates

Non-contagious agents (anthrax or botulinum toxin) or a large scale attack require “weaponization” - making the agents in a form that causes maximum infection and/or death. “Weaponization” involves making particles of just the correct small size (a few microns) to 1) “float” in air instead of settling, 2) evade body’s “particle” defenses, and 3) penetrate deep into lungs. The germs or toxins are adsorbed onto inert particles of the appropriate size. This process is the “secret” of germ warfare. Miller, J. et al., “Germs”, Simon and Schuster, 2001

Why is anthrax a popular bioweapon? Anthrax is natural bacteria found in environment - easy to get Has two states: quickly growing bacteria that kill mammalian host spore state that survives in environment for decades. Spores can be mass produced using common equipment Anthrax is infectious, but not contagious so killing can be more easily “controlled” Does 8,000 – 10,000 spores sound like a lot? You would probably not even be able to see them with the naked eye. In weaponized form, as few as 8, ,000 inhaled spores will kill a healthy adult. Meselson, M. et al., Science, 1994, 266, Mock, M. and Fouet, A., Ann. Rev. Microb., 2001, 55,

An Abbreviated History
Biological Weapons have been contemplated since antiquity Image of trebuchet on left: Trebuchet by Kolderer, c1507 Image on right: Tartars from the Crimea, The History of Costume, By Braun & Schneider c Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are two often cited examples: 14th Century - Tartars’ siege of the city of Kaffa. The Tartars catapult cadavers infected with plague into city see image of Kaffa at Suggested images: 17th Century Woodcut of Kaffa, Copyright the Institute of Nautical Archaeology Trebuchet see image of trebuchet at Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are two often cited examples: 14th Century - Tartars’ siege of the city of Kaffa. The Tartars catapult cadavers infected with plague into city Although plague did eventually lead to the surrender of Kaffa, most experts doubt the cadavers were effective This plague was a highly fatal infectious disease caused by the bacterium Yersinia (syn. Pasturella) pestis, transmitted primarily by the bite of a rat flea. It was also referred to as “Black Death.” Plague epidemic and infected rodents were already in the area (hence the cadavers), the fleas that transmit the disease greatly prefer plague infected rodents to cadavers Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are two often cited examples: 18th Century - British commander Sir Jeffrey Amherst orders blankets used in smallpox clinic given to Native Americans as gifts General Jeffrey Amherst Information about General Amherst: Portrait of Amherst by Sir Joshua Reynolds in Sir Gilbert Parker's Old Quebec (New York: MacMillan, 1903). Sir Jeffrey Amherst Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are two often cited examples: 18th Century - British commander Sir Jeffrey Amherst orders blankets used in smallpox clinic given to Native Americans as gifts Smallpox is spread most effectively through direct inhalation of respiratory droplets (coughing), blankets were likely not that contagious Although a devastating smallpox epidemic did occur among Native Americans at this time, previous contact with early settlers is likely the major cause Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are confirmed military examples: Various native warriors dipped arrows and spears in biological poisons (ex. “arrow frogs” of South America). Photo credits: copyright 2002 Nelson Guda Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are confirmed military examples: In WWI, Germans targeted livestock and cavalry horses in various European countries using animal specific diseases. This was apparently very successful. Suggested image: Cavalry in mist of gas Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are confirmed military examples: In WWII, Japanese admitted to launching at least 11 attacks on Chinese cities using pathogens including Anthrax, Cholera, Salmonella, and Plague. Photo on left: Control Number NLR-PHOCO-A-65386(36), Media Photographs, Descr. Level Item, Collection PHOCO, Series A Item65386(36), Title Camouflaged and poorly equipped Chinese soldiers repell a charge of 50,000 Japanese along the Salween River near Burma, Production Date ca.06/1943, Creating Indiv Roosevelt, Franklin D., Subject Ref WWII; World War II, Geographic Ref China; Burma, Access Unrestricted, Use Restrictions None, Contact Franklin D. Roosevelt Library (NLR), 4079 Albany Post Road, Hyde Park, NY PHONE: FAX: Photo on right: Control Number NWDNS-306-PS-51(7134) Media Photographs, Descr. Level Item, Record Group 306, Series PS Item 51(7134), Title At the United Nations' prisoner-of-war camp at Pusan, prisoners are assembled in one of the camp compounds. The camp contains both North Korean and Chinese Communist prisoners., Production Date 04/1951, Creating Org. United States Information Agency, General Note Use War and Conflict Number 1496 when ordering a reproduction or requesting information about this image, Contributors photographer, Gahn, Access Unrestricted, Use Restrictions None, Contact Still Picture Branch (NWDNS), National Archives at College Park, 8601 Adelphi Road, College Park, MD PHONE: FAX: Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are confirmed military examples: In WWII, Japanese admitted to launching at least 11 attacks on Chinese cities using pathogens including Anthrax, Cholera, Salmonella, and Plague. Unknown civilian casualty numbers, but believed to be in the tens of thousands, in addition to over 10,000 deaths of Chinese prisoners used for experiments! Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity The main impact of biological weapons has been propaganda: China, the Soviet Union, and North Korea accused the US of using biological weapons in the Korean war. Photo on left: SC An American mortar crew fires on the Communist North Korean invaders. 11 July Near Chochiwan, Korea. Signal Corps Photo #FEC (Turnbull) Photo in center: US Army Photo on right: SC KOREAN CONFLICT A .50 Cal. Machine gun squad of Co. E, 2nd Battalion, 7th Regiment, 1st Cavalry Division, fires on North Korean patrols along the north bank of the Naktong River, Korea. 26 August 1950. Korea. Signal Corps Photo #8A/FEC (Sfc. Riley) Leitenberg, M. Crit. Rev. Microb., 1998, 24, Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity The main impact of biological weapons has been propaganda: Allegations have now been shown to be fraudulent, but this proved to be extremely effective campaign that took the US years to overcome. During the Cold War, superpowers traded accusations without substantiation …….until Soviet defections..…..These were very successful and powerful propaganda campaigns since you do not need a “smoking gun” i.e. a missile or bomb to make accusation. Leitenberg, M. Crit. Rev. Microb., 1998, 24, Christopher, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are confirmed recent terrorist/criminal examples: 1984 in rural Oregon a religious cult infected 751 residents with food poisoning through Salmonella contamination at 10 restaurants in an attempt to win local elections. Christopher, et al., JAMA, 1997, 278, Torok, et al., JAMA, 1997, 278, Kolovic, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are confirmed recent terrorist/criminal examples: Early 1990’s the Japanese Aum Shrinrikyo cult released Anthrax in Tokyo, but no known victims. Apparently, this was not “weaponized” correctly. Christopher, et al., JAMA, 1997, 278, Torok, et al., JAMA, 1997, 278, Kolovic, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are confirmed recent terrorist/criminal examples: 1996 the pathogen that causes dysentery was introduced into pastries in the break room of the St. Paul’s Medical Center in Dallas, infecting 45. Christopher, et al., JAMA, 1997, 278, Torok, et al., JAMA, 1997, 278, Kolovic, et al., JAMA, 1997, 278,

