**Botulinum Neurotoxins**

Robert P. Webb1, Virginia I. Roxas-Duncan1 and Leonard A. Smith2 *1Integrated Toxicology Division, US Army Medical Research Institute of Infectious Diseases, 2Senior Research Scientist (ST) for Medical Countermeasures Technology, Office of Chief Scientist, US Army Medical Research Institute of Infectious Diseases, Frederick, USA* 

#### **1. Introduction**

106 Bioterrorism

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spleen cell cultures and bronchial washings and protects immunized animals

Botulism is a neuroparalytic disease caused by neurotoxin produced from the bacterium *Clostridium botulinum*. The botulinum neurotoxins (BoNTs) are among the most potent known biological toxins and have an estimated human median lethal dose (LD50) in the nanogram/kilogram range. Botulinum toxins have historically been employed as biological weapons (BW) through state-sponsored programs in Japan, Germany, the United States, Russia and Iraq as well as by independent terrorist organizations. The extreme potency of the toxin, its persistence within affected neurons, the need for protracted intensive care, and the lack of an effective post-intoxication therapeutic intervention make BoNTs a potentially deadly offensive biological weapon.

#### **2. Botulinum neurotoxins as biological weapons**

#### **2.1 Historical applications of botulinum toxins in biological warfare**

There have been reports of botulinum neurotoxin being used as an offensive weapon since the early 1900s. Anecdotal accounts describe the implementation of crude anaerobic fermenters made by burying canteens filled with water, green beans and slivers of meat to facilitate *C. botulinum* production for use against Mexican federal troops in 1910 (Carrus, 2001). Documented state sponsored programs utilizing BoNT as a potential BW were reported as early as the 1930s when the military medical commander of Unit 731, General Shiro Ishii, confessed to feeding food contaminated with lethal cultures of *C. botulinum* to prisoners of war (Williams & Wallace, 1989). In the early 1940s, the US developed a BoNTbased BW program in response to Allied intelligence reports that Germany was attempting to develop the neurotoxin as an offensive weapon to be used against an invasion force (Franz et al., 1997). The initial efforts of the US program were primarily directed at the isolation and purification of the toxin and the elucidation of the mechanism of pathogenicty of BoNT, then referred to as agent "X" (Cochrane, 1947). In response to the potential threat from Germany, over 1 million doses of a botulinum toxoid vaccine were prepared for Allied troops involved in the D-Day invasion in Normandy (Bryden, 1989). The 1972 Convention on the Prohibition of the Development, Production, and Stockpiling of Bacteriological

al. 2005).

Botulinum Neurotoxins 109

unsuccessful when conventional municipal water treatment methods are employed (Notermans & Havelaar, 1980; Wannemacher, at al., 1993). Another consideration is that large-capacity reservoirs undergo a relatively slow turn-over rate and so a comparably large inoculum of botulinum toxin would be needed (Burrows et al., 1997). Given the technical difficulties involved in producing such a large amount of toxin, this route seems unlikely. Small-scale (personal-use) water filtration systems utilizing ceramic or membrane filters with a 0.2 to 0.4 m pore size were found to be insufficient to effectively remove BoNT/B introduced into drinking water. However, a reverse osmosis device was found to remove BoNT levels below the detection limit of the mouse bioassay (Hörman, et

American agriculture has been described as being "concentrated, highly accessible, vertically integrated" and as such, susceptible to the malicious introduction of biological toxins or human pathogens at processing or distribution points that would reach consumers before a significant threat was realized (Parker, 2002). Wein and Liu published a mathematical model to predict the effects of the deliberate introduction of botulinum toxin at various points through the nine-stage milk collection, processing and distribution network in the US, as it is associated with a single processing facility (Wein and Liu 2005). The study has a number of variables such as the amount of toxin employed, the total volume of the milk, the specific effect of the pasteurization process on the toxin and variable delivery time and consumptions rates, but all scenarios support a significant outbreak of up to 100,000 individuals. The report generated considerable controversy but the editors of the publishing journal defended the release, citing its potential contribution to developing defensive counter-measures as well as informing federal and state governments of the putative threat (Alberts, 2005). The devastating potential of an intentional bioterrorism attack using this avenue was illustrated in 1985 when almost 200,000 people were infected with an antibiotic-resistant strain of *Salmonella* caused by an inadvertent contamination at a

The potential susceptibility of centralized food distribution platforms in the US can be illustrated by outbreaks attributed to improper processing of mass distributed consumable products. In September 2006, a total of 6 individuals from 2 US states and 1 Canadian province were admitted to local hospitals with cranial neuropathies and flaccid paralysis necessitating mechanical ventilation (CDC, 2007). All of the individuals tested positive for BoNT/A and one patient eventually died. The outbreaks were traced to a commercially produced carrot juice manufactured in a single plant which was distributed under three different brand labels. The intoxications were attributed to a lapse in refrigeration during transport or storage and a lack of chemical barriers to *C. botulinum* germination during processing. In the summer of 2007, 8 cases of botulism in 3 US states led to a massive recall of canned meat products. The outbreaks were traced to product from a single production line in a single plant that was packaged and distributed under 90 different labels (NCFPD, 2008) and which necessitated the recall of tens of millions of cans of suspect food. While these outbreaks represent unintentional distribution of BoNT, they do reveal the complexity of the food production and distribution network in the US and how it might be compromised by a BW attack using a

single northern Illinois dairy processing plant (Ryan, et al., 1987).

potent toxin such as BoNT.

(Biological) and Toxin Weapons and on their Destruction signed by President Richard M. Nixon went into effect in March of 1975, effectively terminating the US BW efforts. All of the biological agent stockpiles created in the US offensive program, including botulinum neurotoxins, were destroyed. Despite being signatories on the convention, the Soviet Union continued not only to pursue its biowarfare program, including research on botulinum neurotoxins, but expanded their program during the post-Soviet era (UNSC, 1995; Bozheyeva et al., 1999). Botulinum toxins were reportedly one of several biological agents tested at the Soviet Aralsk-7 site on Vozrozhdeniye (Renaissance) Island in the Aral Sea. The Soviets were also believed to have attempted to use recombinant DNA technology to introduce BoNT genes into alternative strains of bacteria for the purpose of enhancing toxin production (Alibek & Handleman, 1999). Iraq, which also ratified the 1975 convention, reportedly also significantly expanded its biological weapons program (UNSC, 1995). After the Persian Gulf War, Iraq admitted to a United Nations Special Commission inspection team that approximately 4,900 gallons of concentrated botulinum neurotoxins had been produced for use in specially designed bombs, missiles and tank dispersion instruments in 1989 (Zilinskas, 1997). Iraq maintains that no biological weapons were employed during the Persian Gulf War or Operation Iraqi Freedom and that its stockpiles have since been destroyed (Blix, 2004).

The Japanese cult Aum Shrinkyo (now referred to as Aleph) attempted to develop both chemical and biological weapons after its political aspirations were defeated in the 1990 Japanese Diet elections. Formed in 1987 by Shoko Asahara, this group developed rapidly and was reported to have 50,000 members and over US\$ 1 billion in financial resources by 1995 (Sugishima, 2003). The Aum executed a deadly sarin nerve gas attack in Matsumoto City on June 27, 1994 which killed seven people. On March 20, 1995 they perpetrated a sarin gas assault on a Tokyo subway which killed 12 people and injured over 1000 individuals. Less than a month later they attempted a cyanide gas release in a restroom of the subway's Shinjuku Station. Senior Aum members obtained soil samples in an attempt to isolate toxinproducing strains of *C. botulinum*. Despite the cult having members with a variety of scientific expertise, they are believed to have experienced difficulty cultivating toxigenic strains of *C. botulinum* and were most likely unable to produce any substantial amount of toxin (Sugishima, 2003; Leitenberg, 1999).

#### **2.2 Domestic threat targets**

The possibility of dissemination of toxins such as BoNT through municipal water supplies or centralized agricultural or food distribution hubs has been explored in a number of scenarios. There has never been a confirmed case of waterborne botulism. In 1980, Notermans & Havelaar reported on the stability of BoNT/A, /B and /E when introduced into samples of reservoir surface water, drinking water prepared by sand-filtration, and sterile distilled water. Toxicity was reduced 99% in just over 3 days in the surface water, 9 days in filtered water and relatively stable in sterile distilled water. Treatment with FeCl3, a common coagulant processing step employed in municipal water treatments to remove iron, removed 75-95% of the toxins. Treatment with 1 mg of ozone per liter for 2 min destroyed ≥99% of the toxins and 0.3-0.5 mg/L sodium hypochlorite ablated 99% of the toxicity in 30 seconds. Despite the extreme potency of BoNTs, their application as a bioterrorism weapon by introduction into a water supply in the US would most likely be

(Biological) and Toxin Weapons and on their Destruction signed by President Richard M. Nixon went into effect in March of 1975, effectively terminating the US BW efforts. All of the biological agent stockpiles created in the US offensive program, including botulinum neurotoxins, were destroyed. Despite being signatories on the convention, the Soviet Union continued not only to pursue its biowarfare program, including research on botulinum neurotoxins, but expanded their program during the post-Soviet era (UNSC, 1995; Bozheyeva et al., 1999). Botulinum toxins were reportedly one of several biological agents tested at the Soviet Aralsk-7 site on Vozrozhdeniye (Renaissance) Island in the Aral Sea. The Soviets were also believed to have attempted to use recombinant DNA technology to introduce BoNT genes into alternative strains of bacteria for the purpose of enhancing toxin production (Alibek & Handleman, 1999). Iraq, which also ratified the 1975 convention, reportedly also significantly expanded its biological weapons program (UNSC, 1995). After the Persian Gulf War, Iraq admitted to a United Nations Special Commission inspection team that approximately 4,900 gallons of concentrated botulinum neurotoxins had been produced for use in specially designed bombs, missiles and tank dispersion instruments in 1989 (Zilinskas, 1997). Iraq maintains that no biological weapons were employed during the Persian Gulf War or Operation Iraqi Freedom and that its stockpiles have since been

The Japanese cult Aum Shrinkyo (now referred to as Aleph) attempted to develop both chemical and biological weapons after its political aspirations were defeated in the 1990 Japanese Diet elections. Formed in 1987 by Shoko Asahara, this group developed rapidly and was reported to have 50,000 members and over US\$ 1 billion in financial resources by 1995 (Sugishima, 2003). The Aum executed a deadly sarin nerve gas attack in Matsumoto City on June 27, 1994 which killed seven people. On March 20, 1995 they perpetrated a sarin gas assault on a Tokyo subway which killed 12 people and injured over 1000 individuals. Less than a month later they attempted a cyanide gas release in a restroom of the subway's Shinjuku Station. Senior Aum members obtained soil samples in an attempt to isolate toxinproducing strains of *C. botulinum*. Despite the cult having members with a variety of scientific expertise, they are believed to have experienced difficulty cultivating toxigenic strains of *C. botulinum* and were most likely unable to produce any substantial amount of

The possibility of dissemination of toxins such as BoNT through municipal water supplies or centralized agricultural or food distribution hubs has been explored in a number of scenarios. There has never been a confirmed case of waterborne botulism. In 1980, Notermans & Havelaar reported on the stability of BoNT/A, /B and /E when introduced into samples of reservoir surface water, drinking water prepared by sand-filtration, and sterile distilled water. Toxicity was reduced 99% in just over 3 days in the surface water, 9 days in filtered water and relatively stable in sterile distilled water. Treatment with FeCl3, a common coagulant processing step employed in municipal water treatments to remove iron, removed 75-95% of the toxins. Treatment with 1 mg of ozone per liter for 2 min destroyed ≥99% of the toxins and 0.3-0.5 mg/L sodium hypochlorite ablated 99% of the toxicity in 30 seconds. Despite the extreme potency of BoNTs, their application as a bioterrorism weapon by introduction into a water supply in the US would most likely be

destroyed (Blix, 2004).

toxin (Sugishima, 2003; Leitenberg, 1999).

**2.2 Domestic threat targets** 

unsuccessful when conventional municipal water treatment methods are employed (Notermans & Havelaar, 1980; Wannemacher, at al., 1993). Another consideration is that large-capacity reservoirs undergo a relatively slow turn-over rate and so a comparably large inoculum of botulinum toxin would be needed (Burrows et al., 1997). Given the technical difficulties involved in producing such a large amount of toxin, this route seems unlikely. Small-scale (personal-use) water filtration systems utilizing ceramic or membrane filters with a 0.2 to 0.4 m pore size were found to be insufficient to effectively remove BoNT/B introduced into drinking water. However, a reverse osmosis device was found to remove BoNT levels below the detection limit of the mouse bioassay (Hörman, et al. 2005).

American agriculture has been described as being "concentrated, highly accessible, vertically integrated" and as such, susceptible to the malicious introduction of biological toxins or human pathogens at processing or distribution points that would reach consumers before a significant threat was realized (Parker, 2002). Wein and Liu published a mathematical model to predict the effects of the deliberate introduction of botulinum toxin at various points through the nine-stage milk collection, processing and distribution network in the US, as it is associated with a single processing facility (Wein and Liu 2005). The study has a number of variables such as the amount of toxin employed, the total volume of the milk, the specific effect of the pasteurization process on the toxin and variable delivery time and consumptions rates, but all scenarios support a significant outbreak of up to 100,000 individuals. The report generated considerable controversy but the editors of the publishing journal defended the release, citing its potential contribution to developing defensive counter-measures as well as informing federal and state governments of the putative threat (Alberts, 2005). The devastating potential of an intentional bioterrorism attack using this avenue was illustrated in 1985 when almost 200,000 people were infected with an antibiotic-resistant strain of *Salmonella* caused by an inadvertent contamination at a single northern Illinois dairy processing plant (Ryan, et al., 1987).

The potential susceptibility of centralized food distribution platforms in the US can be illustrated by outbreaks attributed to improper processing of mass distributed consumable products. In September 2006, a total of 6 individuals from 2 US states and 1 Canadian province were admitted to local hospitals with cranial neuropathies and flaccid paralysis necessitating mechanical ventilation (CDC, 2007). All of the individuals tested positive for BoNT/A and one patient eventually died. The outbreaks were traced to a commercially produced carrot juice manufactured in a single plant which was distributed under three different brand labels. The intoxications were attributed to a lapse in refrigeration during transport or storage and a lack of chemical barriers to *C. botulinum* germination during processing. In the summer of 2007, 8 cases of botulism in 3 US states led to a massive recall of canned meat products. The outbreaks were traced to product from a single production line in a single plant that was packaged and distributed under 90 different labels (NCFPD, 2008) and which necessitated the recall of tens of millions of cans of suspect food. While these outbreaks represent unintentional distribution of BoNT, they do reveal the complexity of the food production and distribution network in the US and how it might be compromised by a BW attack using a potent toxin such as BoNT.

Botulinum Neurotoxins 111

forming a protein channel that facilitates the translocation of the LC out of the endosomal lumen and into the cytosol. The LC is a zinc-containing endopeptidase that target SNARE (soluble *N*-ethylmaleamide sensitive factor attachment protein receptors) proteins that form the synaptonemal fusion complex in a serotype dependent fashion. The SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDa) is cleaved at different sites by BoNT/A, /C, and /E, synaptobrevin (also referred to as VAMP; vesicle-associated membrane protein), is cleaved at different sites by BoNT/B, /D, /F, and /G, and syntaxin is cleaved by BoNT/C (Simpson, 2004). Proteolytic cleavage of SNARE proteins prevents the release of acetylcholine across the synaptic cleft of the neuromuscular junction, resulting in a

Botulism is typically reported in four clinical categories. *Foodborne botulism* is caused by the ingestion of the pre-formed toxin in contaminated food (CDC, 1998). Most outbreaks are associated with home-canned foods in which inadequate processing results in *C. botulinum* spores germinating, reproducing and producing the toxin (Shapiro, et al., 1998). These conditions include an anaerobic environment with temperatures ranging from 4ºC to 40°C, a pH range from 4.6 to 7.0, and water activity greater than 0.94 (aW is intensity with which water associates with various non-aqueous constituents and solids) (Baird-Parker & Freame, 1967; Stringer, et al., 2005). Despite increased educational awareness of the non-permissive conditions for food preparation, foodborne botulism remains a persistent threat. There were, on average, approximately 24 reported cases per year from 1900-2000, but there have been a smaller number of cases (average of 18 year) reported from 2001-2009 (CDC-NBS). In the period from 1950-1996, there were 444 outbreaks in which one or more cases of botulism from a contaminated food source was implicated. Of these outbreaks, 37.6% were caused by type A, 13.7% by type B, 15.1% by type E and 0.7% by type F while 32.9% of the incidents were caused by unidentified serotype(s) (CDC, 1998). Improvements in both differential diagnostic methods and the technology utilized in serotyping have resulted in a decrease in unidentified serotypes. In the United Stated from 1990-2000 BoNT/A was responsible for 50% of all cases of botulism, where types B and E were responsible for 10% and 37% of the intoxications, respectively; only 3.6% of the cases were from an unidentified strain (CDC,

*Infant botulism* is a toxicoinfection caused by inhalation or ingestion of clostridial spores that can colonize and produce toxin in the intestinal tract of infants less than 12 months of age (Shapiro, 1998). The ability of the bacteria to thrive and elicit toxin is thought to be attributed in part to a deficiency in protective gastrointesintal bacterial flora and the relatively low levels of inhibitory bile-aid found in children under 12 months (CDC, 1998). This form of the disease was officially recognized in a 1976 report in which two infants presenting with acute infantile hypotonoia and weakness were diagnosed with botulism (Pickett, et al., 1976). This has been the most commonly reported form of the disease in the United States since 1980, with an average of approximately 80-100 cases confirmed annually (Shapiro, 1998). In the US between 1976 and 1996, there were 1442 individual cases of infant botulism reported to the CDC (CDC, 1998). Of these, 46.5% were caused by type A, 51.9% by type B. Interestingly, the incidence of infant botulism in the US has a geographical component with 47.6% of the reported cases occurring in California with Delaware, Hawaii,

flaccid muscle paralysis which is the primary clinical sign of botulism.

