Transcript Botulinum toxin as a bioterrorist weapon.

Death
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Death results from loss of diaphragmatic and
accessory respiratory muscle function 
insufficient tidal volume, dyspnea or ventilary
failure
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Death may also result from loss of pharyngeal
muscle function  airway muscle paralysis,
airway obstruction
Lethal Factor
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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
Differential Diagnosis:
Botulism is commonly misdiagnosed as
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Guillan-Barre syndrome
Myasthenia gravis
Eaton-Lambert syndrome
Stroke syndrome
Carbon monoxide intoxication
Tick paralysis
Food poisoning
Differential Diagnosis: Ancillary
Testing
Procedure:
Result Consistent Eliminates possibility
of:
with Botulism
MRI/CT scan (brain
imaging)
Normal
Stroke syndrome
Lumbar puncture
Normal
Guillain-Barre syndrome
Edrophonium chloride
test
Negative
Myasthenia gravis
Electromyography
Decreased amplitude of
action potentials in
involved muscle groups
N/A
Rapid repetitive
electromyography (2050 Hz)
Facilitation (increasing
pattern of action
potential amplitude)
N/A
Confirmatory Tests for C.
Botulinum / BoNT
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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
Confirmatory Tests for BoNT
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Culturing of stool specimens for C. botulinum
Other prospects include PCR, ELISA, timeresolved fluorescence
PCR of various strains of
C. botulinum
MW A B E
F A B A -
B
E
B
F
F
E,F
Botulism as a Biological
Weapon
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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.
Botulism as a Biological
Weapon
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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.
Botulism as a Biological
Weapon
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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)
Botulinum: The Deadly
Assassin
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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.
Botulinum: The Deadly
Assassin
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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.
Botulinum: The Deadly
Assassin
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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.
Purification of Botulinum Toxin
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Toxin is easily purified using general
biochemical purification techniques.
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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.
Botulinum Toxin
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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.
Botulinum toxin as a
bioterrorist weapon.
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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)
Botulinum toxin as a
bioterrorist weapon.
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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.
Medical Uses for Botox
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Temporary removal of wrinkles
Treatments for muscular spasms
Therapy for cerebral palsy, parkinsons,
migraines, and overactive bladder.
Other Interesting Botox Facts
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Botox from drugstore.com
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One vial $442.68
Three vials $1,327.03
Recently, “Botox Parties” have become popular
means for doctors to increase their clients and “it’s
a great way for people to meet each other and it’s a
comfortable way for patients to get Botox.”
Prophylaxis
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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
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Monovalent vaccine that protects against BoNT
serotype F also available as Investigational
New Drug
Therapy: Step 1 – Antitoxin
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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
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Therapy: Step 1 – Antitoxin
(cntd.)
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Investigational heptavalent (ABCDEFG) antitoxin held by US Army
for dissemination to patients affected by botulinum toxin types other
than A, B, E
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Amount of neutralizing AB in both antitoxins far exceeds (over 100fold greater than) highest amount of circulating antitoxin ever
measured at CDC
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Single, 10mL vial; no additional doses necessary
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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
Therapy: Step 2 – Supportive
Care
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Mechanical
ventilation
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Treatment of
secondary
infections
Therapy: Step 2 – Supportive
Care (cntd.)
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Reverse Trendelenburg
positioning of
nonventilated patients
for reduced oral
secretions in airway and
improved respiratory
mechanics
Recovery
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Time can range from several days to several
weeks or longer, depending on extent of toxin
activity
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Involves regeneration of motor nerve endings/
axons of affected neurons
Recovery: Regeneration of
Axon Terminals
Decontamination
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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
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Mass Immunization
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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
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Current Vaccine Production:
Shortcomings
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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
Current Research/Future Strategies
in Vaccine Development
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Recombinant vaccine
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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
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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
Current Research/Future Strategies
in Vaccine Development (cntd.)
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Use of Venezuelan equine encephalitis virus replicon vector (Lee
and coworkers)
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Design of synthetic genes encoding non-toxic, carboxy-terminal
fragments of C. botulinum neurotoxins (Byrne and coworkers)
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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
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
Research Need: Alternate Therapy via
Enteral detoxification
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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
Structure-based drug design
Figure from Arnon, et al,
2001
Targets: Binding Site of BoNT or
Gangliosides Themselves
Binding
Targets: cleavage sites on SNARE
proteins or BoNT catalytic site
Cleavage
of SNARE
proteins
by zincdependent
catalytic
site
Research Need: Immunity via
Recombinant oligoclonal
antibodies
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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
This Just In: Botulism Beaten!
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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