Biological Weapons have been contemplated since antiquity There are confirmed recent terrorist/criminal examples: September of 2001 Anthrax laden letters sent to several locations in US. 22 confirmed cases of anthrax were reported, 11 cases of inhalation anthrax, 5 deaths. photo credits: FBI Inglesby, et al., JAMA, 2002, 287,

Biological Weapons have been contemplated since antiquity There are confirmed recent terrorist/criminal examples: September of 2001 Anthrax laden letters sent to several locations in US. 22 confirmed cases of anthrax were reported, 11 cases of inhalation anthrax, 5 deaths. A remarkable level of publicity surrounded the attack, leading to a widespread awareness and fear of bioterrorism that persists today. With relatively little effort, a relatively small number of victims, and minimal risk of apprehension, perpetrator generated a great deal of terror. Inglesby, et al., JAMA, 2002, 287,

Military Production of Biological Weapons
During/After WWII, many nations carried out research and produced offensive biological weapons. (Belgium, Canada, France, Great Britain, Italy, Japan, the Netherlands, Poland, USSR, US) Fidler, D. Microb. And Infect., 1999, 1,

Currently 12 nations are thought to have offensive biological weapons (aka “poor man’s nuclear weapons”), including Cuba, North Korea, Libya, Syria, Iran, and Iraq. Image credit: Iona Williams Fidler, D. Microb. And Infect., 1999, 1,