**3.2 Epidemiology** 

1998).

#### **3. Microbiology and BoNT toxicity**

#### **3.1 The organism and its toxins**

*Clostridium spp*. are gram-positive, spore-forming, obligate anaerobic bacteria that are ubiquitous in soil and both freshwater and marine sediment (Dunbar, 1990) . BoNTs are produced primarily in *C. botulinum* but may also be produced in the closely related *C. butyricum*, *C. baratii* and *C. argentinense* (Hatheway, 1993). *C. botulinum* strains are classified into four groups (denoted I to IV) based on metabolic activity (Hatheway, 1988) and genetic composition (Collins, 1998; Hill et al., 2007). Group I includes type A strains and proteolytic strains of types B and F; Group II includes type E strains and nonproteolytic strains of types B and F; Group III includes nonproteolytic strains of types C and D; and Group IV includes only strains that produce type G. There are seven immunologically distinct serotypes of BoNTs designated by the letters A through G (BoNT/A to BoNT/G) (Smith & Sugiyama, 1988). BoNT/A, based on animal studies, has a lethal human dose (LD50), assuming 70 kg weight, of approximately 0.09 - 0.15 g by intravenous administration, 0.7 – 0.9 g by inhalation, and 70 g by oral administration (Scott & Suzuki, 1988; Arnon et al., 2001). The toxicity for other serotypes is unknown but all have been shown to uniformly fatal in animals studies. Human botulism is caused primarily by BoNT/A, /B and /E (Arnon et al., 2001), and rarely by BoNT/F (Barash et al., 2005; Gupta et al., 2005). BoNT/C and /D primarily cause botulism in animals. BoNT/G, produced by *C. argentinense*, has been associated with sudden death but not neuroparalytic illness in a few patients in Switzerland (Sonnabend et al., 1981). All seven serotypes can cause inhalational botulism in primates (Middlebrook & Franz, 1997). Recent characterization of an increasing number of unique BoNT subtypes has revealed small to significant variations at the amino acid level (Smith, et al., 2005; Hill, et al. 2007; Smith, et al., 2007). These variations impact binding and protection of neutralizing antibodies (Smith, et al., 2005) and raise concerns that they may prove problematic to the development of prophylactic and therapeutic agents developed against a dissimilar subtype.

BoNTs are produced as a part of a protein complex in which the toxin is non-covalently bound to two or more protein components. This includes the well characterized hemagglutinins (HA) and a nontoxin, nonhemagglutinin (NTNH). The complexes can be distinguished on the basis of their size and serotype association and include the M-(300 kDa in types A-F), L-(500 kDa in serotypes A, B, C, D and G) and LL-(900 kDa in serotype A) forms (Collins & East, 1988). Although neither accessory protein has been implicated in the toxin-mediated blockade of neurotransmitter release, they are believed to protect the toxin against the harsh environment of the gastro-intestinal tract (Simpson, 2004). The neurotoxin is initially produced as a 150-kDa single-chain protoxin that is proteolytically cleaved into an N-terminal 50-kDa light chain (LC) and a C-terminal 100-kDa heavy chain (HC) di-chain that is linked by a single disulfide bond (DasGupta, 1989). The BoNT HC is further delineated into two domains; the N-fragment or translocation domain (Hn) and the Cfragment or receptor-binding domain (Hc). These three domains mediate intoxication of the neuron in a defined tripartite mechanism. The toxin is introduced into the nerve cell by receptor-mediated endocytosis through binding of the Hc domain to specific ectoreceptors on peripheral cholinergic nerve cells (Dong et al., 2006; Rummel et al., 2007). The acidic pH of the endosome initiates a conformation change in the dichain toxin that results in the Hn

*Clostridium spp*. are gram-positive, spore-forming, obligate anaerobic bacteria that are ubiquitous in soil and both freshwater and marine sediment (Dunbar, 1990) . BoNTs are produced primarily in *C. botulinum* but may also be produced in the closely related *C. butyricum*, *C. baratii* and *C. argentinense* (Hatheway, 1993). *C. botulinum* strains are classified into four groups (denoted I to IV) based on metabolic activity (Hatheway, 1988) and genetic composition (Collins, 1998; Hill et al., 2007). Group I includes type A strains and proteolytic strains of types B and F; Group II includes type E strains and nonproteolytic strains of types B and F; Group III includes nonproteolytic strains of types C and D; and Group IV includes only strains that produce type G. There are seven immunologically distinct serotypes of BoNTs designated by the letters A through G (BoNT/A to BoNT/G) (Smith & Sugiyama, 1988). BoNT/A, based on animal studies, has a lethal human dose (LD50), assuming 70 kg weight, of approximately 0.09 - 0.15 g by intravenous administration, 0.7 – 0.9 g by inhalation, and 70 g by oral administration (Scott & Suzuki, 1988; Arnon et al., 2001). The toxicity for other serotypes is unknown but all have been shown to uniformly fatal in animals studies. Human botulism is caused primarily by BoNT/A, /B and /E (Arnon et al., 2001), and rarely by BoNT/F (Barash et al., 2005; Gupta et al., 2005). BoNT/C and /D primarily cause botulism in animals. BoNT/G, produced by *C. argentinense*, has been associated with sudden death but not neuroparalytic illness in a few patients in Switzerland (Sonnabend et al., 1981). All seven serotypes can cause inhalational botulism in primates (Middlebrook & Franz, 1997). Recent characterization of an increasing number of unique BoNT subtypes has revealed small to significant variations at the amino acid level (Smith, et al., 2005; Hill, et al. 2007; Smith, et al., 2007). These variations impact binding and protection of neutralizing antibodies (Smith, et al., 2005) and raise concerns that they may prove problematic to the development of prophylactic and therapeutic agents developed against a dissimilar

BoNTs are produced as a part of a protein complex in which the toxin is non-covalently bound to two or more protein components. This includes the well characterized hemagglutinins (HA) and a nontoxin, nonhemagglutinin (NTNH). The complexes can be distinguished on the basis of their size and serotype association and include the M-(300 kDa in types A-F), L-(500 kDa in serotypes A, B, C, D and G) and LL-(900 kDa in serotype A) forms (Collins & East, 1988). Although neither accessory protein has been implicated in the toxin-mediated blockade of neurotransmitter release, they are believed to protect the toxin against the harsh environment of the gastro-intestinal tract (Simpson, 2004). The neurotoxin is initially produced as a 150-kDa single-chain protoxin that is proteolytically cleaved into an N-terminal 50-kDa light chain (LC) and a C-terminal 100-kDa heavy chain (HC) di-chain that is linked by a single disulfide bond (DasGupta, 1989). The BoNT HC is further delineated into two domains; the N-fragment or translocation domain (Hn) and the Cfragment or receptor-binding domain (Hc). These three domains mediate intoxication of the neuron in a defined tripartite mechanism. The toxin is introduced into the nerve cell by receptor-mediated endocytosis through binding of the Hc domain to specific ectoreceptors on peripheral cholinergic nerve cells (Dong et al., 2006; Rummel et al., 2007). The acidic pH of the endosome initiates a conformation change in the dichain toxin that results in the Hn

**3. Microbiology and BoNT toxicity** 

**3.1 The organism and its toxins** 

subtype.

forming a protein channel that facilitates the translocation of the LC out of the endosomal lumen and into the cytosol. The LC is a zinc-containing endopeptidase that target SNARE (soluble *N*-ethylmaleamide sensitive factor attachment protein receptors) proteins that form the synaptonemal fusion complex in a serotype dependent fashion. The SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDa) is cleaved at different sites by BoNT/A, /C, and /E, synaptobrevin (also referred to as VAMP; vesicle-associated membrane protein), is cleaved at different sites by BoNT/B, /D, /F, and /G, and syntaxin is cleaved by BoNT/C (Simpson, 2004). Proteolytic cleavage of SNARE proteins prevents the release of acetylcholine across the synaptic cleft of the neuromuscular junction, resulting in a flaccid muscle paralysis which is the primary clinical sign of botulism.

#### **3.2 Epidemiology**

Botulism is typically reported in four clinical categories. *Foodborne botulism* is caused by the ingestion of the pre-formed toxin in contaminated food (CDC, 1998). Most outbreaks are associated with home-canned foods in which inadequate processing results in *C. botulinum* spores germinating, reproducing and producing the toxin (Shapiro, et al., 1998). These conditions include an anaerobic environment with temperatures ranging from 4ºC to 40°C, a pH range from 4.6 to 7.0, and water activity greater than 0.94 (aW is intensity with which water associates with various non-aqueous constituents and solids) (Baird-Parker & Freame, 1967; Stringer, et al., 2005). Despite increased educational awareness of the non-permissive conditions for food preparation, foodborne botulism remains a persistent threat. There were, on average, approximately 24 reported cases per year from 1900-2000, but there have been a smaller number of cases (average of 18 year) reported from 2001-2009 (CDC-NBS). In the period from 1950-1996, there were 444 outbreaks in which one or more cases of botulism from a contaminated food source was implicated. Of these outbreaks, 37.6% were caused by type A, 13.7% by type B, 15.1% by type E and 0.7% by type F while 32.9% of the incidents were caused by unidentified serotype(s) (CDC, 1998). Improvements in both differential diagnostic methods and the technology utilized in serotyping have resulted in a decrease in unidentified serotypes. In the United Stated from 1990-2000 BoNT/A was responsible for 50% of all cases of botulism, where types B and E were responsible for 10% and 37% of the intoxications, respectively; only 3.6% of the cases were from an unidentified strain (CDC, 1998).

*Infant botulism* is a toxicoinfection caused by inhalation or ingestion of clostridial spores that can colonize and produce toxin in the intestinal tract of infants less than 12 months of age (Shapiro, 1998). The ability of the bacteria to thrive and elicit toxin is thought to be attributed in part to a deficiency in protective gastrointesintal bacterial flora and the relatively low levels of inhibitory bile-aid found in children under 12 months (CDC, 1998). This form of the disease was officially recognized in a 1976 report in which two infants presenting with acute infantile hypotonoia and weakness were diagnosed with botulism (Pickett, et al., 1976). This has been the most commonly reported form of the disease in the United States since 1980, with an average of approximately 80-100 cases confirmed annually (Shapiro, 1998). In the US between 1976 and 1996, there were 1442 individual cases of infant botulism reported to the CDC (CDC, 1998). Of these, 46.5% were caused by type A, 51.9% by type B. Interestingly, the incidence of infant botulism in the US has a geographical component with 47.6% of the reported cases occurring in California with Delaware, Hawaii,

Botulinum Neurotoxins 113

The clinical presentation of botulism is characterized by distinctive neurological symptoms of the voluntary motor and autonomic cholinergic-associated junctions in the infant, wound and intestinal forms of the disease (CDC, 1998). Foodborne botulism is often accompanied by acute gastrointestinal distress including nausea, abdominal cramps, vomiting and diarrhea that precede the neurological symptoms (Hughes et al, 1981), particularly in types B and E. The initial symptoms of food botulism can manifest anywhere from within a few hours to several days postintoxication. The time to onset of symptoms, the severity and the duration of the disease is largely dictated by the exposure dose and the serotype (Arnon, 2001; Woodruff et al., 1992). Infant botulism may present with constipation, poor feeding, diminished suckling, neck and peripheral weakness, weak crying increased drooling (Corblath, et al., 1993) and ventilatory failure (Arnon, 1992; Long et al., 1985). Wound botulism does not display the gastrointestinal symptoms observed in the foodborne form.

Fever, if present, is generally attributed to a wound infection rather than botulism.

symptom and serotype as shown in Table 1 (Bleck, 2000).

The neuroparalytic effects of botulism present as an acute, afebrile, descending, bilateral flaccid paralysis. The initial neurological symptoms generally involve cranial nerves III, IV and VI (Sobel, 2005) and include ocular disorders such as blurred vision, diplopia, ptosis and photophobia (Terranova et al., 1979). This is generally followed by dysfunction of cranial nerves VII and IX which cause, dysphagia, dysphonia and dysarthia. The neurological impairment may then spread to the upper extremities, the trunk and then the lower extremities. Respiratory distress can be caused by a weakened glottis that tends to obstruct the airway during attempted inspiration or from paralytic weakness of the diaphragm and parasternal and intercostas muscles. Fatigue, sore throat, dry mouth, constipation and dizziness have also been reported to be associated with botulinum intoxications (Hughes, 1981). Fatalities are most often the result of respiratory failure or secondary infections typically associated with prolonged mechanical ventilation. Bleck summarized the clinical findings of several published reports of foodborne botulism by

The primary neurological disorders associated with botulism are common to the foodborne, intestinal, and wound forms of the disease (Sobel, 2005). The duration and severity of the neuroparalytic effects of the disease can be influenced by both the amount and serotype of the toxin introduced into the system. Insight into the impact of the different serotypes in humans has only recently been investigated and much of the data comes from studies involving the therapeutic applications of BoNTs. Of the three most prevalent serotypes involved in human incidences of botulism, BoNT/A has been shown to have the most persistent action and can last 12-16 weeks when used in therapeutic applications (Eleopra, 2004). Serotype B has also been used in therapeutic applications but only exhibits the comparable efficacy to BoNT/A when used in higher doses (Sloop, et al., 1997; Settler, 2001). Electrophysiological studies conducted in juvenile monkeys using purified BoNT/A (BOTOX, Allergan, Irvine, CA) and BoNT/B (Neurobloc, Elan, Shannon, Ireland) indicated BoNT/A diffusion was more pronounced (Arezzo, 2001). The few studies pertaining to serotype C in humans (Eleopra et al., 1997; Eleopra et al., 1998a, Eleopra et al., 1998b) indicate that it is similar to BoNT/A in terms of the toxicity and duration of activity. A 2004 study evaluated the electrophysical responses to human volunteers injected with low doses

**3.3 Clinical symptoms** 

and Utah also experiencing high incidence (CDC, 1988). The correlation between the increased incidences of infant botulism outbreaks associated with certain geographical locations has not been elucidated.

*Wound botulism* (WB) results from the growth of *C. botulinum* spores in a contaminated wound with in vivo toxin production. This form of the disease was first reported in 1951 as a relatively rare illness associated with post-operative complications (Davis et al., 1951). Historically, approximately 75% of the wound botulism cases in the US have been reported in California (Weber, et al., 1993) and this represents over 90% of the reported incidences in the world (Benson, 2001). There were 127 cases of wound botulism in California from 1951 to 1998. Of these, 105 were attributed to intravenous drug users and all but one were admitted black-tar heroin users (Benson, 2001). The increased incidence of WB in California has not abated and between 1993 and 2006 an additional 17 cases were identified in intravenous drug users; 16 of which were diagnosed with one or more recurrent episodes (Yuan et al., 2011).

*Adult intestinal toxemia botulism* is a rare toxicoinfection that occurs in older children and adults with abnormal gastrointestinal tract physiology, such as colitis or intestinal surgical procedures (Freeman et al., 1986; Fenecia et al., 1999). The disease has also been correlated with alteration of protective endogenous microflora by broad-spectrum antibiotics after inflammatory intestinal disease or surgery (Chia et al., 1986).

In addition to the four naturally occurring forms of the disease described previously, there are two additional forms of *inadvertent botulism* that result from the application of the purified toxin. *Iatrogenic botulism* results from the injection of BoNT for either cosmetic or therapeutic purposes. Two adult patients developed symptoms of botulism when given therapeutic doses of BoNT/A drawn from two different production lots (Bakheit, 1997). These cases have since been attributed to either patient sensitivity to the drug or the inadvertent injection of the toxin directly into the vascular capillaries. An adolescent being treated for spastic quadriparesis with Myobloc (botulinum toxin serotype B) developed clinical signs of a systemic BoNT injection (Partikian, 2007). While there are no clear dosing guidelines for BoNT formulations for therapeutic interventions in children, the clinical diagnosis of botulism was again believed to have been the result of inadvertent injection into a blood vessel or diffusion from nearby muscle sites. In November of 2007, four adults were given cosmetic injections of undiluted BoNT/A intended for laboratory research (Chertow et al., 2006). Serial dilutions of two of the individual's serum samples indicated they were given approximately 21 to 43 estimated human lethal doses. All four patients survived but only after prolonged hospitalization with anti-toxin treatment and ventilator support ranging from 40 to 104 days.

