Death

1. Death • Death results from loss of diaphragmatic and accessory respiratory muscle function  insufficient tidal volume, dyspnea or ventilary failure • Death may also result from loss of pharyngeal muscle function  airway muscle paralysis, airway obstruction

2. Lethal Factor • Foodborne • 6% mortality rate in treated patients • Early deaths – failure to diagnose/recognize severity of disease • Death after 2 weeks from complications of long-term mechanical ventilatory management • Wound • 15% case fatality rate in treated patients • Lethal dose on the order of ng per kg body weight • .7-.9 μg = lethal dose for 70 kg individual if inhaled; ricin: 3-5 μg/kg • Cited as 100,000 to 3,000,000 times as potent as nerve gas sarin (WWII) • Serotypes A, B, E most associated with botulism outbreaks in humans, but also F

3. Differential Diagnosis:Botulism is commonly misdiagnosed as • Guillan-Barre syndrome • Myasthenia gravis • Eaton-Lambert syndrome • Stroke syndrome • Carbon monoxide intoxication • Tick paralysis • Food poisoning

4. Differential Diagnosis: Ancillary Testing

5. Confirmatory Tests for C. Botulinum / BoNT • Mouse Bioassay for toxin • Sample from serum, feces, or food in • question • Administer sample to mouse with and • without type-specific anti-toxin • Determine response level • Results in 1-2 days, can determine • presence/absence and type of BoNT by • seeing which anti-toxin type • neutralizes biological activity of toxic • sample • Cumbersome, and with animal subjects

6. Confirmatory Tests for BoNT • Culturing of stool specimens for C. botulinum • Other prospects include PCR, ELISA, time-resolved fluorescence PCR of various strains of C. botulinum MW A B E F A B A - B E B F F E,F

7. Botulism as a Biological Weapon • Starting in the 1930’s, the Japanese during their occupation of Manchuria admitted to feeding cultures of C. botulinum to prisoners. • The US had produced botulism toxin for use in warfare during WWII and were equipped with the vaccine during the D-Day invasion. • Despite the 1972 Biological and Toxin Weapons Convention prohibition of offensive research and production of biological weapons, the Soviet Union and Iraq have successfully created these types of weapons.

8. Botulism as a Biological Weapon • The Soviet Union had attempted to splice out the botulinum toxin gene into other bacteria. • Iran, Iraq, North Korea, and Syria are all believed by the US to be researching or have developed botulinum toxin as a weapon.

9. Botulism as a Biological Weapon • After the Persian Gulf War, Iraq admitted to having produced 19,000L of the concentrated toxin, 10,000L of which were fitted to military weapons. • Iraq had also outfitted several missiles with botulinum toxin, anthrax, and aflatoxin; (likely to be aerosolized and enter the bloodstream through the lungs)

10. Botulinum: The Deadly Assassin • Botulinum toxin can be implicated with the assassination of Reinhard Heydrich, head of the Gestapo and the SS during WWII • Shrapnel injuries from a bomb delivered by Czech patriots • Post-operation, his condition was satisfactory; until after the seventh day where his condition suddenly became worse and he died the next morning.

11. Botulinum: The Deadly Assassin • Czech patriots were trained and equipped by the British government, who were developing botulinum toxin as a weapon. • Heydrich’s chart suggest massive pulmonary embolism, however his heart and lungs were normal. • The head of the British biological warfare program has made remarks that imply that he had a hand in the assassination. • The bomb was specially modified, possibly to include the botulinum toxin.

12. Botulinum: The Deadly Assassin • During WWII, the US Office of Strategic Services has developed a plan for Chinese prostitutes to assassinate high-ranking Japanese officers in occupied Chinese cities. • Gelatin capsules with a lethal dose of botulinum toxin were to be slipped into food or a drink.

13. Purification of Botulinum Toxin • Toxin is easily purified using general biochemical purification techniques. • Toxin separated from extract by salting out, acid precipitation, gel filtration on Sephadex G-200, chromatography on SP-Sephadex, and a second purification step of gel filtration. • 10 L of culture produced 22.9 mg of toxin with1.1x108 LD50.

14. Botulinum Toxin • LD50 is approximately 1ng/kg via intravenously, subcutaneously,or intraperitonealy and about 3ng/kg by inhalation. • The average person is 70-100kg, so a lethal dose would require only 70-300ng per person.

15. Botulinum toxin as a bioterrorist weapon. • Splicing the toxin gene into other bacteria to allow for growth in more permissive conditions or to increase the transmission rate. • Genetic modifications that removes the effectiveness of the vaccine (altering the antigens by mutagenic PCR assay)

16. Botulinum toxin as a bioterrorist weapon. • The goal of bioterrorism is to create terror, not necessarily with casualties. • A bioterrorist group could easily purify botulinum toxin and contaminate food in order to affect thousands of people. • Hospital care is required for weeks to months which would quickly exceed the capacity for proper care.