Currently 12 nations are thought to have offensive biological weapons (aka “poor man’s nuclear weapons”), including Cuba, North Korea, Libya, Syria, Iran, and Iraq. There have been several international attempts to stop the proliferation of biological weapons with treaties, but none have proven effective. Image credit: Iona Williams Fidler, D. Microb. And Infect., 1999, 1,

U.S. Bioweapons Research
Military Production of Biological Weapons During the Cold War, the US had an active bioweapons program until Richard Nixon unilaterally dismantled it in 1969 and 1970 since it was not militarily significant and was a propaganda liability. U.S. Bioweapons Research Through 1970 BC* *”Before Cloning” In this building in Fort Detrick, Maryland, anthrax spores for weapon production were manufactured during the offensive US biological weapon program. Photo: van Aken/Sunshine. Additional photo of scientists working at Fort Detrick: Miller, J. et al., “Germs”, Simon and Schuster, 2001

“High Tech” Russian Bioweapons Research
Military Production of Biological Weapons “High Tech” Russian Bioweapons Research Through 2002 AC Photo on left: In this building in Fort Detrick, Maryland, anthrax spores for weapon production were manufactured during the offensive US biological weapon program. Photo: van Aken/Sunshine. Photos (on right) credit: CBC/SRC

The Russians built an immense bioweapons production program, at one time employing ~60,000 scientists, engineers, and technicians. Photo credit: CBC/SRC Miller, J. et al., “Germs”, Simon and Schuster, 2001

The Russian bioweapon manufacturing and research capacity defied all reason, and was apparently underappreciated by US government until two key defections V. Pasechnik (1989) and K. Alibekov (1992). Photo of Pasechnik credit: CBC/SRC Interview with K. Alibekov: Pasechnik Miller, J. et al., “Germs”, Simon and Schuster, 2001

The Russian bioweapon manufacturing and research capacity defied all reason, and was apparently underappreciated by US government until two key defections V. Pasechnik (1989) and K. Alibekov (1992). Photo of Pasechnik credit: CBC/SRC Bioweapons facilities could be used for legitimate pharmaceuticals, so impossible to confirm via spy photos. US was fooled for decades! Pasechnik Miller, J. et al., “Germs”, Simon and Schuster, 2001

TONS PER YEAR (Yes, this is in TONS)
Military Production of Biological Weapons Soviet Bioweapons Production listed as TONS PER YEAR (Yes, this is in TONS) E. tularensis (Tularemia) 1,500 variola virus (Smallpox) 100 Yersinia pestis (Plague) 1,500 Marburg virus (Hemorrhagic) Photo of Pasechnik credit: CBC/SRC B. anthracis (Anthrax) 4,500 Pasechnik

TONS PER YEAR (Yes, this is in TONS)
Military Production of Biological Weapons Soviet Bioweapons Production listed as TONS PER YEAR (Yes, this is in TONS) E. tularensis (Tularemia) 1,500 variola virus (Smallpox) 100 Yersinia pestis (Plague) 1,500 Marburg virus (Hemorrhagic) Photo of Pasechnik credit: CBC/SRC B. anthracis (Anthrax) 4,500 Pasechnik Enough to kill 7.8 billion people per year!

In 1979 a few milligrams to 1 gram of weaponized anthrax were released from manufacturing plant in Sverdlovsk, Russia. 1 gram is about the size of 1 or 2 aspirin tablets. Images copyright: Reprinted with permission from Meselson, M., Science 266, 1202 (1994). Copyright 1994 American Association for the Advancement of Science Reprinted with permission from Meselson, M., Science 266, 1202 (1994). Copyright 1994 American Association for the Advancement of Science

In 1979 a few milligrams to 1 gram of weaponized anthrax were released from manufacturing plant in Sverdlovsk, Russia. Downwind of the accidental release, 66 people died of 77 known patients. The U.S. was not able to confirm this until 1994. 1 gram is about the size of 1 or 2 aspirin tablets. Meselson, M., Science, 1994, 266,

Iraq built a bioweapons program in about 5 years, and completely hid it from the outside world. It took UNSCOM’s inspectors 4-5 years to find the Iraqi program after they were already inside Iraq!!! At the start of the Gulf war, Iraq had enough anthrax and botulinum toxin to seriously hurt allied forces, but apparently no good way to disperse lethal aerosols. Zilinskas, R., JAMA, 1997, 278, Zilinskas, R., JAMA, 1997, 278,