*Inhalational botulism* is intoxication by an inhalational exposure of the aerosolized toxin. The only reported human inhalation incidence occurred in Germany in 1962 during a necropsy when three laboratory workers were exposed to animals subjected to aerosolization of a highly purified, lyophilized BoNT/A (Holzer, 1962). The patients were hospitalized five days postexposure, administered equine antitoxin, and discharged after 9 days. While not a naturally occurring form of the disease, inhalational botulism has implications as a potential weapon of bioterrorism (Middlebrook & Franz 1997; Zilinskas, 1997). An aerosol dispersion of BoNT could create a toxic gas cloud that could encompass a large area and is considered to be a likely scenario for a terrorism attack.

and Utah also experiencing high incidence (CDC, 1988). The correlation between the increased incidences of infant botulism outbreaks associated with certain geographical

*Wound botulism* (WB) results from the growth of *C. botulinum* spores in a contaminated wound with in vivo toxin production. This form of the disease was first reported in 1951 as a relatively rare illness associated with post-operative complications (Davis et al., 1951). Historically, approximately 75% of the wound botulism cases in the US have been reported in California (Weber, et al., 1993) and this represents over 90% of the reported incidences in the world (Benson, 2001). There were 127 cases of wound botulism in California from 1951 to 1998. Of these, 105 were attributed to intravenous drug users and all but one were admitted black-tar heroin users (Benson, 2001). The increased incidence of WB in California has not abated and between 1993 and 2006 an additional 17 cases were identified in intravenous drug users; 16 of which were diagnosed with one or more recurrent episodes

*Adult intestinal toxemia botulism* is a rare toxicoinfection that occurs in older children and adults with abnormal gastrointestinal tract physiology, such as colitis or intestinal surgical procedures (Freeman et al., 1986; Fenecia et al., 1999). The disease has also been correlated with alteration of protective endogenous microflora by broad-spectrum antibiotics after

In addition to the four naturally occurring forms of the disease described previously, there are two additional forms of *inadvertent botulism* that result from the application of the purified toxin. *Iatrogenic botulism* results from the injection of BoNT for either cosmetic or therapeutic purposes. Two adult patients developed symptoms of botulism when given therapeutic doses of BoNT/A drawn from two different production lots (Bakheit, 1997). These cases have since been attributed to either patient sensitivity to the drug or the inadvertent injection of the toxin directly into the vascular capillaries. An adolescent being treated for spastic quadriparesis with Myobloc (botulinum toxin serotype B) developed clinical signs of a systemic BoNT injection (Partikian, 2007). While there are no clear dosing guidelines for BoNT formulations for therapeutic interventions in children, the clinical diagnosis of botulism was again believed to have been the result of inadvertent injection into a blood vessel or diffusion from nearby muscle sites. In November of 2007, four adults were given cosmetic injections of undiluted BoNT/A intended for laboratory research (Chertow et al., 2006). Serial dilutions of two of the individual's serum samples indicated they were given approximately 21 to 43 estimated human lethal doses. All four patients survived but only after prolonged hospitalization with anti-toxin treatment and ventilator

*Inhalational botulism* is intoxication by an inhalational exposure of the aerosolized toxin. The only reported human inhalation incidence occurred in Germany in 1962 during a necropsy when three laboratory workers were exposed to animals subjected to aerosolization of a highly purified, lyophilized BoNT/A (Holzer, 1962). The patients were hospitalized five days postexposure, administered equine antitoxin, and discharged after 9 days. While not a naturally occurring form of the disease, inhalational botulism has implications as a potential weapon of bioterrorism (Middlebrook & Franz 1997; Zilinskas, 1997). An aerosol dispersion of BoNT could create a toxic gas cloud that could encompass a large area and is considered

inflammatory intestinal disease or surgery (Chia et al., 1986).

locations has not been elucidated.

support ranging from 40 to 104 days.

to be a likely scenario for a terrorism attack.

(Yuan et al., 2011).

#### **3.3 Clinical symptoms**

The clinical presentation of botulism is characterized by distinctive neurological symptoms of the voluntary motor and autonomic cholinergic-associated junctions in the infant, wound and intestinal forms of the disease (CDC, 1998). Foodborne botulism is often accompanied by acute gastrointestinal distress including nausea, abdominal cramps, vomiting and diarrhea that precede the neurological symptoms (Hughes et al, 1981), particularly in types B and E. The initial symptoms of food botulism can manifest anywhere from within a few hours to several days postintoxication. The time to onset of symptoms, the severity and the duration of the disease is largely dictated by the exposure dose and the serotype (Arnon, 2001; Woodruff et al., 1992). Infant botulism may present with constipation, poor feeding, diminished suckling, neck and peripheral weakness, weak crying increased drooling (Corblath, et al., 1993) and ventilatory failure (Arnon, 1992; Long et al., 1985). Wound botulism does not display the gastrointestinal symptoms observed in the foodborne form. Fever, if present, is generally attributed to a wound infection rather than botulism.

The neuroparalytic effects of botulism present as an acute, afebrile, descending, bilateral flaccid paralysis. The initial neurological symptoms generally involve cranial nerves III, IV and VI (Sobel, 2005) and include ocular disorders such as blurred vision, diplopia, ptosis and photophobia (Terranova et al., 1979). This is generally followed by dysfunction of cranial nerves VII and IX which cause, dysphagia, dysphonia and dysarthia. The neurological impairment may then spread to the upper extremities, the trunk and then the lower extremities. Respiratory distress can be caused by a weakened glottis that tends to obstruct the airway during attempted inspiration or from paralytic weakness of the diaphragm and parasternal and intercostas muscles. Fatigue, sore throat, dry mouth, constipation and dizziness have also been reported to be associated with botulinum intoxications (Hughes, 1981). Fatalities are most often the result of respiratory failure or secondary infections typically associated with prolonged mechanical ventilation. Bleck summarized the clinical findings of several published reports of foodborne botulism by symptom and serotype as shown in Table 1 (Bleck, 2000).

The primary neurological disorders associated with botulism are common to the foodborne, intestinal, and wound forms of the disease (Sobel, 2005). The duration and severity of the neuroparalytic effects of the disease can be influenced by both the amount and serotype of the toxin introduced into the system. Insight into the impact of the different serotypes in humans has only recently been investigated and much of the data comes from studies involving the therapeutic applications of BoNTs. Of the three most prevalent serotypes involved in human incidences of botulism, BoNT/A has been shown to have the most persistent action and can last 12-16 weeks when used in therapeutic applications (Eleopra, 2004). Serotype B has also been used in therapeutic applications but only exhibits the comparable efficacy to BoNT/A when used in higher doses (Sloop, et al., 1997; Settler, 2001). Electrophysiological studies conducted in juvenile monkeys using purified BoNT/A (BOTOX, Allergan, Irvine, CA) and BoNT/B (Neurobloc, Elan, Shannon, Ireland) indicated BoNT/A diffusion was more pronounced (Arezzo, 2001). The few studies pertaining to serotype C in humans (Eleopra et al., 1997; Eleopra et al., 1998a, Eleopra et al., 1998b) indicate that it is similar to BoNT/A in terms of the toxicity and duration of activity. A 2004 study evaluated the electrophysical responses to human volunteers injected with low doses


DTRs, deep tendon reflexes; NA, not available.

Table 1. Summary of Symptoms of Patients with Botulism Caused by Serotypes A, B an E. from Springer Scientific Publishing.

Botulinum Neurotoxins 115

of BoNT/A, /B, /C or /F (Eleopra et al., 2004). The results were consistent with other research efforts in that BoNT/B, used in higher doses, and BoNT/C have a similar profile as BoNT/A. As reported in previous studies (Mezaki et al., 1995; Chen et al., 1998), BoNT/F

A rapid and accurate identification of botulism is not difficult when the disease is strongly suspected, such as found in the setting of a large outbreak. But because cases of naturally occurring botulism most often occur singularly, the individual diagnosis may prove more challenging. Botulism is thought to be substantially underdiagnosed (CDC, 1988) and with low-level exposure, minor neurological manifestations of the disease may resolve without medical intervention. A differential diagnosis of botulism without concurrent knowledge of a confirmed outbreak can be difficult and other paralytic illnesses may need to be excluded. These include Guillain-Barre syndrome, myasthenia gravis, tick paralysis, and Eaton-Lambert syndrome (Dembek, et al., 2007). Less likely conditions such as tetrodotoxin and shellfish poisoning, aminoglycoside toxicity and a variety of other neurotoxic products and neurological abnormalities may initially present with similar symptoms. However, a thorough medical examination of the patient and their medical history can generally exclude any competing diagnosis. A patient presenting with an acute, bilateral, descending flaccid paralysis that is afebrile and has normal sensorium should suggest a clinical case of

The most reliable method for the detection of BoNTs and for diagnosing botulism is the mouse bioassay. This test can be performed by the Centers for Disease Control and Prevention (CDC) or state public health laboratories. The assay involves injecting mice with samples collected from patients displaying symptoms of botulism. Mice will typically begin showing signs of botulism within 8 h. The serotype of samples can also be ascertained in this manner by neutralizing the toxin with serotype-specific antibodies prior to injecting the mice (Shapiro et al., 1998). In cases of foodborne or infant botulism, stool samples can also be cultured to look for *C. botulinum*. Samples of the suspected food should also be cultured

Standard therapy for botulism involves administration of botulinum antitoxin in an attempt to prevent neurologic progression of a moderately progressive illness, or to reduce the duration of respiratory failure in individuals with a severe, rapidly progressive illness. This is done in conjuction with careful monitoring of respiratory vital capacity and aggressive ventilatory care for individuals that display respiratory failure. The only specific pharmacological treatment for botulism is administration of equine-derived botulinum antitoxin. On March 12, 2010, a new heptavalent botulinum antitoxin (HBAT, Cangene Corp.) became available through a US CDC-sponsored FDA Investigational New Drug (IND) protocol for the treatment of naturally acquired non-infant botulism (CDC, 2010). This antitoxin replaced the previously FDA-approved equine bivalent botulinum antitoxin AB and an investigational monovalent equine antitoxin E (BAT-AB and BAT-E,

was found to have a shorter duration compared to BoNT/A.

anaerobically with heat or alcohol treatment to select for spores.

**4. Medical countermeasures 4.1 Current medical intervention** 

**3.4 Diagnosis of botulism** 

botulism.

Type A (%) Type B (%) Type E (%)

Dysphagia 96 97 82 Dry mouth 83 100 93 Diplopia 90 92 39 Dysarthia 100 69 50 Upper extremity weakness 86 64 NA Lower extremity weakness 76 64 NA Blurred vision 100 42 91 Dyspnea 91 34 88 Paresthesiae 20 12 NA

Constipation 73 73 52 Nausea 73 57 84 Vomiting 70 50 96 Abdominal cramps 33 46 NA Diarrhea 35 8 39

Fatigue 92 69 84 Sore throat 75 39 38 Dizziness 86 30 63

Ptosis 96 55 46 Diminished gag reflex 81 54 NA Ophthalmoparesis 87 46 NA Facial paresis 84 48 NA Tongue weakness 21 31 66 Pupils fixed or dilated 33 56 75 Nystagmus 44 4 NA Upper extremity weakness 91 62 NA Lower extremity weakness 82 59 NA Ataxia 24 13 NA DTRs diminished or absent 54 29 NA DTRs hyperactive 12 0 NA

Alert 88 93 27 Lethargic 4 4 73 Obtunded 8 4 0

Table 1. Summary of Symptoms of Patients with Botulism Caused by Serotypes A, B an E.

*Neurological symptoms* 

*Gastrointestinal Symptoms* 

*Miscellaneous symptoms* 

*Neurological Findings* 

*Initial mental status* 

DTRs, deep tendon reflexes; NA, not available.

from Springer Scientific Publishing.

of BoNT/A, /B, /C or /F (Eleopra et al., 2004). The results were consistent with other research efforts in that BoNT/B, used in higher doses, and BoNT/C have a similar profile as BoNT/A. As reported in previous studies (Mezaki et al., 1995; Chen et al., 1998), BoNT/F was found to have a shorter duration compared to BoNT/A.

#### **3.4 Diagnosis of botulism**

A rapid and accurate identification of botulism is not difficult when the disease is strongly suspected, such as found in the setting of a large outbreak. But because cases of naturally occurring botulism most often occur singularly, the individual diagnosis may prove more challenging. Botulism is thought to be substantially underdiagnosed (CDC, 1988) and with low-level exposure, minor neurological manifestations of the disease may resolve without medical intervention. A differential diagnosis of botulism without concurrent knowledge of a confirmed outbreak can be difficult and other paralytic illnesses may need to be excluded. These include Guillain-Barre syndrome, myasthenia gravis, tick paralysis, and Eaton-Lambert syndrome (Dembek, et al., 2007). Less likely conditions such as tetrodotoxin and shellfish poisoning, aminoglycoside toxicity and a variety of other neurotoxic products and neurological abnormalities may initially present with similar symptoms. However, a thorough medical examination of the patient and their medical history can generally exclude any competing diagnosis. A patient presenting with an acute, bilateral, descending flaccid paralysis that is afebrile and has normal sensorium should suggest a clinical case of botulism.

The most reliable method for the detection of BoNTs and for diagnosing botulism is the mouse bioassay. This test can be performed by the Centers for Disease Control and Prevention (CDC) or state public health laboratories. The assay involves injecting mice with samples collected from patients displaying symptoms of botulism. Mice will typically begin showing signs of botulism within 8 h. The serotype of samples can also be ascertained in this manner by neutralizing the toxin with serotype-specific antibodies prior to injecting the mice (Shapiro et al., 1998). In cases of foodborne or infant botulism, stool samples can also be cultured to look for *C. botulinum*. Samples of the suspected food should also be cultured anaerobically with heat or alcohol treatment to select for spores.

#### **4. Medical countermeasures**

#### **4.1 Current medical intervention**

Standard therapy for botulism involves administration of botulinum antitoxin in an attempt to prevent neurologic progression of a moderately progressive illness, or to reduce the duration of respiratory failure in individuals with a severe, rapidly progressive illness. This is done in conjuction with careful monitoring of respiratory vital capacity and aggressive ventilatory care for individuals that display respiratory failure. The only specific pharmacological treatment for botulism is administration of equine-derived botulinum antitoxin. On March 12, 2010, a new heptavalent botulinum antitoxin (HBAT, Cangene Corp.) became available through a US CDC-sponsored FDA Investigational New Drug (IND) protocol for the treatment of naturally acquired non-infant botulism (CDC, 2010). This antitoxin replaced the previously FDA-approved equine bivalent botulinum antitoxin AB and an investigational monovalent equine antitoxin E (BAT-AB and BAT-E,

Botulinum Neurotoxins 117

neutralizing antibodies, it also displayed a number of localized and systemic reactogenic effects (Reames et al., 1947). An improved product in which the A and B toxins were purified to 10-15% homomgeneity by acid-precipitation of the culture supernatants and adjuvanted onto aluminum phosphate was found to be well-tolerated in humans, displayed only minor localized reactogenic effects and produced significantly improved antibody titers over the previous bivalent toxoid (Fiock et al., 1961). A pentavalent ABCDE toxoid vaccine with 1% thymerosol as a preservative prepared by Parke, Davis and Company was administered to approximately 400 individuals (Fiock et al., 1963). The vaccine displayed minor reactogenic effects and elicted detectable antibody titers to all five serotypes. This product was administered to over 1600 individuals from 1970 to 1981 under an Investigational New Drug (IND) application. The Michigan Department of Public Health produced a pentavalent botulism (ABCDE) toxoid (PBT) using similar procedures as the Parke Davis product, but which contained roughly 50% of the formaldehyde and only 0.01% thymerosol, that has been administered under an IND to at-risk laboratory workers and military personnel. However, the declining immunogenicity, dwindling supplies and local reactogenic effects of the PBT (Rusnak & Smith, 2009; CDC, 2009) have led to recent efforts

More contemporary toxoid vaccines have been created by chemical neutralization of the purified toxin (Keller, 2008; Jones, et al., 2008). However, large scale production of these products would require a secure, CDC-licensed facility to both propagate large amounts the bacterium and manipulate the purified toxin. Subsequent efforts have largely relied on the expression of recombinant protein antigens encoding one or more of the BoNT domains.

Codon optimized genes encoding the BoNT (Hc) antigens produced in a *P. pastoris* expression platform have been successfully developed as a recombinant subunit vaccine against serotypes A-F and have been demonstrated to elicit protective immunity in both rodent (Smith, 2009; Rusnak & Smith 2009) and non-human primates (Boles et al., 2006; Morefield, 2008). In February of 2011, the Dynport Vaccine Company announced that phase II clinical trials of a recombinant bivalent Hc vaccine against serotypes A and B (rBV A/B) had been completed and that full licensure would be sought. (CSC, 2011). Recombinant, catalytically inactive BoNT holoproteins made by mutagenesis of key amino acids residues have also been employed as potential vaccine candidates. A recombinant BoNT/C protein with active site mutations H229G, E230T and H223N produced in *E. coli* displayed no catalytic activity and elicited protective immunity against the parental toxin when delivered either subcutaneously or orally (Kiyatkin et al., 1997). Pier described the expression of a recombinant BoNT/A gene bearing R363A and Y365F mutations in the LNT01 nontoxigenic strain of *C. botulinum* (Pier et al., 2008). The recombinant protein was unable to cleave SNAP-25 and elicited protective immunity in mice when challenged with BoNT/A. Recombinant BoNT/A with active site mutations H223A, E224A and H227A produced in *P. pastoris* was found to be completely non-toxic and provided protective immunity in mice against 1000 MLD50 of not only the parental toxin, but against subtypes /A2 and /A3 as

Once the neurotoxic LC is endocytosed within the cytosol of peripheral cholinergic neurons, circulatory antibodies will no longer be effective at neutralizing the catalytic

to create new vaccines.

well (Webb et al., 2009).

