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 DiagnosisBotulism 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 1930s, 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.
• Heydrichs 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.

17
Medical Uses for Botox

• Temporary removal of wrinkles
• Treatments for muscular spasms
• Therapy for cerebral palsy, parkinsons,
  migraines, and overactive bladder.

18
Other Interesting Botox Facts

• Botox from drugstore.com
• One vial 442.68
• Three vials 1,327.03
• Recently, Botox Parties have become popular
  means for doctors to increase their clients and
  its a great way for people to meet each other
  and its a comfortable way for patients to get
  Botox.

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 85C 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