Iraq built a bioweapons program in about 5 years, and completely hid it from the outside world. It took UNSCOM’s inspectors 4-5 years to find the Iraqi program after they were already inside Iraq!!! Experts believe Iraq could rebuild its bioweapons manufacturing capabilities in around 6 months. Zilinskas, R., JAMA, 1997, 278, UNSCOM: United Nations Special Commission sent to investigate bioweapons production in Iraq Zilinskas, R., JAMA, 1997, 278,

Iraq built a bioweapons program in about 5 years, and completely hid it from the outside world. It took UNSCOM’s inspectors 4-5 years to find the Iraqi program after they were already inside Iraq!!! Iraq’s current missile and aerosol dispersal capabilities are unknown. Zilinskas, R., JAMA, 1997, 278, UNSCOM: United Nations Special Commission sent to investigate bioweapons production in Iraq Zilinskas, R., JAMA, 1997, 278,

Iraq built a bioweapons program in about 5 years, and completely hid it from the outside world. It took UNSCOM’s inspectors 4-5 years to find the Iraqi program after they were already inside Iraq!!! UNSCOM left Iraq in 1998. Zilinskas, R., JAMA, 1997, 278, UNSCOM: United Nations Special Commission sent to investigate bioweapons production in Iraq Zilinskas, R., JAMA, 1997, 278,

Take Home Lessons So Far:
Biological weapons are cheap to make and easy to conceal. They have been of little military significance thus far, but of tremendous value from a propaganda perspective Points 1 and 2 make biological weapons ideal for terrorism

What American Scientists Are Doing About It:
A lot! Better detectors/surveillance systems and medical testing to quickly identify biological agent threats Two different approaches to defend against a bioweapons attack: Immunization and Treatment

Immunizations work. HOWEVER… The problem with immunizations (was the basis for Russian strategy): Takes around 1 year to develop a new bioweapon,and takes US 10 years to develop and get FDA approval of vaccine! Vaccines must be approved on a case by case basis. Stephan Johnston of UT Southwestern may have found the answer: a generalizable strategy for vaccine development Tang and Johnston, Nature, 1992, 356, 152-4

Johnston’s Approach: Immunize with genetic material from virus or bacteria, not the whole organism Revolutionary idea that works. No risk of infection, since only part of genetic material used Simply change sequence of genetic material for new organism, so new vaccines are much, much faster to develop, produce, and FDA approve!

We are using antibodies that have been modified with state-of-the-art genetic engineering to fight bacteria after a patient is infected. Photo credits: All photos by Charles Tischler, UT College of Engineering

We are using antibodies that have been modified with state-of-the-art genetic engineering to fight bacteria after a patient is infected. The approach is general and could be applied to other bacteria and toxins alike. The idea is an old one, we have just improved upon it by incorporating engineered antibodies that can take on even infections as deadly as anthrax

Anthrax: The Deadly Toxins Kill You, Not the Bacteria
In a 1974 report, the World Health Organization panel concluded that 50 kgs of anthrax spores released from a small plane under proper atmospheric conditions would kill 95,000 out of a 500,000 population center. Spores are inhaled, then they germinate. (8, ,000 inhaled spores are fatal). Vegetative bacteria multiply to very high levels- “I thought it was the flu”. Initial symptoms mild, so a fatal infection is present BEFORE unsuspecting patient seeks treatment Anthrax [AN-thracks] is a bacterial disease that mainly affects animals. In rare cases, it can spread to people and cause life-threatening illness. Anthrax is most common in areas where people raise livestock and where public health programs are lax. Animals get anthrax by grazing on soils contaminated with anthrax spores. Anthrax in people is usually the result of a work-related exposure to infected animals or contaminated animal products. To prevent anthrax, avoid contact with livestock and animal products when in countries where anthrax is common. A vaccination is available for people at high risk for work-related exposures. Bacteria release toxins - rapidly leads to septic shock-like symptoms and patient dies

Adenylate cyclase that leads to swelling in cutaneous anthrax
The Three Anthrax Toxins PA Toxin Punches hole in target cell membrane - allows other toxins to enter cell LF Toxin Protease that cleaves important proteins inside cell - leads to septic shock-like symptoms, death EF Toxin Adenylate cyclase that leads to swelling in cutaneous anthrax What is the infectious agent that causes anthrax? Anthrax is caused by Bacillus anthracis, a bacterium that lives naturally in certain types of soil. The bacterium produces spores. Spores are hardy forms of the bacterium that can survive in soil or on contaminated objects for years.