**4.3 Post-intoxication interventions** 

Sanofi Pasteur). HBAT contains equine-derived antibody against all BoNT serotypes: 7,500 U anti-A; 5,500 U anti-B; 5,000 U anti-C; 1,000 U anti-D; 8,500 U anti-E; 5,000 U anti-F; and 1,000 U anti-G per vial. This antitoxin is composed of <2% intact immunoglobulin G (IgG) and ≥90% Fab and F(ab')2 immunoglobulin fragments created by despeciation (CDC, 2010). The recommended adult dosing is one 20 mL vial of HBAT (McLaughlin and Funk, 2010). BabyBIG®, human botulism immune globulin intravenous (BIG-IV), is an FDA-approved drug for the treatment of infant botulism types A and B. Available through the California Infant Botulism Treatment and Prevention Program, BabyBIG® is obtained from the pooled plasma of adults vaccinated with the pentavalent (A-E) botulinum toxoid who displayed high titers of neutralizing antibodies against BoNT/A and /B. Because BabyBIG® is of human origin, it does not carry the risk for anaphylaxis inherent with equine products, nor does BabyBIG® demonstrate a risk for possible lifelong hypersensitivity to equine antigens. BabyBIG® has been shown to significantly shorten the hospitalization period and reduce treatment costs up to \$75,000.00 per incident (Thompson et al., 2005; Fox, 2005).

Antitoxin can neutralize toxin molecules that are not yet bound to nerve endings and may limit the progression of the disease and prevent further nerve damage by clearing them from circulation. Thus, the antitoxin should be administered immediately upon a definitive diagnosis of botulism, preferably within 24 h after the onset of symptoms (Tacket et al., 1984; Chang and Ganguly, 2003). Compared to standard care alone, immediate administration of antitoxin has been shown to shorten time on respiratory support, and also reduced the hospitalization period (Shapiro et al., 1997; Tacket et al., 1984). It is not clear how long the toxin can persist in the bloodstream before clearance. Ravichandran reported that BoNT/A had a serum half-life of approximately 4 hours in small animal studies (Ravichandran et al., 2005). This study also suggested that blood does not sequester or modify the toxin in any detectable way. Thus, the blood may act as a reservoir for the toxin until it either enters the target cells or is eliminated from the body. Detectable levels of toxin were observed in one of the four patients in the Florida outbreak 8 days after receiving a massive overdose of an unlicensed preparation of BoNT/A during a cosmetic procedure (Chertow et al., 2006). Under such circumstances, antitoxin administration, even if delayed, may still be effective in limiting the duration of the illness. The use of equine-based antitoxins, but not the human product, has been associated with symptoms of hypersensitivity (including urticaria, serum sickness, and anaphylaxis). Hence, dermal testing is required before antitoxin administration. Due to the risk of adverse reactions, prophylactic antitoxin is not recommended in patients who are exposed to BoNT but have no symptoms. These patients may undergo gastric lavage or induced vomiting in an attempt to eliminate the toxin before absorption (Chan-Tack & Bartlett, 2010).

#### **4.2 Prophylaxis**

In the United States, there is currently no FDA-licensed prophylactic product against botulism. Some of the earliest vaccine efforts were initiated during the second World War due to concerns that the toxin might be employed as an offensive biological weapon against the allied forces by Germany. A bivalent A/B toxoid vaccine was produced from 3-day culture autolysates of *C. botulinum* that were chemically neutralized with formaldehyde, filtered and adsorbed onto an alum adjuvant. While the vaccine did elicit the production of

Sanofi Pasteur). HBAT contains equine-derived antibody against all BoNT serotypes: 7,500 U anti-A; 5,500 U anti-B; 5,000 U anti-C; 1,000 U anti-D; 8,500 U anti-E; 5,000 U anti-F; and 1,000 U anti-G per vial. This antitoxin is composed of <2% intact immunoglobulin G (IgG) and ≥90% Fab and F(ab')2 immunoglobulin fragments created by despeciation (CDC, 2010). The recommended adult dosing is one 20 mL vial of HBAT (McLaughlin and Funk, 2010). BabyBIG®, human botulism immune globulin intravenous (BIG-IV), is an FDA-approved drug for the treatment of infant botulism types A and B. Available through the California Infant Botulism Treatment and Prevention Program, BabyBIG® is obtained from the pooled plasma of adults vaccinated with the pentavalent (A-E) botulinum toxoid who displayed high titers of neutralizing antibodies against BoNT/A and /B. Because BabyBIG® is of human origin, it does not carry the risk for anaphylaxis inherent with equine products, nor does BabyBIG® demonstrate a risk for possible lifelong hypersensitivity to equine antigens. BabyBIG® has been shown to significantly shorten the hospitalization period and reduce treatment costs up to \$75,000.00 per

Antitoxin can neutralize toxin molecules that are not yet bound to nerve endings and may limit the progression of the disease and prevent further nerve damage by clearing them from circulation. Thus, the antitoxin should be administered immediately upon a definitive diagnosis of botulism, preferably within 24 h after the onset of symptoms (Tacket et al., 1984; Chang and Ganguly, 2003). Compared to standard care alone, immediate administration of antitoxin has been shown to shorten time on respiratory support, and also reduced the hospitalization period (Shapiro et al., 1997; Tacket et al., 1984). It is not clear how long the toxin can persist in the bloodstream before clearance. Ravichandran reported that BoNT/A had a serum half-life of approximately 4 hours in small animal studies (Ravichandran et al., 2005). This study also suggested that blood does not sequester or modify the toxin in any detectable way. Thus, the blood may act as a reservoir for the toxin until it either enters the target cells or is eliminated from the body. Detectable levels of toxin were observed in one of the four patients in the Florida outbreak 8 days after receiving a massive overdose of an unlicensed preparation of BoNT/A during a cosmetic procedure (Chertow et al., 2006). Under such circumstances, antitoxin administration, even if delayed, may still be effective in limiting the duration of the illness. The use of equine-based antitoxins, but not the human product, has been associated with symptoms of hypersensitivity (including urticaria, serum sickness, and anaphylaxis). Hence, dermal testing is required before antitoxin administration. Due to the risk of adverse reactions, prophylactic antitoxin is not recommended in patients who are exposed to BoNT but have no symptoms. These patients may undergo gastric lavage or induced vomiting in an attempt to eliminate the toxin before absorption (Chan-Tack &

In the United States, there is currently no FDA-licensed prophylactic product against botulism. Some of the earliest vaccine efforts were initiated during the second World War due to concerns that the toxin might be employed as an offensive biological weapon against the allied forces by Germany. A bivalent A/B toxoid vaccine was produced from 3-day culture autolysates of *C. botulinum* that were chemically neutralized with formaldehyde, filtered and adsorbed onto an alum adjuvant. While the vaccine did elicit the production of

incident (Thompson et al., 2005; Fox, 2005).

Bartlett, 2010).

**4.2 Prophylaxis** 

neutralizing antibodies, it also displayed a number of localized and systemic reactogenic effects (Reames et al., 1947). An improved product in which the A and B toxins were purified to 10-15% homomgeneity by acid-precipitation of the culture supernatants and adjuvanted onto aluminum phosphate was found to be well-tolerated in humans, displayed only minor localized reactogenic effects and produced significantly improved antibody titers over the previous bivalent toxoid (Fiock et al., 1961). A pentavalent ABCDE toxoid

vaccine with 1% thymerosol as a preservative prepared by Parke, Davis and Company was administered to approximately 400 individuals (Fiock et al., 1963). The vaccine displayed minor reactogenic effects and elicted detectable antibody titers to all five serotypes. This product was administered to over 1600 individuals from 1970 to 1981 under an Investigational New Drug (IND) application. The Michigan Department of Public Health produced a pentavalent botulism (ABCDE) toxoid (PBT) using similar procedures as the Parke Davis product, but which contained roughly 50% of the formaldehyde and only 0.01% thymerosol, that has been administered under an IND to at-risk laboratory workers and military personnel. However, the declining immunogenicity, dwindling supplies and local reactogenic effects of the PBT (Rusnak & Smith, 2009; CDC, 2009) have led to recent efforts to create new vaccines.

More contemporary toxoid vaccines have been created by chemical neutralization of the purified toxin (Keller, 2008; Jones, et al., 2008). However, large scale production of these products would require a secure, CDC-licensed facility to both propagate large amounts the bacterium and manipulate the purified toxin. Subsequent efforts have largely relied on the expression of recombinant protein antigens encoding one or more of the BoNT domains.

Codon optimized genes encoding the BoNT (Hc) antigens produced in a *P. pastoris* expression platform have been successfully developed as a recombinant subunit vaccine against serotypes A-F and have been demonstrated to elicit protective immunity in both rodent (Smith, 2009; Rusnak & Smith 2009) and non-human primates (Boles et al., 2006; Morefield, 2008). In February of 2011, the Dynport Vaccine Company announced that phase II clinical trials of a recombinant bivalent Hc vaccine against serotypes A and B (rBV A/B) had been completed and that full licensure would be sought. (CSC, 2011). Recombinant, catalytically inactive BoNT holoproteins made by mutagenesis of key amino acids residues have also been employed as potential vaccine candidates. A recombinant BoNT/C protein with active site mutations H229G, E230T and H223N produced in *E. coli* displayed no catalytic activity and elicited protective immunity against the parental toxin when delivered either subcutaneously or orally (Kiyatkin et al., 1997). Pier described the expression of a recombinant BoNT/A gene bearing R363A and Y365F mutations in the LNT01 nontoxigenic strain of *C. botulinum* (Pier et al., 2008). The recombinant protein was unable to cleave SNAP-25 and elicited protective immunity in mice when challenged with BoNT/A. Recombinant BoNT/A with active site mutations H223A, E224A and H227A produced in *P. pastoris* was found to be completely non-toxic and provided protective immunity in mice against 1000 MLD50 of not only the parental toxin, but against subtypes /A2 and /A3 as well (Webb et al., 2009).

#### **4.3 Post-intoxication interventions**

Once the neurotoxic LC is endocytosed within the cytosol of peripheral cholinergic neurons, circulatory antibodies will no longer be effective at neutralizing the catalytic

Botulinum Neurotoxins 119

Diseases (NIAID), antibody combinations that effectively protect against multiple BoNT/A, /B, and /E subtypes are currently being produced and tested to support FDA licensure. Antibodies that protect against the four remaining toxin serotypes (BoNT/C, /D, /F, and

While a majority of the BoNT immunotherapy research has been focused on antibodies that bind the HC, efforts were also directed to explore the potential for antibodies that bind the enzymatic LC. Using a novel hybridoma method for cloning human antibodies (Adekar et al., 2008; Dessain et al., 2004), a fully human antibody specific for the BoNT/A LC was isolated which potently inhibited BoNT/A in vitro and in vivo, via mechanisms not previously associated with BoNT-neutralizing antibodies (Adekar et al., 2008). In another study, Dong et al. (2010) created a library of non-immune llama single-domain VHH (camelid heavy-chain variable regions) displayed on the surface of the yeast *Saccharomyces cerevisiae*. Library selections against BoNT/A LC yielded 15 yeast-displayed VHHs, eight of which inhibited the cleavage of substrate SNAP-25 by BoNT/A LC. The most potent VHH (Aa1) had a solution K(d) of 1.47 x 10-10 M and an IC50 of 4.7 x 10-10 M. X-ray crystal structure of the BoNT/A LC-Aa1 VHH complex revealed that the Aa1 VHH binds the alpha-exosite region of BoNT/A LC. Recently, Tremblay et al. (2010) reported on the selection of small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B from libraries of VHHs or nanobodies cloned from vaccinated alpacas. Several VHHs were demonstrated to exhibit high affinity (KD near 1 nm) and were potent inhibitors of BoNT/A LC (Ki near 1 nM); a VHH inhibitor of BoNT/A LC was able to protect BoNT/A-mediated SNAP25

Several compounds that inhibit the acidification process of endosomes by various mechanisms have been evaluated for both toxicity and ability to inhibit BoNT-induced synaptic failure. Lysosomotropic agents ammonium chloride and methylamine hydrochloride have been shown to antagonize the toxin internalization step by delaying the time-to-block of nerve-evoked muscle contractions after exposure to BoNT/A, /B, /C1, and TeNT (Simpson, 1983). However, these amines act by inhibiting the acidification process of endosomes; they do not selectively inactivate the toxins nor irreversibly modify tissue function at concentrations that inhibit the onset of BoNT-induced paralysis. Other candidates that have been examined were uncouplers of oxidative phosphorylation CCCP, FCCP (Adler et al., 1994), and vesicle H+-ATPase inhibitors including Bafilomycin A, which was shown to antagonize BoNTs A-G (Simpson et al., 1994). Some of these compounds were toxic or had low safety margins, hence they were deemed unsuitable as therapeutic

Anti-malarial compounds chloroquine and hydroxychloroquine have also been evaluated for potentially inhibiting BoNT-mediated internalization (Simpson, 1982). The efficacy of these agents was found similar to that of ammonium chloride and methylamine hydrochloride; both groups also exhibited a comparable therapeutic window. Deshpande and co-workers (1997) extended the studies on antimalarial agents by examining a large

/G) are also in development.

cleavage.

candidates.

**4.5 Antibodies specific for the catalytic light chain** 

**4.6 Inhibitors of internalization and translocation** 

activity and ablating the toxicity. A number of recent research efforts have focused on the development of small-molecule inhibitors to reduce or eliminate the cleavage of SNARE proteins in neurons. The drawbacks associated with the use of peptides as drug candidates (e.g., poor tissue penetration, serum resistance, oral bioavailability, and quick elimination), and the potential usefulness of small molecules as pre- and post-exposure therapeutic agents have led many laboratories to instead pursue small-molecule approaches. To date, research has focused predominantly on developing small-molecule inhibitors that target the BoNT/A LC protease, due to the serotype's persistence and highly toxic nature. A number of BoNT/A small-molecule inhibitors, identified using conventional and novel approaches, have been reported that exhibit varying degrees of inhibitory capacity (Burnett et al., 2003; 2009; Boldt et al., 2006; Park et al., 2006; Tang et al., 2007; Capkova et al., 2007; 2009; Moe et al., 2009; Roxas-Duncan et al., 2009; Burnett et al., 2010). Most of these studies were conducted in vitro using truncated forms of the LC which might not be structurally representative of the intracellular form of BoNT. The inhibitors reported by Roxas-Duncan et al (2009), identified via a hierarchical screening strategy, were evaluated in vitro using both forms of LC (truncated and full-length), and ex vivo using mouse phrenic nerve hemidiaphragm preparations. Despite intensive efforts on small-molecule inhibitor discovery and development, no compound has been identified that would be suitable for preclinical testing. At present, there are only two reports of BoNT/A small-molecule inhibitors that have been tested in vivo. Janda and coworkers described a mouse toxicity bioassay in which two different inhibitors were injected intravenously immediately after an injection of 5-10 i.p. LD50 of BoNT/A. One compound, given at a 2.5 mM dose, showed a 36% increase in time to death, while the other, given at a 1 mM dose, resulted in a 16% overall survival rate (Eubanks et al., 2007). In another study, a single dose of three different inhibitors was administered i.p. at 2 mg/kg 30 min prior to a challenge of 5 MLD50 of BoNT/A (Pang et al., 2010). The control mice died within 12 hrs. All 3 inhibitors provided 100% protection at 12 hrs and one compound provided 70% and 60% survival rates at 24 and 48 hrs; all three inhibitors provided a 10% overall survival rate with no signs of botulism at 5 days.

Recently, other regions of the LC, in addition to the BoNT active site, generated attention as potential targets for inhibition. Merrino et al., (2006) has focused on a family of bisimidazole BoNT/A inhibitors targeting the peripheral sites of substrate binding. Silhar et al., (2010) reported on D-chicoric acid, a natural product isolated from Echinacea, that inhibits BoNT/A LC by binding to an exosite.

#### **4.4 Human recombinant monoclonal antibodies**

Currently, immunotherapy is deemed as the most effective immediate response to BoNT exposure. However, BabyBig® is exclusively approved for use in infants, and equine antisera can induce serum sickness and anaphylaxis (Arnon et al., 2001; Arnon, 2004). Monoclonal antibody (mAb) combinations (oligoclonal antibodies) may be viable substitutes for polyclonal antisera (Nowakowski et al., 2002; Razai et al., 2005). Construction of scFv phage Ab libraries has enabled the generation of large panels of high-affinity binding monoclonal antibodies. Mouse neutralization studies revealed that effective protection is observed only when combinations of three or more mAbs are used (Nowakowski et al., 2002; Marks, 2004). Under the aegis of the National Institute of Allergy and Infectious

activity and ablating the toxicity. A number of recent research efforts have focused on the development of small-molecule inhibitors to reduce or eliminate the cleavage of SNARE proteins in neurons. The drawbacks associated with the use of peptides as drug candidates (e.g., poor tissue penetration, serum resistance, oral bioavailability, and quick elimination), and the potential usefulness of small molecules as pre- and post-exposure therapeutic agents have led many laboratories to instead pursue small-molecule approaches. To date, research has focused predominantly on developing small-molecule inhibitors that target the BoNT/A LC protease, due to the serotype's persistence and highly toxic nature. A number of BoNT/A small-molecule inhibitors, identified using conventional and novel approaches, have been reported that exhibit varying degrees of inhibitory capacity (Burnett et al., 2003; 2009; Boldt et al., 2006; Park et al., 2006; Tang et al., 2007; Capkova et al., 2007; 2009; Moe et al., 2009; Roxas-Duncan et al., 2009; Burnett et al., 2010). Most of these studies were conducted in vitro using truncated forms of the LC which might not be structurally representative of the intracellular form of BoNT. The inhibitors reported by Roxas-Duncan et al (2009), identified via a hierarchical screening strategy, were evaluated in vitro using both forms of LC (truncated and full-length), and ex vivo using mouse phrenic nerve hemidiaphragm preparations. Despite intensive efforts on small-molecule inhibitor discovery and development, no compound has been identified that would be suitable for preclinical testing. At present, there are only two reports of BoNT/A small-molecule inhibitors that have been tested in vivo. Janda and coworkers described a mouse toxicity bioassay in which two different inhibitors were injected intravenously immediately after an injection of 5-10 i.p. LD50 of BoNT/A. One compound, given at a 2.5 mM dose, showed a 36% increase in time to death, while the other, given at a 1 mM dose, resulted in a 16% overall survival rate (Eubanks et al., 2007). In another study, a single dose of three different inhibitors was administered i.p. at 2 mg/kg 30 min prior to a challenge of 5 MLD50 of BoNT/A (Pang et al., 2010). The control mice died within 12 hrs. All 3 inhibitors provided 100% protection at 12 hrs and one compound provided 70% and 60% survival rates at 24 and 48 hrs; all three inhibitors

provided a 10% overall survival rate with no signs of botulism at 5 days.