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19. Prophylaxis • Passive immunity – pentavalent (ABCDE) botulinum toxoid • Distributed by CDC for laboratory workers at high risk of exposure to botulinum toxin • Distributed by military for protection of troops against attack • Used for more than 30 years to immunize more than 3000 laboratory workers in many countries • Monovalent vaccine that protects against BoNT serotype F also available as Investigational New Drug

20. Therapy: Step 1 – Antitoxin • Passive immunization with equine trivalent antitoxin • Antibodies for BoNTs/A, B, E • Neutralizes toxin molecules that are not yet bound to nerve endings • Therefore should be administered early (within 24 hours of toxin exposure) and • Will minimize subsequent nerve damage and severity of disease but will not reverse existent paralysis • Available from CDC via state and local health departments

21. Therapy: Step 1 – Antitoxin (cntd.) • Investigational heptavalent (ABCDEFG) antitoxin held by US Army for dissemination to patients affected by botulinum toxin types other than A, B, E • Amount of neutralizing AB in both antitoxins far exceeds (over 100-fold greater than) highest amount of circulating antitoxin ever measured at CDC • Single, 10mL vial; no additional doses necessary • As of June 1998, human botulism immune globin (a human-derived antitoxin product) is available only for infant botulism patients, under a Treatment Investigational New Drug protocol

22. Therapy: Step 2 – Supportive Care • Mechanical ventilation • Treatment of secondary infections

23. Therapy: Step 2 – Supportive Care (cntd.) • Reverse Trendelenburg positioning of nonventilated patients for reduced oral secretions in airway and improved respiratory mechanics

24. Recovery • Time can range from several days to several weeks or longer, depending on extent of toxin activity • Involves regeneration of motor nerve endings/ axons of affected neurons

25. Recovery: Regeneration of Axon Terminals

26. Decontamination • Foodborne: heating to internal temperature of 85˚C for 5 minutes will detoxify food or drink • Aerosolized: • Persistence at site of intentional release determined by atmospheric conditions and particle size of aerosol • Toxin degraded by extremes of temperature and humidity • Decay rate weather dependent, but estimated at less than 1%-4% per minute • At 1% decay rate, toxin significantly inactivated 2 days after aerosolization

27. Mass Immunization • Pro argument: could theoretically eliminate threat posed by botulinum toxins A through E • Con argument: • Scarcity of toxoid • Rarity of natural disease • Elimination of potential therapeutic benefits of medicinal botulinum toxin • 3 injections + annual booster delivers immunity

28. Current Vaccine Production: Shortcomings • C. botulinum is a spore-former; need to renovate or build facility for manufacture toxin-based product – many resources needed • Yields of toxin production from C. botulinum relatively low • Safety precautions to handle large quantities of toxin increase cost of manufacturing • Pentavalent toxin consists of relatively crude extract of clostridial proteins that may influence immunogenicity/reactivity of vaccine • Formaldehyde used to inactivate toxin; residual amounts used to prevent reactivation of toxin, but formaldehyde is reactogenic

29. Current Research/Future Strategies in Vaccine Development • Recombinant vaccine • No new research facility needed, no culturing of large quantities of hazardous toxin-producing bacteria  fewer manufacturing costs  less expensive vaccine • 3 functional domains: binding, internalization, catalytic; fragment would not possess all three  no need for formalin to maintain deactivation • Synthetic vaccine – Atassi and coworkers • Investigators synthesized peptides overlapping Hc region of BoNT serotype A • BoNT/A antibodies and T-lymphocytes used to map these epitopes (of Hc region) • Synthesized BoNT epitopes could potentially be delivered as vaccine, to induce endogenous immunity Mike Meagher, contracted by US Army to develop BoNT vaccine

30. Current Research/Future Strategies in Vaccine Development (cntd.) • Use of Venezuelan equine encephalitis virus replicon vector (Lee and coworkers) • Introduction of nontoxic Hc region of BoNT/A into vector yields high levels of Hc (demonstrated in mice) • Protective antigens against BoNT produced in vivo • Design of synthetic genes encoding non-toxic, carboxy-terminal fragments of C. botulinum neurotoxins (Byrne and coworkers) • gene products used to illicit immunity • demonstrated protective immunity in mice and non-human primates against high levels of toxin • Oral vaccine – gene/product modified to alter toxicity without changing penetrability, specificity, immunogenicity; fraction of oral dose would be degraded, but enough absorbed

31. Research Need: Alternate Therapy via Enteral detoxification • Distribution of antitoxin to local hospitals takes several hours • Standard detoxification can be administered immediately • Reduce absorption through GI tract or reduce circulating levels of botulinum toxin through osmotic catharsis • Perhaps more useful in foodborne than in aerosolized botulism  limited value

32. Structure-based drug design Figure from Arnon, et al, 2001

33. Targets: Binding Site of BoNT or Gangliosides Themselves Binding

34. Targets: cleavage sites on SNARE proteins or BoNT catalytic site Cleavage of SNARE proteins by zinc-dependent catalytic site

35. Research Need: Immunity via Recombinant oligoclonal antibodies • Half life of 1 month, as opposed to 5-8 days (for equine) • Oligoclonal human antibodies = theoretic protection against toxins ABCDEFG for months • Not entirely foreign source  perhaps less reactogenicity than equine products • Already shown effective against HIV and Anthrax • Stockpiles could deter terrorist attacks • Prophylaxis or treatment

36. This Just In: Botulism Beaten! • USCF Research Team led by James Mark, August, 2002, published in Nature Reviews Immunology • 3 recombinant, oligoclonal antibodies that neutralize toxin • Will protect against toxin within 1-2 hours after delivered • Effective up to 2 days after exposure to toxin • Protection can last 3-6 months • “The drug neutralizes the toxin better than the most potent natural immune response.” – Marks, BBC News Online