Anthrax PA Toxin - The Key Toxin
7 Fully assembled, active form of PA toxin Seven individual PA toxin molecules assemble themselves into a large ring structure that is the active form. The ring structure forms a pore through which the other toxins (EF and LF toxins) enter the interior of the target cell in a process that is not yet completely understood. Once in the cell’s interior, the EF and LF toxins cause damage and ultimately death. Thus, blocking the action of the PA toxin will prevent the other toxins from getting to where they can do damage. Structure of anthrax PA toxin

How The Anthrax Toxins Work (And How To Stop Them)
Block receptor binding This is a complicated cartoon that describes all the known details of how the PA toxin operates. For our purposes, the essential feature is that the PA toxin molecules bind to the target cells (step 2), especially immune system cells called macrophages, then seven PA toxin molecules form a ring structure (step 4). At this point, the EF or LF toxins bind to the ring (step 5), and after a couple of steps that are not fully understood (steps 6 and 7), the EF or LF toxin molecules are transported into the cell’s interior, where they start wreaking havoc. The bottom line is that the EF and the LF toxins are the ones that do all the damage, but they are harmless unless they are transported into the cell by the assembled PA toxin ring. In essence, blocking PA effectively neutralizes all of the anthrax toxins. Figure adapted from Miller et al., Biochem, 38:10432 (1999) *Little et al., Infect Immun, 56:1807 (1988) Bradley et al., Nature 414:225 (2001)

How To Treat Late Stage Anthrax
Antibiotics kill the bacteria, but do nothing to neutralize the toxin already present - Explains why 5 people died after reaching the hospital last fall. They did not feel sick enough to enter hospital until they already had a fatal amount of toxin in them. New approach: give specific agent that neutralizes the toxins along with antibiotics - an extremely powerful antibody, should save these late stage patients. What are the signs and symptoms of anthrax? In the body, anthrax spores produce a powerful toxin (poison) that causes the signs and symptoms of illness. The signs and symptoms vary depending on how a person was infected. Infection by skin contact: Most cases occur by skin contact. Skin infection begins as a raised itchy bump that looks like an insect bite. Within 1-2 days, it develops into a boil-like sore and then a painless ulcer with a characteristic dark (dying) area in the center. The infection can also cause swelling of the lymph glands near the site. About 20% of untreated cases will result in death. With proper treatment, deaths from this type of anthrax are rare. Infection by inhalation: People who get anthrax by breathing in spores have symptoms that are like a common cold. After several days, the symptoms can progress to severe breathing problems and shock. This type of anthrax usually results in death in 1-2 days after the start of severe symptoms. Infection by ingestion: Intestinal infections from eating contaminated meat are rare. The infection causes severe inflammation of the intestinal tract. The first signs are nausea, loss of appetite, vomiting, and fever, followed by abdominal pain, vomiting of blood, and severe diarrhea. Intestinal anthrax results in death in 25% to 60% of cases. How soon after exposure do symptoms appear? Symptoms usually appear within 7 days. How is anthrax diagnosed? Anthrax is usually diagnosed by isolating the bacterium from the blood, skin lesions, or respiratory discharges. NOTES CONTINUED ON NEXT PAGE

Antibodies Carry out the “friend vs. foe” recognition mission of the immune system The most exquisite and versatile family of recognition molecules known Extremely useful for detection purposes - form the basis of many medical diagnostics Have two binding sites binding site (active site): a specific region of an enzyme where a substrate binds and catalysis takes place NOTES FROM PREVIOUS SLIDE (cont) Who is at risk for anthrax? When anthrax affects humans, it is usually due to a work-related exposure to infected animals or their products. Workers who are exposed to dead animals and animal products from countries where anthrax is common can become infected. There is little risk to most U.S. travelers to other countries. The greatest risk comes from handling rugs and handicrafts made from goat skin or goat hair. Spores can live indefinitely in wool, blankets, and other animal products. How common is anthrax? Anthrax is most common in animals in agricultural regions of the world. Anthrax is rare in humans. An estimated 20,000 to 100,000 cases occur yearly worldwide, mostly in developing countries. Anthrax is very rare in the United States and in other countries where animals are inspected before and after slaughter. Anthrax is also rare in U.S. travelers, although certain handicrafts might be contaminated and should be avoided. Where can I find more information about anthrax? Agencies CDC, NCID, Division of Bacterial and Mycotic Diseases, Meningitis and Special Pathogens Branch Websites Information regarding anthrax and the recent bioterrorism activities is available at: Genetic engineering can be used to create molecules that only contain the binding sites. These have many advantages

with the binding sites highlighted
Antibodies The antibody molecule The antibody molecule with the binding sites highlighted