BoNT/A LC by binding to an exosite.

**4.4 Human recombinant monoclonal antibodies** 

Recently, other regions of the LC, in addition to the BoNT active site, generated attention as potential targets for inhibition. Merrino et al., (2006) has focused on a family of bisimidazole BoNT/A inhibitors targeting the peripheral sites of substrate binding. Silhar et al., (2010) reported on D-chicoric acid, a natural product isolated from Echinacea, that inhibits

Currently, immunotherapy is deemed as the most effective immediate response to BoNT exposure. However, BabyBig® is exclusively approved for use in infants, and equine antisera can induce serum sickness and anaphylaxis (Arnon et al., 2001; Arnon, 2004). Monoclonal antibody (mAb) combinations (oligoclonal antibodies) may be viable substitutes for polyclonal antisera (Nowakowski et al., 2002; Razai et al., 2005). Construction of scFv phage Ab libraries has enabled the generation of large panels of high-affinity binding monoclonal antibodies. Mouse neutralization studies revealed that effective protection is observed only when combinations of three or more mAbs are used (Nowakowski et al., 2002; Marks, 2004). Under the aegis of the National Institute of Allergy and Infectious Diseases (NIAID), antibody combinations that effectively protect against multiple BoNT/A, /B, and /E subtypes are currently being produced and tested to support FDA licensure. Antibodies that protect against the four remaining toxin serotypes (BoNT/C, /D, /F, and /G) are also in development.

#### **4.5 Antibodies specific for the catalytic light chain**

While a majority of the BoNT immunotherapy research has been focused on antibodies that bind the HC, efforts were also directed to explore the potential for antibodies that bind the enzymatic LC. Using a novel hybridoma method for cloning human antibodies (Adekar et al., 2008; Dessain et al., 2004), a fully human antibody specific for the BoNT/A LC was isolated which potently inhibited BoNT/A in vitro and in vivo, via mechanisms not previously associated with BoNT-neutralizing antibodies (Adekar et al., 2008). In another study, Dong et al. (2010) created a library of non-immune llama single-domain VHH (camelid heavy-chain variable regions) displayed on the surface of the yeast *Saccharomyces cerevisiae*. Library selections against BoNT/A LC yielded 15 yeast-displayed VHHs, eight of which inhibited the cleavage of substrate SNAP-25 by BoNT/A LC. The most potent VHH (Aa1) had a solution K(d) of 1.47 x 10-10 M and an IC50 of 4.7 x 10-10 M. X-ray crystal structure of the BoNT/A LC-Aa1 VHH complex revealed that the Aa1 VHH binds the alpha-exosite region of BoNT/A LC. Recently, Tremblay et al. (2010) reported on the selection of small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B from libraries of VHHs or nanobodies cloned from vaccinated alpacas. Several VHHs were demonstrated to exhibit high affinity (KD near 1 nm) and were potent inhibitors of BoNT/A LC (Ki near 1 nM); a VHH inhibitor of BoNT/A LC was able to protect BoNT/A-mediated SNAP25 cleavage.

#### **4.6 Inhibitors of internalization and translocation**

Several compounds that inhibit the acidification process of endosomes by various mechanisms have been evaluated for both toxicity and ability to inhibit BoNT-induced synaptic failure. Lysosomotropic agents ammonium chloride and methylamine hydrochloride have been shown to antagonize the toxin internalization step by delaying the time-to-block of nerve-evoked muscle contractions after exposure to BoNT/A, /B, /C1, and TeNT (Simpson, 1983). However, these amines act by inhibiting the acidification process of endosomes; they do not selectively inactivate the toxins nor irreversibly modify tissue function at concentrations that inhibit the onset of BoNT-induced paralysis. Other candidates that have been examined were uncouplers of oxidative phosphorylation CCCP, FCCP (Adler et al., 1994), and vesicle H+-ATPase inhibitors including Bafilomycin A, which was shown to antagonize BoNTs A-G (Simpson et al., 1994). Some of these compounds were toxic or had low safety margins, hence they were deemed unsuitable as therapeutic candidates.

Anti-malarial compounds chloroquine and hydroxychloroquine have also been evaluated for potentially inhibiting BoNT-mediated internalization (Simpson, 1982). The efficacy of these agents was found similar to that of ammonium chloride and methylamine hydrochloride; both groups also exhibited a comparable therapeutic window. Deshpande and co-workers (1997) extended the studies on antimalarial agents by examining a large

the cytosol.

to translocate a ligated cargo into the cytosol.

could be potentially used to deliver conjugated therapeutic cargoes.

**5. Emergency preparedness and public response** 

Botulinum Neurotoxins 121

internalization into the endosomes were observed, but minimal levels were detected in the cytosol. The movement of a small, membrane-permeable dye from the endosome into the cytosol is hypothesized to be due to passive diffusion instead of an active translocation event. Ho et al (2010) reported on a recombinant BoNT/A HC with an amino terminal GFP fusion that was internalized into mouse neurons; however, the GFP cargo was observed to be almost exclusively limited to endocytotic vesicles, with little detectable translocation into

Although the of BoNT heavy chain comprises the domains necessary for binding and internalization into endosomes, recent studies suggest that all or part of the LC is essential

Recombinant BoNT/D fusion proteins bearing amino terminal GFP, luciferase, dihydrofolate reductase or BoNT/A LC protein were found to promote translocation of cargo proteins into the cytosol in an enzymatically active form (Bade et al., 2004). A recombinant neutralized BoNT/A bearing an E224A E262A double mutation, labeled with Alexa-488, has been shown to specifically bind and internalize into human SH-SY5Y neuroblastoma cells (Sing et al., 2010). Additionally, this protein was found to marginally bind to the surface of human rhabdomyosarcoma cells with a toxicity limit of 1 µg in mice. No additional information is available regarding the drug conjugation study on this protein. Moreover, recombinant versions of full-length BoNT/A holotoxin devoid of catalytic activity were recently developed (Pier et al., 2008; Webb et al., 2009; Yang et al., 2008) which

The 2007 anthrax attacks in the United States illustrated the need to develop a comprehensive preparedness plan for identifying and managing healthcare resources in the event of a biological attack. Anthrax, plague, botulinum toxins, smallpox, tularemia, and viral hemorrhagic fever viruses have been identified as having a significant potential for use in a bioterrorism attack because they can be easily disseminated or transmitted, have high morbidity or mortality rates and would cause widespread social disruption (Rotz et al., 2002). A bioterrorism attack involving dissemination of BoNT by existing food or water distribution networks is theoretically possible but this route is associated with significant logistical difficulties (Franz, et al., 1997; Zilinskas, 1999) and most experts believe that an aerosol dispersion poses the greatest threat (Arnon, 2002). Because botulism is a relatively rare disease, clinicians and the healthcare infrastructure in general have limited experience in diagnosis of any form of the disease. A 2005 study reported that of 631 physicians participating in a mock bioterrosim event, slightly less than half provided an accurate diagnosis and plan of management for botulism (Cosgrove, 2005). In the event of a biological attack with BoNT, a rapid and accurate diagnosis of the agent by physicians and clinical laboratories would be crucial to an equally rapid response and mobilization of equipment and biological agents for management. Health care providers who suspect botulism should immediately call their state health department's emergency 24-hour emergency telephone number. The state health department will contact the CDC to report suspected botulism cases, arrange for a clinical consultation by telephone and, if indicated, request release of botulinum antitoxin. State health departments should call the CDC 24 hour telephone number at 770-488-7100. The call will be taken by the CDC Emergency

group of 4- and 8-aminoquinolines. Unfortunately, these compounds failed to extend the therapeutic window. The most effective compounds were 4-aminoquinolines and quinacrine that delayed BoNT/A-induced neuromuscular block by more than threefold compared to the control (toxin only) values. Maximum protection was solely achieved when the tissues were exposed to the compounds before or at the same time as the toxin treatment; a delay of >20 min abolished the inhibitory capacity of these compounds.

An additional approach to prevent or reduce BoNT internalization has been attempted by treating nerve-muscle preparations with the protein ionophores nigericin and monensin (Adler et al., 1994; Sheridan, 1996). These ionophores block vesicle acidification by acting as H+ shunts to neutralize pH gradients, thereby interfering with the delivery of active LC in the cytosol. Though the efficacy of these ionophores was observed to be comparable to other inhibitors of internalization, they were more toxic; high concentrations resulted in a depression of neuromuscular transmission (Adler et al., 1994; Sheridan, 1996).

#### **4.7 Compounds that restore neuronal function**

A known potassium channel blocker, 3,4-diaminopyridine (3,4-DAP) was evaluated for its ability to antagonize BoNT-induced depression of tension in rat diaphragm muscles (Adler et al., 1995). BoNT-induced paralysis was nearly completely inhibited after addition of 100 uM of 3,4-DAP, and this effect was sustained even after 4 h of treatment. The antagonistic effects of 3,4-DAP was also demonstrated in vivo, provided its concentration in the plasma is maintained at ~30 M during the course of intoxication (Adler et al., 2000). However, 3,4- DAP is generally toxic, thus, its high drug concentration requirement prohibits routine therapeutic use (Millard, 2006).

Mastoparan, a phospholipase activator, was evaluated for its ability to attenuate BoNT intoxication. Addition of mastoparan and 80 mM K+ completely prevented BoNT inhibition of radiolabeled acetylcholine in PC12 cells, but this effect was blocked by either EGTA or the N-type calcium channel blocker ω-conotoxin (Ray et al., 1999). These findings imply that the effects of mastoparan are dependent on Ca2+ influx via the neuronal type voltage-sensitive Ca2+ channels.

#### **4.8 Drug delivery vehicle research**

One significant challenge in the development of BoNT small-molecule therapeutics is the delivery of the compounds to the cytosol of peripheral cholinergic nerve cells (PCNC), which are the sites of BoNT action. In combination with the discovery and development of BoNT small-molecule inhibitors, cell-specific intracellular targeting is critical to increase the therapeutic index and minimize potential systemic toxicity associated with the drug treatment. Several studies have examined the potential of using specific recombinant BoNT domains or a neutralized full-length BoNT holoprotein as drug delivery system. Goodnough et al. observed that BoNT/A and unlabeled rHC were able to compete for binding, implicating specific neuronal targeting (Goodnough et al., 2002). The efficiency of neuron-specific cargo delivery into the cytosol was evaluated by coupling labeled dextran to a recombinant BoNT/A HC using a 3-(2-pyridylthio)-propionyl hydrazide linker (Zhang et al., 2009). A florescent tracking dye was conjugated to the dextran moiety and incubated in mouse cultured mouse spinal cord neurons. The binding of the fluorescent tag and its

group of 4- and 8-aminoquinolines. Unfortunately, these compounds failed to extend the therapeutic window. The most effective compounds were 4-aminoquinolines and quinacrine that delayed BoNT/A-induced neuromuscular block by more than threefold compared to the control (toxin only) values. Maximum protection was solely achieved when the tissues were exposed to the compounds before or at the same time as the toxin treatment; a delay of

An additional approach to prevent or reduce BoNT internalization has been attempted by treating nerve-muscle preparations with the protein ionophores nigericin and monensin (Adler et al., 1994; Sheridan, 1996). These ionophores block vesicle acidification by acting as H+ shunts to neutralize pH gradients, thereby interfering with the delivery of active LC in the cytosol. Though the efficacy of these ionophores was observed to be comparable to other inhibitors of internalization, they were more toxic; high concentrations resulted in a

A known potassium channel blocker, 3,4-diaminopyridine (3,4-DAP) was evaluated for its ability to antagonize BoNT-induced depression of tension in rat diaphragm muscles (Adler et al., 1995). BoNT-induced paralysis was nearly completely inhibited after addition of 100 uM of 3,4-DAP, and this effect was sustained even after 4 h of treatment. The antagonistic effects of 3,4-DAP was also demonstrated in vivo, provided its concentration in the plasma is maintained at ~30 M during the course of intoxication (Adler et al., 2000). However, 3,4- DAP is generally toxic, thus, its high drug concentration requirement prohibits routine

Mastoparan, a phospholipase activator, was evaluated for its ability to attenuate BoNT intoxication. Addition of mastoparan and 80 mM K+ completely prevented BoNT inhibition of radiolabeled acetylcholine in PC12 cells, but this effect was blocked by either EGTA or the N-type calcium channel blocker ω-conotoxin (Ray et al., 1999). These findings imply that the effects of mastoparan are dependent on Ca2+ influx via the neuronal type voltage-sensitive

One significant challenge in the development of BoNT small-molecule therapeutics is the delivery of the compounds to the cytosol of peripheral cholinergic nerve cells (PCNC), which are the sites of BoNT action. In combination with the discovery and development of BoNT small-molecule inhibitors, cell-specific intracellular targeting is critical to increase the therapeutic index and minimize potential systemic toxicity associated with the drug treatment. Several studies have examined the potential of using specific recombinant BoNT domains or a neutralized full-length BoNT holoprotein as drug delivery system. Goodnough et al. observed that BoNT/A and unlabeled rHC were able to compete for binding, implicating specific neuronal targeting (Goodnough et al., 2002). The efficiency of neuron-specific cargo delivery into the cytosol was evaluated by coupling labeled dextran to a recombinant BoNT/A HC using a 3-(2-pyridylthio)-propionyl hydrazide linker (Zhang et al., 2009). A florescent tracking dye was conjugated to the dextran moiety and incubated in mouse cultured mouse spinal cord neurons. The binding of the fluorescent tag and its

depression of neuromuscular transmission (Adler et al., 1994; Sheridan, 1996).

>20 min abolished the inhibitory capacity of these compounds.

**4.7 Compounds that restore neuronal function** 

therapeutic use (Millard, 2006).

**4.8 Drug delivery vehicle research** 

Ca2+ channels.

internalization into the endosomes were observed, but minimal levels were detected in the cytosol. The movement of a small, membrane-permeable dye from the endosome into the cytosol is hypothesized to be due to passive diffusion instead of an active translocation event. Ho et al (2010) reported on a recombinant BoNT/A HC with an amino terminal GFP fusion that was internalized into mouse neurons; however, the GFP cargo was observed to be almost exclusively limited to endocytotic vesicles, with little detectable translocation into the cytosol.

Although the of BoNT heavy chain comprises the domains necessary for binding and internalization into endosomes, recent studies suggest that all or part of the LC is essential to translocate a ligated cargo into the cytosol.

Recombinant BoNT/D fusion proteins bearing amino terminal GFP, luciferase, dihydrofolate reductase or BoNT/A LC protein were found to promote translocation of cargo proteins into the cytosol in an enzymatically active form (Bade et al., 2004). A recombinant neutralized BoNT/A bearing an E224A E262A double mutation, labeled with Alexa-488, has been shown to specifically bind and internalize into human SH-SY5Y neuroblastoma cells (Sing et al., 2010). Additionally, this protein was found to marginally bind to the surface of human rhabdomyosarcoma cells with a toxicity limit of 1 µg in mice. No additional information is available regarding the drug conjugation study on this protein. Moreover, recombinant versions of full-length BoNT/A holotoxin devoid of catalytic activity were recently developed (Pier et al., 2008; Webb et al., 2009; Yang et al., 2008) which could be potentially used to deliver conjugated therapeutic cargoes.

#### **5. Emergency preparedness and public response**

The 2007 anthrax attacks in the United States illustrated the need to develop a comprehensive preparedness plan for identifying and managing healthcare resources in the event of a biological attack. Anthrax, plague, botulinum toxins, smallpox, tularemia, and viral hemorrhagic fever viruses have been identified as having a significant potential for use in a bioterrorism attack because they can be easily disseminated or transmitted, have high morbidity or mortality rates and would cause widespread social disruption (Rotz et al., 2002). A bioterrorism attack involving dissemination of BoNT by existing food or water distribution networks is theoretically possible but this route is associated with significant logistical difficulties (Franz, et al., 1997; Zilinskas, 1999) and most experts believe that an aerosol dispersion poses the greatest threat (Arnon, 2002). Because botulism is a relatively rare disease, clinicians and the healthcare infrastructure in general have limited experience in diagnosis of any form of the disease. A 2005 study reported that of 631 physicians participating in a mock bioterrosim event, slightly less than half provided an accurate diagnosis and plan of management for botulism (Cosgrove, 2005). In the event of a biological attack with BoNT, a rapid and accurate diagnosis of the agent by physicians and clinical laboratories would be crucial to an equally rapid response and mobilization of equipment and biological agents for management. Health care providers who suspect botulism should immediately call their state health department's emergency 24-hour emergency telephone number. The state health department will contact the CDC to report suspected botulism cases, arrange for a clinical consultation by telephone and, if indicated, request release of botulinum antitoxin. State health departments should call the CDC 24 hour telephone number at 770-488-7100. The call will be taken by the CDC Emergency

Botulinum Neurotoxins 123

targeting pathways involved in the cellular response to BoNT intoxication, are being explored. An understanding of these mechanisms may provide insight into the design and development of innovative and effective therapeutic strategies to counteract BoNT

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intoxications.