Anti-toxin Antibodies
Old technology: Polyclonal anti-toxins and anti-venoms, “tried and true” Developed 1890s, and widely used before antibiotics Block toxin function Behring & Kitasato, Dtsche Med Woch 49: 1113 (1890) Lucchesi & Gildersleeve, JAMA 116: 1506 (1941) Cassadevall, Clin Immun 93:5 (1999) Photo credits (from left to right): Title: Western Diamondback Rattlesnake, Creator: Stolz, Gary M., Source: WO , Publisher: U.S. Fish and Wildlife Service, Contributor: DIVISION OF PUBLIC AFFAIRS, Language: EN – ENGLISH, Rights: (public domain) Title: Texas Diamon-backed rattlesnakes, Creator: Carley, J., Source: WO , Publisher: U.S. Fish and Wildlife Service, Contributor: DIVISION OF PUBLIC AFFAIRS, Language: EN – ENGLISH, Rights: (public domain) Title: Cottonmouth Snake, Creator: Perry, Matthew, Source: WO_2428, Publisher: U.S. Fish and Wildlife Service, Contributor: DIVISION OF PUBLIC AFFAIRS, Language: EN – ENGLISH, Rights: (public domain) Title: Coral Snake, Creator: Goldman, Luther C., Source: WO-416, Publisher: U.S. Fish and Wildlife Service, Contributor: DIVISION OF PUBLIC AFFAIRS, Language: EN – ENGLISH, Rights: (public domain)

Old technology: Polyclonal anti-toxins and anti-venoms, “tried and true” Developed 1890s, and widely used before antibiotics Block toxin function Drawbacks Serum sickness, Lot-to-lot variability,Contamination, Low titre, Expensive, Limited targets, Not powerful enough for some of the most deadly toxins Behring & Kitasato, Dtsche Med Woch 49: 1113 (1890) Lucchesi & Gildersleeve, JAMA 116: 1506 (1941) Cassadevall, Clin Immun 93:5 (1999) New technology: Use much more powerful “engineered” monoclonal antibodies

Antibody X Cell Toxin The basic idea behind our approach is that the antibody binds to the anthrax PA toxin before the toxin can bind the cell. If the antibody can be engineered to not let go of the toxin before the toxin is cleared from the body, then anthrax symptoms and death can hopefully be avoided. The general scheme

The Idea: Bind The PA Toxin And Don’t Let Go
Key cellular recognition site Engineered antibody PA toxin A proposed scheme for anthrax PA binding by a therapeutic engineered antibody. The antibody binds to the part of the PA toxin molecule that attaches to the target cell, thereby preventing PA toxin action. Such antibodies, if powerful enough, should serve as an antidote to late stage anthrax infections. Antibody neutralizes PA toxin by blocking key site

Engineered Antibody Fragments
Whole IgG Fab scAb scFv Genetic engineering can be used to create various antibody fragments that still retain the full binding properties of an intact antibody. By making the antibody fragments, as opposed to whole antibodies, researchers can control various aspects of antibody behavior, providing many technical advantages. For a variety of reasons, we have initially investigated anthrax therapeutic antibodies in the so-called scFv and scAb forms.

Whole antibody scAb scFv
Two different representations of scAb and scFv antibody fragments, with a whole antibody shown above for reference.

Antibodies Are Very Complex. How Do We Improve Them?
This is an antibody binding site showing all the atoms as small spheres and bonds as sticks. With this representation, it is clear that the antibody binding site is complicated indeed. In fact, despite intense effort, the complicated nature of the antibody has made it impossible to reproducibly design better antibodies from first principles as a designer would engineer a new device or integrated circuit. Instead, scientists must engineer antibodies using powerful methods modeled after natural selection using a form of natural selection that is controlled in the laboratory.

How To Improve An Antibody: Evolution
Charles Darwin: Random DNA changes, then natural selection of organisms that happen to have beneficial changes The process takes a very long time in Nature The idea will work with molecules like antibodies (as opposed to whole organisms) if we use molecular biology to speed up the process in the laboratory

The Wonders of Natural Evolution
Photo credits: Dr. Brent Iverson Photos: Top Left: Hawaiian scorpion fish, taken off the Kona Coast of the Big Island, Hawaii; Top Right: Queen angelfish, taken off of Tobago, West Indies: Bottom: Gray reef shark, taken off of Rangiroa, French Polynesia.

We isolate millions of copies of the DNA that codes for our antibody that binds to the anthrax PA toxin Error-prone PCR PCR Shuffle

We isolate millions of copies of the DNA that codes for our antibody that binds to the anthrax PA toxin We use genetic engineering techniques to make a few random changes on each piece of antibody DNA (gene) Randomized Gene pool

We isolate millions of copies of the DNA that codes for our antibody that binds to the anthrax PA toxin We use genetic engineering techniques to make a few random changes on each piece of antibody DNA (gene) We use viruses produced in host bacteria to produce the antibodies from the genes scFv DNA scFv Phage Display image: Smith, Lerner, Winter Phage Display

We isolate millions of copies of the DNA that codes for our antibody that binds to the anthrax PA toxin We use genetic engineering techniques to make a few random changes on each piece of antibody DNA (gene) We use viruses produced in host bacteria to produce the antibodies from the genes We find the antibodies that happen to bind more strongly to the anthrax toxin through a process referred to as “panning” Phage Display image: Smith, Lerner, Winter

We isolate millions of copies of the DNA that codes for our antibody that binds to the anthrax PA toxin We use genetic engineering techniques to make a few random changes on each piece of antibody DNA (gene) We use viruses produced in host bacteria to produce the antibodies from the genes We find the antibodies that happen to bind more strongly to the anthrax toxin through a process referred to as “panning” We can repeat the entire process if necessary

Directed Evolution (Technology)
Error-prone PCR PCR Shuffle Phage Display Smith, Lerner, Winter scFv DNA Stemmer scFv

(more fit) the antibody binding
Directed Evolution Technology: Phage Display “Panning” The harsher the wash, the stronger (more fit) the antibody binding Make phage “library” Antigen Wash Step 1: We use genetic engineering techniques to make a few random changes on each piece of antibody DNA (gene) Step 2: We use viruses produced in host bacteria to produce the antibodies from the genes. We find the antibodies that happen to bind more strongly to the anthrax toxin through a process referred to as “panning” Step 3: We can repeat the entire process if necessary Antigen Incubate with antigen Randomized Gene pool Repeat

How Powerful Did We Make Them?
Antibody Variant kon (*105 M-1 sec-1) koff (*10-4 sec-1) Kd (nM) L97 scFv 3.1   14B7 scFv 3.0   A2E scFv 3.2   1H scFv 6.4   14B7 scAb 2.8   1H scAb 6.1   14B7 Fab 2.9   14B7 mAb 5.7   PA-Receptor* *Escuyer et al, Infect & Immun 59:3381 (1991) We measured the ability of our various engineered antibodies to bind PA toxin. These measurements revealed that our best antibody, 1H, binds to PA 50 times stronger than the best antibody previously known (called 14B7). In addition, we were able to determine that 1H stays bound to PA on average for 100 minutes. This is significant, because the PA toxin only remains in an animal for about 35 minutes. Thus, the 1H antibody should be able stay bound to, and prevent the activity of, PA toxin until it is naturally flushed from the animal’s system. Jennifer Maynard Nat. Biotech., 2002, 20,

Antibody Variant kon (*105 M-1 sec-1) koff (*10-4 sec-1) Kd (nM) L97 scFv 3.1   14B7 scFv 3.0   A2E scFv 3.2   1H scFv 6.4   14B7 scAb 2.8   1H scAb 6.1   14B7 Fab 2.9   14B7 mAb 5.7   PA-Receptor* *Escuyer et al, Infect & Immun 59:3381 (1991) t1/2 ~100 minutes see notes on previous slide Jennifer Maynard Nat. Biotech., 2002, 20,

Our engineered antibody can stay bound to the PA toxin for 100 minutes on average We found that the PA toxin is naturally flushed from an animal after 35 minutes Our engineered antibody should be able to bind, and hold onto, the PA toxin long enough to block its activity until it is cleared from the animal.