**7. References** 

0022-1759

ISSN 0027-8424

ISSN 1351-5101

Philadelphia, PA, USA.

1070, ISSN 0098-7484

Philadelphia, PA, USA

3042

0781702267, New York, NY, USA.

New NY.

Operations Center, which will page the Foodborne and Diarrheal Diseases Branch medical officer on call. The CDC established the Laboratory Response Network (LRN) in 1999; a national network of about 150 federal, state, local and military labs that can respond to biological and chemical terrorism, and other public health emergencies (CDC-EPR). The LRN hierarchy also includes *reference labs* that can perform tests to detect and confirm the presence of a threat agent to ensure a rapid local response in the event of a bioterrorism incident without having to rely on confirmation from CDC labs. *Sentinel labs* are comprised of thousands of hospital-based labs that have direct contact with patients. These labs might well be the first facility to spot a suspicious specimen and refer it to the appropriate reference lab (Sentinel, ASM).

The ability of the government and healthcare infrastructure to respond to a bioterroism attack using BoNT in a densely populated urban environment would largely dictated be by the ventilator and antitoxin supplies and how quickly they could be mobilized to the outbreak area. Approximately 120,000 of the 200,000 doses of the HBAT contracted by the Biomedical Advanced Research and Development Authority (BARDA) have been delivered for use in the US national strategic stockpile and the remaining 80,000 doses are scheduled for delivery by 2014. A 2010 study estimated that there are approximately 63,066 fullfeatured mechanical ventilators in the US, 46.4% of which were capable of ventilating pediatric and neonatal patients (Rubinson et al, 2010). Additionally, there was an estimated 82,775 additional positive pressure ventilators (PPV) that could be brought on-line in response to an acute respiratory failure (ARF) surge event. This study may represent a conservative estimate as it only accounted for survey responders and it did not consider the various units rented by hospitals, those facilities who failed to respond to the survey or those at nursing facilities or schools.

#### **6. Conclusion**

BoNT has been investigated as a BW by a number of different state-sponsored initiatives and terrorist organizations and is considered a biological threat to both our military and the public. Effective medical countermeasures against BoNT intoxication are limited. Currently, the only available treatment other than supportive care is a new investigational botulinum heptavalent equine-based antitoxin. However, antitoxin cannot intervene in the pathogenesis of the disease once the toxin enters the nerve cell, and cannot support all those infected in the event of a biological terrorist attack. Hence, there is a critical need for postintoxication therapy that can be administered rapidly and effectively to a large infected population. Because small molecules provide an opportunity to treat botulism both before and after cellular intoxication has occurred, considerable research efforts have been devoted to the development of these types of inhibitors. A number of strong contributions to the field have been made, yet no small molecule inhibitor was identified that possesses the appropriate characteristics (safety, efficacy, solubility) required to be a pharmaceutical intervention.

Research studies addressing these obstacles are underway. Continuing efforts will be facilitated particularly by the availability of structural information and by knowledge of the mechanism of the BoNT LC-mediated proteolysis of SNARE proteins. Additionally, potential novel strategies to therapeutic development, e.g., host-directed therapeutics, and

Operations Center, which will page the Foodborne and Diarrheal Diseases Branch medical officer on call. The CDC established the Laboratory Response Network (LRN) in 1999; a national network of about 150 federal, state, local and military labs that can respond to biological and chemical terrorism, and other public health emergencies (CDC-EPR). The LRN hierarchy also includes *reference labs* that can perform tests to detect and confirm the presence of a threat agent to ensure a rapid local response in the event of a bioterrorism incident without having to rely on confirmation from CDC labs. *Sentinel labs* are comprised of thousands of hospital-based labs that have direct contact with patients. These labs might well be the first facility to spot a suspicious specimen and refer it to the appropriate

The ability of the government and healthcare infrastructure to respond to a bioterroism attack using BoNT in a densely populated urban environment would largely dictated be by the ventilator and antitoxin supplies and how quickly they could be mobilized to the outbreak area. Approximately 120,000 of the 200,000 doses of the HBAT contracted by the Biomedical Advanced Research and Development Authority (BARDA) have been delivered for use in the US national strategic stockpile and the remaining 80,000 doses are scheduled for delivery by 2014. A 2010 study estimated that there are approximately 63,066 fullfeatured mechanical ventilators in the US, 46.4% of which were capable of ventilating pediatric and neonatal patients (Rubinson et al, 2010). Additionally, there was an estimated 82,775 additional positive pressure ventilators (PPV) that could be brought on-line in response to an acute respiratory failure (ARF) surge event. This study may represent a conservative estimate as it only accounted for survey responders and it did not consider the various units rented by hospitals, those facilities who failed to respond to the survey or

BoNT has been investigated as a BW by a number of different state-sponsored initiatives and terrorist organizations and is considered a biological threat to both our military and the public. Effective medical countermeasures against BoNT intoxication are limited. Currently, the only available treatment other than supportive care is a new investigational botulinum heptavalent equine-based antitoxin. However, antitoxin cannot intervene in the pathogenesis of the disease once the toxin enters the nerve cell, and cannot support all those infected in the event of a biological terrorist attack. Hence, there is a critical need for postintoxication therapy that can be administered rapidly and effectively to a large infected population. Because small molecules provide an opportunity to treat botulism both before and after cellular intoxication has occurred, considerable research efforts have been devoted to the development of these types of inhibitors. A number of strong contributions to the field have been made, yet no small molecule inhibitor was identified that possesses the appropriate characteristics (safety, efficacy, solubility) required to be a pharmaceutical

Research studies addressing these obstacles are underway. Continuing efforts will be facilitated particularly by the availability of structural information and by knowledge of the mechanism of the BoNT LC-mediated proteolysis of SNARE proteins. Additionally, potential novel strategies to therapeutic development, e.g., host-directed therapeutics, and

reference lab (Sentinel, ASM).

those at nursing facilities or schools.

**6. Conclusion** 

intervention.

targeting pathways involved in the cellular response to BoNT intoxication, are being explored. An understanding of these mechanisms may provide insight into the design and development of innovative and effective therapeutic strategies to counteract BoNT intoxications.

#### **7. References**


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Virginia I. Roxas-Duncan1 and Leonard A. Smith2

*U.S. Army Medical Research Institute of Infectious Diseases,* 

*2Senior Research Scientist (ST) for Medical Countermeasures Technology,* 

*Office of Chief Scientist, U.S. Army Medical Research Institute of Infectious Diseases,* 

Ricin is one of the most toxic and easily produced plant toxins. It is derived from the castor plant, *Ricinus communis* (Fig. 1)*,* a shrub native to Africa but currently is being cultivated in many areas of the world for its co mmercial products, primarily castor oil. The seeds of *R. communis*, commonly called beans (although not a true bean) (Fig. 2), are oblong, brown, and have a thick mottled shell sometimes used to make decorative necklaces and bracelets. Castor seeds contain 40 to 60% vegetable oil that is rich in triglycerides, mainly ricinolein (McKeon et al., 1999). Castor oil is used in a number of products. For many years, purified castor oil has been ingested as a human nutritional supplement, emetic, or purgative worldwide (Scarpa and Guerci, 1982; Caupin, 1997; Olsnes, 2004). In addition to castor oil production, castor plants are also being grown for aesthetic (ornamental garden bush) and ecological values. It is used extensively as a decorative plant in parks and other public areas. Ecologically, despite the ricin being poisonous to humans and many animals, *R. communis* is the host plant of insects including moths and butterflies, and is also used as a food plant by

Fig. 1. Castor plant, *Ricinus communis*, a large shrub having large palmate leaves and spiny

capsules containing seeds that are the source of castor oil and ricin (Adapted from

http://dtirp.dtra.mil/images/RicinusCommunis.jpg)

**1. Introduction**

some Lepidopteran larvae and birds.

*1Integrated Toxicology Division,* 

*USA* 

Zilinkas, R.A. (1997). Iraq's biological weapons: the past as future? *JAMA*, Vol.278, No.5, pp. 418-424, ISSN 0098-7484 **7** 

Virginia I. Roxas-Duncan1 and Leonard A. Smith2

*1Integrated Toxicology Division, U.S. Army Medical Research Institute of Infectious Diseases, 2Senior Research Scientist (ST) for Medical Countermeasures Technology, Office of Chief Scientist, U.S. Army Medical Research Institute of Infectious Diseases, USA* 

#### **1. Introduction**

132 Bioterrorism

Zilinkas, R.A. (1997). Iraq's biological weapons: the past as future? *JAMA*, Vol.278, No.5, pp.

Ricin is one of the most toxic and easily produced plant toxins. It is derived from the castor plant, *Ricinus communis* (Fig. 1)*,* a shrub native to Africa but currently is being cultivated in many areas of the world for its co mmercial products, primarily castor oil. The seeds of *R. communis*, commonly called beans (although not a true bean) (Fig. 2), are oblong, brown, and have a thick mottled shell sometimes used to make decorative necklaces and bracelets. Castor seeds contain 40 to 60% vegetable oil that is rich in triglycerides, mainly ricinolein (McKeon et al., 1999). Castor oil is used in a number of products. For many years, purified castor oil has been ingested as a human nutritional supplement, emetic, or purgative worldwide (Scarpa and Guerci, 1982; Caupin, 1997; Olsnes, 2004). In addition to castor oil production, castor plants are also being grown for aesthetic (ornamental garden bush) and ecological values. It is used extensively as a decorative plant in parks and other public areas. Ecologically, despite the ricin being poisonous to humans and many animals, *R. communis* is the host plant of insects including moths and butterflies, and is also used as a food plant by some Lepidopteran larvae and birds.

Fig. 1. Castor plant, *Ricinus communis*, a large shrub having large palmate leaves and spiny capsules containing seeds that are the source of castor oil and ricin (Adapted from http://dtirp.dtra.mil/images/RicinusCommunis.jpg)

In recent centuries, however, natural castor oil was at first identified as a laxative and as a lubricant for the wheels of wagons and carts, as well as aircrafts during World War I (WWI). Today, castor oil (extracted minus the ricin) has a wide variety of commercial applications (International Castor Oil Association, ICOA, 1992). It is used for medicinal purposes both internally, as a strong and effective purgative or cathartic, and externally to treat corns, among other purposes (Sims & Frey, 2005). Castor oil and its derivatives also have numerous industrial merits, being used in a wide variety of products, such as the basic ingredient in racing motor oil for high-performance engines, a fuel additive for two-cycle engines, a primary raw material in the production of nylons and other resins and fibers, and a component in paint and varnish, insulation, fabric coatings, soap, ink, plastics, hydraulic fluids, lubricants, guns, insecticidal oils, cosmetics, and antifungal compounds (Brugsch, 1960; Caupin, 1997; McKeon et al., 1999; Sims & Frey, 2005). Because of its economic benefits and myriads of uses, castor seeds are currently being produced in more than 30 countries in the world. In 2008, world's production of castor oil totaled to 1,605,362 metric tons (MT) (Food and Agricultural Organization of the United Nations Statistical Database, FAOSTAT, 2011); the leading producers include India (1,171,000 MT), China (190,000 MT), and Brazil (122,140 MT). After oil extraction and inactivation of ricin, the defatted waste "mash" (also called castor bean meal) is used as animal feed while the seed husks are used as high

Ricin toxin was discovered in 1888 by Hermann Stillmark, a student at the Dorpat University in Estonia (Stillmark, 1888, as cited in Franz and Jaax, 1997). During Stillmark's extensive research, he also observed that ricin caused agglutination of erythrocytes and precipitation of serum proteins (Olsnes, 2004). Olsnes and Phil (1972) demonstrated that ricin inhibited protein synthesis, and suggested that the effects resulted from restricted elongation of nascent polypeptide chain. Subsequent studies revealed that the molecular

In the last decade, immunotoxins using the ricin A-chain chemically-linked to monoclonal antibodies have been used as an alternative in therapies against cancer, AIDS and other illnesses (Engert et al., 1997; Schnell et al., 2003; Youn et al., 2005). Ricin-based immunotoxins, some of which contained deglycosylated ricin A chain conjugated to either the anti-CD22 antibody RFB4 (Amlot et al., 1993; Sausville et al., 1995), or its Fab fragment (Vitetta et al., 1991) have also been shown to provide enhanced therapeutic efficacy and resulted in improved antitumor activity (Li et al., 2005; Kreitman et al., 2005; Vitetta, 2006). However, the U.S. Food and Drug Administration (FDA) has placed a hold on the clinical testing of RTA-based immunotoxins because they caused vascular leak syndrome (VLS) in

During WWI, ricin was investigated as a potential offensive biological weapon. Two methods of weaponizing the toxin were explored, i.e., bullets and shrapnel coated with ricin, or a 'dust cloud' of toxin inhaled into the lungs (Smart, 1997). Nonetheless, the thermal instability of ricin constrained its initial use in exploding shells, and ethical and treaty issues limited its use as a poison or blinding agents (Hunt et al., 1918, as cited in Millard &

target of the toxin was the 60S ribosomal subunit (Olsnes and Phil, 1982).

nitrogen fertilizer (Kole, 2011).

**2.1 Biological warfare and terrorism** 

**2.1.1 History of ricin as a biological weapon** 

humans.

Castor seeds contain large amounts of ricin, which is also present in lower concentrations throughout the plant. Ricin content significantly varies among cultivars/accessions and may range from 0.1 to 4% of the weight of the seed (Auld et al., 2003; Baldoni et al., 2011; Pinkerton et al., 1999). Ricin is one of the most potent poisons in the plant kingdom (Lee & Wang, 2005). Because of the wide availability of its source plants, ease of production, stability, and lethal potency, ricin toxin is considered to be a bioterrorism threat. Ricin is the most common biological agent used in biocrimes, and has also been reported as one of the most prevalent agents involved in WMD (weapons of mass destructions) investigations (FBI, n.d.). Recent attempted uses of ricin by various extremists and radical groups have heightened concerns regarding ricin's potential for urban terrorism.

Fig. 2. Castor seeds (Adapted from http://www.ars.usda.gov/is/AR/archive/jan01/plant0101.htm)

#### **2. History, biological warfare and terrorism**

The castor plant belongs to the genus *Ricinus* of the Euphorbiaceae or spurge family (Atsmon, 1985). *R. communis* is indigenous to the southeastern Mediterranean region, eastern Africa, and India, but is now widespread throughout temperate and subtropical regions (McKeon et al., 2000; Phillips & Rix, 1999). The gourd mentioned in the Book of Jonah (Jon 4:6-9; Old Testament), bears the Hebrew name kikayon, and is presumed to be the kiki of the Egyptians, the castor-oil plant (Easton's Bible Dictionary, n.d.). It is commonly known as "Palm of Christ" or *Palma Christi*, that derives from castor oil's ability to heal wounds and cure ailments. Castor seeds have been found in Egyptian tombs dating back to 4000 BC, and were used in folk medicines against a wide variety of diseases. The plant has been cultivated for its commercial products, primarily castor oil, for at least 4000 years (Olsnes, 2004). Castor oil was used in rituals of sacrifice to please the gods in early civilizations. In Ancient Egypt and in Europe, it has been used for lighting, body ointments, improving hair growth and texture, and medicinal purposes, where it was regarded as a folk medicine. In India, castor oil has been documented since 2000 BC and was mainly used in lamps and in local medicine as a laxative, purgative, cathartic and other ethnomedical systems. In China, castor seed and its oil have been prescribed for centuries in local medicine for internal use. In Italy, castor oil was used as an instrument of coercion by the Squadristi, the Fascists armed squads of dictator Benito Mussolini; this idea originated from Gabriele D' Annunzio, a controversial nationalist, poet and war veteran (MacDonald, 1999). Political dissidents and regime opponents were forced to ingest the oil in large amounts, triggering severe diarrhea and dehydration that oftentimes led to death (New World Encyclopedia, n.d.).

Castor seeds contain large amounts of ricin, which is also present in lower concentrations throughout the plant. Ricin content significantly varies among cultivars/accessions and may range from 0.1 to 4% of the weight of the seed (Auld et al., 2003; Baldoni et al., 2011; Pinkerton et al., 1999). Ricin is one of the most potent poisons in the plant kingdom (Lee & Wang, 2005). Because of the wide availability of its source plants, ease of production, stability, and lethal potency, ricin toxin is considered to be a bioterrorism threat. Ricin is the most common biological agent used in biocrimes, and has also been reported as one of the most prevalent agents involved in WMD (weapons of mass destructions) investigations (FBI, n.d.). Recent attempted uses of ricin by various extremists and radical groups have

The castor plant belongs to the genus *Ricinus* of the Euphorbiaceae or spurge family (Atsmon, 1985). *R. communis* is indigenous to the southeastern Mediterranean region, eastern Africa, and India, but is now widespread throughout temperate and subtropical regions (McKeon et al., 2000; Phillips & Rix, 1999). The gourd mentioned in the Book of Jonah (Jon 4:6-9; Old Testament), bears the Hebrew name kikayon, and is presumed to be the kiki of the Egyptians, the castor-oil plant (Easton's Bible Dictionary, n.d.). It is commonly known as "Palm of Christ" or *Palma Christi*, that derives from castor oil's ability to heal wounds and cure ailments. Castor seeds have been found in Egyptian tombs dating back to 4000 BC, and were used in folk medicines against a wide variety of diseases. The plant has been cultivated for its commercial products, primarily castor oil, for at least 4000 years (Olsnes, 2004). Castor oil was used in rituals of sacrifice to please the gods in early civilizations. In Ancient Egypt and in Europe, it has been used for lighting, body ointments, improving hair growth and texture, and medicinal purposes, where it was regarded as a folk medicine. In India, castor oil has been documented since 2000 BC and was mainly used in lamps and in local medicine as a laxative, purgative, cathartic and other ethnomedical systems. In China, castor seed and its oil have been prescribed for centuries in local medicine for internal use. In Italy, castor oil was used as an instrument of coercion by the Squadristi, the Fascists armed squads of dictator Benito Mussolini; this idea originated from Gabriele D' Annunzio, a controversial nationalist, poet and war veteran (MacDonald, 1999). Political dissidents and regime opponents were forced to ingest the oil in large amounts, triggering severe diarrhea and dehydration that oftentimes led to death (New World

heightened concerns regarding ricin's potential for urban terrorism.

http://www.ars.usda.gov/is/AR/archive/jan01/plant0101.htm)

**2. History, biological warfare and terrorism** 

Fig. 2. Castor seeds (Adapted from

Encyclopedia, n.d.).

In recent centuries, however, natural castor oil was at first identified as a laxative and as a lubricant for the wheels of wagons and carts, as well as aircrafts during World War I (WWI). Today, castor oil (extracted minus the ricin) has a wide variety of commercial applications (International Castor Oil Association, ICOA, 1992). It is used for medicinal purposes both internally, as a strong and effective purgative or cathartic, and externally to treat corns, among other purposes (Sims & Frey, 2005). Castor oil and its derivatives also have numerous industrial merits, being used in a wide variety of products, such as the basic ingredient in racing motor oil for high-performance engines, a fuel additive for two-cycle engines, a primary raw material in the production of nylons and other resins and fibers, and a component in paint and varnish, insulation, fabric coatings, soap, ink, plastics, hydraulic fluids, lubricants, guns, insecticidal oils, cosmetics, and antifungal compounds (Brugsch, 1960; Caupin, 1997; McKeon et al., 1999; Sims & Frey, 2005). Because of its economic benefits

and myriads of uses, castor seeds are currently being produced in more than 30 countries in the world. In 2008, world's production of castor oil totaled to 1,605,362 metric tons (MT) (Food and Agricultural Organization of the United Nations Statistical Database, FAOSTAT, 2011); the leading producers include India (1,171,000 MT), China (190,000 MT), and Brazil (122,140 MT). After oil extraction and inactivation of ricin, the defatted waste "mash" (also called castor bean meal) is used as animal feed while the seed husks are used as high nitrogen fertilizer (Kole, 2011).

Ricin toxin was discovered in 1888 by Hermann Stillmark, a student at the Dorpat University in Estonia (Stillmark, 1888, as cited in Franz and Jaax, 1997). During Stillmark's extensive research, he also observed that ricin caused agglutination of erythrocytes and precipitation of serum proteins (Olsnes, 2004). Olsnes and Phil (1972) demonstrated that ricin inhibited protein synthesis, and suggested that the effects resulted from restricted elongation of nascent polypeptide chain. Subsequent studies revealed that the molecular target of the toxin was the 60S ribosomal subunit (Olsnes and Phil, 1982).

In the last decade, immunotoxins using the ricin A-chain chemically-linked to monoclonal antibodies have been used as an alternative in therapies against cancer, AIDS and other illnesses (Engert et al., 1997; Schnell et al., 2003; Youn et al., 2005). Ricin-based immunotoxins, some of which contained deglycosylated ricin A chain conjugated to either the anti-CD22 antibody RFB4 (Amlot et al., 1993; Sausville et al., 1995), or its Fab fragment (Vitetta et al., 1991) have also been shown to provide enhanced therapeutic efficacy and resulted in improved antitumor activity (Li et al., 2005; Kreitman et al., 2005; Vitetta, 2006). However, the U.S. Food and Drug Administration (FDA) has placed a hold on the clinical testing of RTA-based immunotoxins because they caused vascular leak syndrome (VLS) in humans.

#### **2.1 Biological warfare and terrorism**

#### **2.1.1 History of ricin as a biological weapon**

During WWI, ricin was investigated as a potential offensive biological weapon. Two methods of weaponizing the toxin were explored, i.e., bullets and shrapnel coated with ricin, or a 'dust cloud' of toxin inhaled into the lungs (Smart, 1997). Nonetheless, the thermal instability of ricin constrained its initial use in exploding shells, and ethical and treaty issues limited its use as a poison or blinding agents (Hunt et al., 1918, as cited in Millard &

*Rickettsia prowazekii*, toxins (e.g., ricin, Staphylococcal enterotoxin B, epsilon toxin of *Clostridium perfringens*), *Chlamydia psittaci*, food safety threats (e.g., *Salmonella* spp., *Escherichia coli* O157:H7, *Shigella*), and water safety threats (e.g. *Vibrio cholerae*, *Cryptosporidium parvum*) (Rotz et al., 2002). Though ricin is not considered an effective weapon of mass destruction, its potential as a weapon of terror cannot be discounted. Further, ricin's notoriety is likely driven by the ready availability of castor beans, press coverage, and popularization on the internet. In the U.S. for example, the use of ricin as a biological agent in bioterrorism and homicides is of particular concern especially after the events of September 11, 2001. Worldwide, numerous cases involving the possession, experimentation, or planned misuse of ricin by bioterrorists and extremist groups have been investigated or prosecuted by law enforcement agencies (Franz and Jaax, 1997; James Martin Center for Nonproliferation Studies (CNS), 2004; Research International, Inc. (RII), 2011).

The following incidents have reportedly involved the use and/or possession of ricin.

2011).

work of the KGB.

 On September 7, 1978 while waiting at a bus stop in London, a Bulgarian dissident, Georgi Markov, felt a jab in the back of his right thigh and saw a man picking up an umbrella (Crompton & Gall, 1980). Markov, a 49-year-old novelist and playwriter had published and broadcasted anticommunist views. An assassin reportedly injected a small pellet of ricin (believed to have been supplied by the KGB), into Markov's right thigh using a weapon in the shape of an umbrella (Maman & Yehezkelli, 2005). He subsequently developed severe gastroenteritis, high fever, and died 3 days later (discussed in detail later in this chapter). At autopsy, a small 1.53-mm metallic sphere that had 2 tiny holes and could hold a volume of 0.28 mm3, was found at the wound site. No specific isolation of any poison was possible. Because of the small volume and rapid demise of the patient, ricin was believed to be the only capable inciting agent. The coroner recreated the scenario by injecting a pig with a somewhat greater dose than Markov had received (Crompton & Gall, 1980). With an illness similar to Markov's, the animal died 26 hours later. Thereafter the coroner was satisfied that Markov had been unlawfully killed by a tiny pellet containing 0.2 to 0.5 mg dose of ricin (Musshoff & Madea, 2009). The KGB denied any involvement although high-profile defectors Oleg Kalugin and Oleg Gordievsky have since confirmed the KGB's involvement (Pearce,

 In 1981, exposed CIA double agent Boris Korczak was reportedly shot with a ricin-laced pellet (Carus, 2002). He survived this assassination attempt which was thought to be the

 In 1982, William A. Chanslor, a Texas attorney was sentenced to jail for 3 years and fined \$5,000 for plotting to kill his 39-year-old wife with ricin. He claimed that he wanted the ricin to assist his wife, paralyzed from the waist down due to a stroke, in

In 1983, two brothers were arrested by the Federal Bureau of Investigation (FBI) for

 In 1983 and 1985, Montgomery Todd Meeks, a high school senior was tried and convicted of attempted murder and solicitation to murder in connection with a plot to kill his father using ricin (Trager, 1985, as cited in Carus (2002). He claimed that the act

 In 1991, four members of the Minnesota Patriots Council, a radical tax-protesting militia organization, acquired castor beans and planned to use ricin to assassinate local deputy

committing suicide (Time Magazine, 16 August 1982).

producing an ounce of pure ricin (RII, 2011).

was motivated by his father's abuse (RII, 2011).

LeClaire, 2007). WWI ended before the toxin could be weaponized and tested. During WWII, ricin was produced in hundreds of kilograms and armed into W bombs (ricincontaining bombs), but apparently was never used in battle (Franz & Jaax, 1997). Although its toxicity made it marginally better over existing agents, ricin was surpassed by the even more potent biological agents of the time. Interest in ricin continued for a short period after WWII, but soon subsided when the U.S. Army Chemical Corps began a program to weaponize sarin. During the Cold War, the Soviet Union also studied ricin as a possible biological weapons agent. Ken Alibek, a former top official involved in Russia's biological weapons program who defected to the U.S. in 1991, claimed that Russia developed ricin toxin as a weapon, and that the ricin toxin used against the Bulgarian dissident Georgi Markov, as well Vladimir Kostov (another Bulgarian exile in Paris), was concocted in Russian laboratories (Maman & Yehezkelli, 2005). During 1989, approximately 10 L of concentrated ricin solution was reportedly manufactured at Salman Park just south of Baghdad, some of which were used in animal testing and as payload in artillery shells (Zilinskas, 1997). More recently, ricin has been used by terrorist organizations (CDC, 2008). In 2002, a report emerged that Ansar al-Islam, a Sunni Islamic group allegedly linked to Osama Bin Laden's al-Qaeda organization, had been testing biological weapons including ricin at a small facility in northern Iraq (BBC News, 20 August 2002). A news item documented evidence of the manufacture of ricin and botulinum toxin in Iraq (Mendenhall, 2003). Syria was also believed to have produced unknown quantities of the toxin. Iran allegedly procured 120 tons of castor beans in 1992, presumably for ricin production (Croddy & Wirtz, 2005). Ricin was also found in Afghanistan after the collapse of the Taliban Government in 2001 (GlobalSecurity.org, n.d.; Barceloux, 2008).

Ricin is currently monitored as a Schedule 1 toxic chemical under the Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on Their Destruction (CWC). Also, the intentional use of ricin or related toxins as weapons is prohibited under the 1972 Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction (BTWC) (Millard & Le Claire, 2007; Poli, 2007). The possession or transfer of ricin, abrin, or genes encoding functional forms of these toxins is also regulated in the U.S. by the Centers for Disease Control and Prevention (CDC) Select Agents and Toxins Program.

Although ricin's potential use as a military weapon was investigated, its utility over conventional weaponry remains disputed. Despite its toxicity, ricin is less potent than other agents such as botulinum neurotoxin or anthrax. Kortepeter & Parker (1999) estimated that eight metric tons of ricin would have to be aerosolized over a 100 km2 area to achieve about 50% casualty, whereas only kilogram quantities of anthrax spores would cause the same effect. Furthermore, dispersal of ricin on a wide scale is logistically impractical. Thus, while ricin is easy to produce, it is not as likely to cause as many casualties as other agents (Schep et al., 2009). However, it has been the agent of choice in numerous biocrimes (see below).

#### **2.1.2 Ricin as a terrorist weapon**

Ricin has been classified by the CDC as a Category B agent. Category B agents are moderately easy to disseminate, can cause morbidity and low mortality, and include *Coxiella burnetii*, *Brucella* spp, *Burkholderia mallei*, *B. pseudomallei*, alphaviruses (VEE, EEE, WEE),

LeClaire, 2007). WWI ended before the toxin could be weaponized and tested. During WWII, ricin was produced in hundreds of kilograms and armed into W bombs (ricincontaining bombs), but apparently was never used in battle (Franz & Jaax, 1997). Although its toxicity made it marginally better over existing agents, ricin was surpassed by the even more potent biological agents of the time. Interest in ricin continued for a short period after WWII, but soon subsided when the U.S. Army Chemical Corps began a program to weaponize sarin. During the Cold War, the Soviet Union also studied ricin as a possible biological weapons agent. Ken Alibek, a former top official involved in Russia's biological weapons program who defected to the U.S. in 1991, claimed that Russia developed ricin toxin as a weapon, and that the ricin toxin used against the Bulgarian dissident Georgi Markov, as well Vladimir Kostov (another Bulgarian exile in Paris), was concocted in Russian laboratories (Maman & Yehezkelli, 2005). During 1989, approximately 10 L of concentrated ricin solution was reportedly manufactured at Salman Park just south of Baghdad, some of which were used in animal testing and as payload in artillery shells (Zilinskas, 1997). More recently, ricin has been used by terrorist organizations (CDC, 2008). In 2002, a report emerged that Ansar al-Islam, a Sunni Islamic group allegedly linked to Osama Bin Laden's al-Qaeda organization, had been testing biological weapons including ricin at a small facility in northern Iraq (BBC News, 20 August 2002). A news item documented evidence of the manufacture of ricin and botulinum toxin in Iraq (Mendenhall, 2003). Syria was also believed to have produced unknown quantities of the toxin. Iran allegedly procured 120 tons of castor beans in 1992, presumably for ricin production (Croddy & Wirtz, 2005). Ricin was also found in Afghanistan after the collapse of the

Taliban Government in 2001 (GlobalSecurity.org, n.d.; Barceloux, 2008).

for Disease Control and Prevention (CDC) Select Agents and Toxins Program.

numerous biocrimes (see below).

**2.1.2 Ricin as a terrorist weapon** 

Ricin is currently monitored as a Schedule 1 toxic chemical under the Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on Their Destruction (CWC). Also, the intentional use of ricin or related toxins as weapons is prohibited under the 1972 Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction (BTWC) (Millard & Le Claire, 2007; Poli, 2007). The possession or transfer of ricin, abrin, or genes encoding functional forms of these toxins is also regulated in the U.S. by the Centers

Although ricin's potential use as a military weapon was investigated, its utility over conventional weaponry remains disputed. Despite its toxicity, ricin is less potent than other agents such as botulinum neurotoxin or anthrax. Kortepeter & Parker (1999) estimated that eight metric tons of ricin would have to be aerosolized over a 100 km2 area to achieve about 50% casualty, whereas only kilogram quantities of anthrax spores would cause the same effect. Furthermore, dispersal of ricin on a wide scale is logistically impractical. Thus, while ricin is easy to produce, it is not as likely to cause as many casualties as other agents (Schep et al., 2009). However, it has been the agent of choice in

Ricin has been classified by the CDC as a Category B agent. Category B agents are moderately easy to disseminate, can cause morbidity and low mortality, and include *Coxiella burnetii*, *Brucella* spp, *Burkholderia mallei*, *B. pseudomallei*, alphaviruses (VEE, EEE, WEE), *Rickettsia prowazekii*, toxins (e.g., ricin, Staphylococcal enterotoxin B, epsilon toxin of *Clostridium perfringens*), *Chlamydia psittaci*, food safety threats (e.g., *Salmonella* spp., *Escherichia coli* O157:H7, *Shigella*), and water safety threats (e.g. *Vibrio cholerae*, *Cryptosporidium parvum*) (Rotz et al., 2002). Though ricin is not considered an effective weapon of mass destruction, its potential as a weapon of terror cannot be discounted. Further, ricin's notoriety is likely driven by the ready availability of castor beans, press coverage, and popularization on the internet. In the U.S. for example, the use of ricin as a biological agent in bioterrorism and homicides is of particular concern especially after the events of September 11, 2001. Worldwide, numerous cases involving the possession, experimentation, or planned misuse of ricin by bioterrorists and extremist groups have been investigated or prosecuted by law enforcement agencies (Franz and Jaax, 1997; James Martin Center for Nonproliferation Studies (CNS), 2004; Research International, Inc. (RII), 2011). The following incidents have reportedly involved the use and/or possession of ricin.