2 Adjacent Mutations Appear Important
L Q55:L L S56:P Both Make the Surface More Hydrophobic Maybe Helps with Stability We were able to determine that the improvement in the 1H antibody is derived from the changes indicated in the movie as the green colored regions. These small changes made the antibody 50 times better. hydrophobic: repelling or not absorbing water Jennifer Maynard Nat. Biotech., 2002, 20,

Does It Work? Animal Model --> Rat
Challenge --> Venous Injection of Toxin (10X Lethal Dose) Literature Protocol --> Pre-Incubate Neutralizing Agent + Toxin Ezzell et al, Infect & Immun, 45:761 (1984) Ivins et al, App & Enviro Micro, 55:2098 (1989), Little et al, Infect & Immun 56: 1807 (1988); 58: 1606 (1990), Mourez et al, Nat Btech 19:958 (2001), Sellman et al, Science 292: 695 (2001) Our Protocol --> Inject 4x and 1.5x Excess of Antibody 5 Minutes Prior to Toxin Jennifer Maynard Nat. Biotech., 2002, 20,

Does It Work? Treatment koff (*10-4 sec-1) TTD (min) * Survivors
PBS ,87,92,97, /5 L97 scFv 190  ,66,67,70, /5 14B7 scFv 32  ,103,112,123, /5 A2E scFv 10  ,242, /5 1H scFv 1.7  , /5 14B7 scAb 30  ,115,140,172, /5 1H scAb 1.6  /5 We injected the animals with our various antibody molecules, then five minutes later, we injected 10 times the lethal dose of anthrax toxins. We used groups of five rats per experiment. We determined how many animals survived. If they died, we recorded how long they survived, reported as Time To Death (TTD) in the table. The exciting result is that our most powerful antibody, 1H, protected all of the rats when we used 4 times as much antibody as toxin, and even protected 4 out of 5 rats when we only used 1.5 times as much antibody as toxin. 1H scAb, 1.6  /5 (1.5X concentration) *Total time of experiment 5 hrs Jennifer Maynard Nat. Biotech., 2002, 20,

Does It Work? Treatment koff (*10-4 sec-1) TTD (min) * Survivors
PBS ,87,92,97, /5 L97 scFv 190  ,66,67,70, /5 14B7 scFv 32  ,103,112,123, /5 A2E scFv 10  ,242, /5 1H scFv 1.7  , /5 14B7 scAb 30  ,115,140,172, /5 1H scAb 1.6  /5 See notes on previous slide 1H scAb, 1.6  /5 (1.5X concentration) *Total time of experiment 5 hrs Jennifer Maynard Nat. Biotech., 2002, 20,

What Is Next? More animal tests to optimize formulation of antibody
With optimized formulation, we need to test antibody with real anthrax spores These animal studies will be carried out at the Southwest Foundation for Biomedical Research, in San Antonio

Take Home Lessons: 1. Biological weapons are cheap to make and easy to conceal. 2. They have been of little military significance thus far, but of tremendous value from a propaganda perspective 3. Points 1. and 2. make biological weapons ideal for terrorism 4. American scientists are still playing “catch-up”, but have created several promising approaches to reduce the threat of biological weapons Where can I find more information about anthrax? Agencies CDC, NCID, Division of Bacterial and Mycotic Diseases, Meningitis and Special Pathogens Branch Websites Information regarding anthrax and the recent bioterrorism activities is available at:

Collaborators at the University of Texas Dr. George Georgiou
Dr. Steve Kornguth Collaborators at the SFBR Dr. Jean Patterson Key students at the University of Texas Dr. Jennifer Maynard Dr. Andrew Hayhurst Dr. Kitty Braat Mr. Robert Mabry DARPA, U.S. Army, ARO/MURI, SBCCOM

Dr. Brent Iverson University Distinguished Teaching Professor
Dr. Brent Iverson is a professor at the University of Texas at Austin. His research area is the production, characterization, and manipulation of large, functional molecules from three different points of view: 1)Antibody and Enzyme Engineering, 2) Artificial macromolecules with defined higher order structure and function, and 3) The chemistry of nucleic acid binding, recognition, and modification.

Outreach Lecture Series Volume 19

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