 In December 2002, six terrorist suspects were arrested in Manchester, England. Their apartment was serving as a "ricin laboratory." Among them was a 27-year-old chemist

 In January 2003, authorities discovered traces of ricin in the apartment of six Algerians in Wood Green, northern London (Hopkins & Branigan, 08 January 2003). They also discovered castor beans and equipment for crushing the beans. Those arrested are believed to be part of a terrorist cell known as the "Chechen network" which may have ties to the Algerian group behind the millennium bomb plots in the U.S. All but one of the suspects was acquitted of charges in April, 2005 (Research

 In October 2003, an envelope with a threatening note and a sealed container that had ricin in it was discovered at a mail processing and distribution facility in Greenville, South Carolina. The note threatened to poison water supplies if demands were not met

 In February 2004, traces of ricin were discovered on an automatic mail sorter in the mailroom of the Dirksen Senate Office building in Washington, D.C which handled mail addressed to Senate Majority Leader Bill Frist (CNN.com, 04 February 2004). In January 2005, the FBI arrested an Ocala, Florida man after agents found ricin and other products in the home he lives in with his mother (CNN.com, 14 January 2005). On October 3, 2006, a survivalist from Phoenix, Arizona was sentenced to 7 years in

 In 2007, traces of ricin had been found at Limerick Prison (Lally, 2007). The ricin was smuggled into Ireland from the U.S. in a contact lens case, to be used in an assassination

 In November 2008, Roger Von Bergendorff was fined and sentenced to 3.5 years in prison for having ricin and unregistered firearm silencers. In February 2008, Bergendorff was living in a motel room in Las Vegas, Nevada when he was taken to a hospital in critical condition. Authorities recovered castor beans, a weapons cache, a copy of "The Anarchist Cookbook" with a page about ricin marked, and 4 crude grams of ricin in his room. Investigators also found respirators, gloves and chemicals that could be used in the production of ricin in one of Bergendorff's storage units in Utah

 In June 2009, a father and son, Ian and Nicky Davison were arrested after the discovery of ricin at a house in County Durham (BBC News, 06 June 2009). Ian Davidson was sentenced to 10 years in May 2010 for preparing acts of terrorism, three counts of possessing material useful to commit acts of terrorism and possessing a prohibited weapon; his son was given 2 years youth detention for possessing material useful to commit acts of terrorism (Wainwright, 2010).In January 2011, The FBI has arrested the owner of a Coventry Township, Ohio home for unlawful possession of

 In June 2011, Michael Crooker, a former Agawam man, was sentenced to 15 years in federal prison for illegally possessing ricin and threatening a prosecutor (Associated Press, AP, 22 June 2011). In another report during the same period, a British citizen, Asim Kauser, was brought to court on charges including possessing instructions for

producing ricin (Global Security Network, GSN, 17 June 2011).

prison for attempting to manufacture ricin (Martens, 2006).

plot. An arrest was made before the ricin could be used (RII, 2011).

who was producing the toxin (CDC, 2003a).

International, 2011).

(Powers, 18 November 2008).

ricin (Sharma, 29 January 2011).

(CDC, 2003b).

sheriffs, U.S. Marshals, and IRS agents. Despite having no specific expertise in biological warfare, they extracted about 0.7 g of 5% ricin, which was enough to kill about 100 people. Two members were convicted in 1994, and the other two in 1995 under the Biological Weapons Anti-Terrorism Act (BWATA) law (RII, 2011).


 On April 21, 1992, the Washington Post published an article regarding the unsuccessful attempt to poison the famous Soviet dissident Alexander Solzhenitsyn with the same lethal chemical (thought to be ricin) used to kill Bulgarian dissident Georgi Markov in

 In December 1995, Thomas Lewis Lavy, an electrician from Valdez, Alaska was arrested in Onia, Arkansas for possession of ricin (Kifner, 2005). In April 1993, he was caught while trying to smuggle 130 g of ricin and other materials from Alaska into Canada, and was then charged under BWATA with possession of a biological toxin with intent to

kill. Lavy killed himself in his prison cell several days after his arrest (RII, 2011). In 1995, a federal case was brought against Dr. Ray W. Mettetal, Jr., a neurologist at Rockingham Memorial Hospital in Harrisonburg, Virginia, after ricin was discovered in

 In 1995, Deborah Green, a non-practicing oncologist from Prairie Village, Kansas, attempted to murder her husband, Michael Farrar, a cardiologist, with ricin (Musick, 25 May 2000; Carus 2002). Green had purchased the castor beans through a special order from a garden center in Kansas City, Missouri, and placed them in Farrar's food. It is unclear if she extracted the ricin or merely added the beans to the food. Later, Farrar had to undergo multiple heart and brain surgeries related to the

 In 1997, a man was indicted under the provisions of BWATA for possessing ricin and nicotine sulfate. He pled guilty to manufacturing ricin and was sentenced to more than

 On August 25, 1998, Dwayne Lee Kuehl was arrested in Escanaba, Michigan, for producing ricin with intent to use it against an Escanaba city housing inspector (RII

 In November 1999, FBI agents apprehended James Kenneth Gluck in Tampa, Florida, for threatening to kill court officials in Jefferson County, Colorado with ricin (The New

 In August 2001, the FSB (Russian Federal Security Service) told the Itar-Tass news service it had intercepted a recorded conversation between two Chechen field commanders (Brigadier General Rizvan Chitigov and field commander Hizir Alhazurov) about instructions on the homemade production of poisons against Russian troops (RII, 2011). Russian authorities reportedly raided Chitigov's home and seized materials, including instructions on how to produce ricin from castor beans, a small chemical laboratory, three homemade explosives, two land mines, and 30 grenades

 In June 2002, Ken Olson, an Agilent software engineer, was convicted of ricin possession (Tizon, 2004). He was given a 13-year, 9-month sentence in April 2004. In August 2002, the Sunni miltant group Ansar al-Islam was reported to be involved in testing biological agents including ricin on barnyard animals and perhaps even an

unwitting human subject (BBC News, 20 August 2002).

his possession and also of providing false information (Carus, 2002).

under the Biological Weapons Anti-Terrorism Act (BWATA) law (RII, 2011).

London in 1978 (Remnick, 21 April 1992).

poisoning (CNS, 2004).

2011).

(Gad, 2007).

12 years in prison (Cordesman, 2002).

York Times, 08 November 1999).

sheriffs, U.S. Marshals, and IRS agents. Despite having no specific expertise in biological warfare, they extracted about 0.7 g of 5% ricin, which was enough to kill about 100 people. Two members were convicted in 1994, and the other two in 1995


Fig. 3. Three-dimensional representation of ricin. The A chain (RTA) is depicted in red, the B chain (RTB) in blue, and the disulfide bond in yellow. (Image courtesy of Dr. Mark A. Olson,

The ricin protein's coding region consists of a 24 amino acid N-terminal signal sequence preceding a 266 amino acid RTA. The RTB has 262 amino acids. A 12-amino acid linker joins the two chains. The nontranslated mRNA regions of ricin and RCA are identical (Roberts, et al., 1985; Lamb et al., 1985). The signal peptide preceding the RTA in both lectins and the linker peptide joining the RTA and RTB are also alike in size and amino acid sequence. Overall, the RCA and RTA chains are 93% homologous (18 amino acid variants) while the corresponding B

The carboxyl-terminal end of the RTA folds into a domain that interacts between the two domains of the B chain (Montfort et al., 1987). A disulfide bond is formed at amino acid 259 of the RTA and amino acid 4 of the RTB (Robertus, 1988; 1991; Lord et al., 1994). Thirty percent of the RTA protein is helical (Fig. 4). The RTA folds into three somewhat arbitrary domains. The active site cleft of the RTA is located at the interface between all three domains. A conformational change occurs in the active site when the RTA is released from the RTB.

Fig. 4. Ribbon representation of RTA. The RTA has three structural domains and exhibits a substantial amount of secondary structure. Color schemes: cyan = helix; magenta = strands; red = coil regions. (Image courtesy of Dr. Mark A. Olson, Integrated Toxicology Division,

Integrated Toxicology Division, USAMRIID, Fort Detrick, MD).

chains differ in 41 amino acids, and are 84% homologous (Lamb et al., 1985).

**3.1.2.1 Primary structure of ricin/RCA** 

**3.1.2.2 Ricin secondary structure** 

USAMRIID, Fort Detrick, MD).

The above events clearly demonstrate that ricin is readily available or accessible, relatively easy to produce, and seemingly, a biological weapon of choice by extremist groups and individuals. Hence, it should be seriously considered as a potential bioterrorism threat agent.

#### **3. Description of the agent**

#### **3.1 Overview of ribosome-inactivating proteins**

Ricin belongs to a diverse family of ribosome-inactivating proteins (RIPs) that include numerous other toxins from a wide variety of plants, as well as potent bacterial Shiga and Shiga-like toxins (Endo et al., 1988). RIPs possess N-glycosidase activity that depurinates a highly conserved adenine residue within the specific 14 nucleotide region (also known as αsarcin/ricin loop) of the 28S ribosomal RNA (rRNA) subunit of 60S ribosome (Endo et al., 1987; Endo & Tsurugi, 1987). There are three types of RIPs. Type 1 RIPs are monomeric Nglycosidase enzymes of approximately 30 kDA molecular mass, and are frequently found in higher plants, e.g., pokeweed antiviral protein, trichosanthin, saporin, and luffin (Nielsen & Boston, 2001). In general, type I RIPs are not cytotoxic because they lack the means of entering the cell (B chain) to inactivate ribosomes (Lord et al., 1994).

Type 2 RIPs are glycosylated heterodimers possessing an N-glycosidase enzyme (denoted A chain) linked through a disulfide bond to a galactose-binding lectin (denoted B-chain) that facilitates endocytosis (Lord et al., 1994; Stirpe & Battelli, 2006). Type 2 RIPs include potent toxins such as ricin, abrin (isolated from the seeds of rosary pea, *Abrus precatorius*), modeccin (from the fruits and roots of *Adenia digitata*), volkensin (from the roots of *A. volkensii*), and viscumins (from mistletoe, *Viscum album*; Lord et al., 1994). Some type 2 RIPs possess an aberrant or non-functional B-chain, hence are relatively nontoxic. These include nigrin b from the elderberry plant (*Sambucus nigra*), lectins from winter aconite (*Eranthis hyemalis*), and ebulin lectins from dwarf elder (*S. ebulus*).

Type 3 RIPs are the least common class and resemble type I plant RIPs in catalytic activity and overall net charge (Nielsen & Boston, 2001). They are synthesized as inactive precursor molecules with a polypeptide insert in the active site region of the A chain domain (Chaudhry et al., 1994).

#### **3.1.2 Biochemistry of ricin**

Ricin is the most well-characterized member of the type 2 RIPs. It consists of two glycoprotein subunits, designated A chain (RTA) and B chain (RTB), of approximately equal molecular mass (~32 kDa) linked by a single disulfide bond (Fig. 3). The ricin toxin is stored in the matrix of the castor seed, together with a 120 kDa lectin called *R. communis* agglutinin I (RCA). RCA is composed of two ricin-like dimers. Although the nucleotide sequences of ricin and RCA are similar, these proteins are products of distinct genes (Kole, 2011); it has been suggested that the ricin gene evolved first and then duplicated to give rise to the RCA gene (Ready et al., 1984). Compared with ricin, RCA is virtually nontoxic (Olsnes et al., 1974) but is a powerful red blood cell agglutinin (Hegde & Podder, 1992).

The above events clearly demonstrate that ricin is readily available or accessible, relatively easy to produce, and seemingly, a biological weapon of choice by extremist groups and individuals. Hence, it should be seriously considered as a potential bioterrorism threat

Ricin belongs to a diverse family of ribosome-inactivating proteins (RIPs) that include numerous other toxins from a wide variety of plants, as well as potent bacterial Shiga and Shiga-like toxins (Endo et al., 1988). RIPs possess N-glycosidase activity that depurinates a highly conserved adenine residue within the specific 14 nucleotide region (also known as αsarcin/ricin loop) of the 28S ribosomal RNA (rRNA) subunit of 60S ribosome (Endo et al., 1987; Endo & Tsurugi, 1987). There are three types of RIPs. Type 1 RIPs are monomeric Nglycosidase enzymes of approximately 30 kDA molecular mass, and are frequently found in higher plants, e.g., pokeweed antiviral protein, trichosanthin, saporin, and luffin (Nielsen & Boston, 2001). In general, type I RIPs are not cytotoxic because they lack the means of

Type 2 RIPs are glycosylated heterodimers possessing an N-glycosidase enzyme (denoted A chain) linked through a disulfide bond to a galactose-binding lectin (denoted B-chain) that facilitates endocytosis (Lord et al., 1994; Stirpe & Battelli, 2006). Type 2 RIPs include potent toxins such as ricin, abrin (isolated from the seeds of rosary pea, *Abrus precatorius*), modeccin (from the fruits and roots of *Adenia digitata*), volkensin (from the roots of *A. volkensii*), and viscumins (from mistletoe, *Viscum album*; Lord et al., 1994). Some type 2 RIPs possess an aberrant or non-functional B-chain, hence are relatively nontoxic. These include nigrin b from the elderberry plant (*Sambucus nigra*), lectins from winter aconite (*Eranthis hyemalis*),

Type 3 RIPs are the least common class and resemble type I plant RIPs in catalytic activity and overall net charge (Nielsen & Boston, 2001). They are synthesized as inactive precursor molecules with a polypeptide insert in the active site region of the A chain domain

Ricin is the most well-characterized member of the type 2 RIPs. It consists of two glycoprotein subunits, designated A chain (RTA) and B chain (RTB), of approximately equal molecular mass (~32 kDa) linked by a single disulfide bond (Fig. 3). The ricin toxin is stored in the matrix of the castor seed, together with a 120 kDa lectin called *R. communis* agglutinin I (RCA). RCA is composed of two ricin-like dimers. Although the nucleotide sequences of ricin and RCA are similar, these proteins are products of distinct genes (Kole, 2011); it has been suggested that the ricin gene evolved first and then duplicated to give rise to the RCA gene (Ready et al., 1984). Compared with ricin, RCA is virtually nontoxic (Olsnes et al., 1974) but is a powerful red blood cell agglutinin (Hegde & Podder,

agent.

**3. Description of the agent** 

**3.1 Overview of ribosome-inactivating proteins** 

and ebulin lectins from dwarf elder (*S. ebulus*).

(Chaudhry et al., 1994).

1992).

**3.1.2 Biochemistry of ricin** 

entering the cell (B chain) to inactivate ribosomes (Lord et al., 1994).

Fig. 3. Three-dimensional representation of ricin. The A chain (RTA) is depicted in red, the B chain (RTB) in blue, and the disulfide bond in yellow. (Image courtesy of Dr. Mark A. Olson, Integrated Toxicology Division, USAMRIID, Fort Detrick, MD).

#### **3.1.2.1 Primary structure of ricin/RCA**

The ricin protein's coding region consists of a 24 amino acid N-terminal signal sequence preceding a 266 amino acid RTA. The RTB has 262 amino acids. A 12-amino acid linker joins the two chains. The nontranslated mRNA regions of ricin and RCA are identical (Roberts, et al., 1985; Lamb et al., 1985). The signal peptide preceding the RTA in both lectins and the linker peptide joining the RTA and RTB are also alike in size and amino acid sequence. Overall, the RCA and RTA chains are 93% homologous (18 amino acid variants) while the corresponding B chains differ in 41 amino acids, and are 84% homologous (Lamb et al., 1985).

#### **3.1.2.2 Ricin secondary structure**

The carboxyl-terminal end of the RTA folds into a domain that interacts between the two domains of the B chain (Montfort et al., 1987). A disulfide bond is formed at amino acid 259 of the RTA and amino acid 4 of the RTB (Robertus, 1988; 1991; Lord et al., 1994). Thirty percent of the RTA protein is helical (Fig. 4). The RTA folds into three somewhat arbitrary domains. The active site cleft of the RTA is located at the interface between all three domains. A conformational change occurs in the active site when the RTA is released from the RTB.

Fig. 4. Ribbon representation of RTA. The RTA has three structural domains and exhibits a substantial amount of secondary structure. Color schemes: cyan = helix; magenta = strands; red = coil regions. (Image courtesy of Dr. Mark A. Olson, Integrated Toxicology Division, USAMRIID, Fort Detrick, MD).

among cell types. Skin testing in mice showed no dermal toxicity, indicating poor

Ingestion and mastication of 3-6 castor beans is the estimated fatal dose in adults; the fatal dose in children is not known, but is likely less (CDC, 2006a). Most cases of castor bean ingestion do not result in poisoning, because: a) it is difficult for ricin to be released from ingested castor beans; b) ricin release requires mastication, and the degree of mastication is likely to be important in determining the extent of poisoning; and c) ricin is not as well absorbed through the gastrointestinal tract when compared to injection or inhalation (CDC,

The lethality of ricin by different routes in several animal species was summarized by Millard & LeClaire (2007). By ingestion, the hens were the least sensitive to the toxin (LD50 = 14 g/kg), and the horses were the most sensitive (LD50 = 0.1 g/kg). By injection, rabbits had an LD50 = 0.1 μg/kg (i.m.), and 0.5 μg/kg (i.v.), while guinea pigs had <1.1 μg/kg (i.v.) and

Limited information is available regarding human toxicity. Based on animal experiments and accidental human exposures, the approximate LD50 and time to death for humans are, respectively, 3 μg/kg and 36-72 hours by inhalation, 30 μg/kg and 6-8 days by ingestion, 3 μg/kg and 36-72 hours by intravenous injection, and 500 μg and 3-6 days by subcutaneous injection (based on Georgi Markov's assassination) (Franz & Jaax, 1997; Maman &

The vulnerability of certain populations (e.g., children, pregnant women, the elderly, those with immunosuppression, or underlying respiratory or gastrointestinal tract disease) to the health effects of ricin exposure is unknown; however, persons with pre-existing tissue

Ricin may adhere to skin, nonetheless, person-to-person transmission through casual contact has not been reported. Ricin is transmitted by the airborne route through release of the toxin in the form of a powder, or a mist, or reaerosolization of ricin into the air from disturbed surfaces (CDC, 2006a). However, to be effective, the toxin would need to be

The clinical signs, symptoms, and pathological manifestations of ricin toxicity vary with the dose and route of exposure, as detailed below. For symptomatic patients, the clinical course presents with the rapid onset of nausea, vomiting, and abdominal pain. Gastrointestinal

irritation or damage may sustain further injury upon ricin exposure (CDC, 2006a).

dispersed in particles smaller than 5 microns (CDC, 2006a).

**4. Clinical symptoms, signs and pathology** 

absorption across the skin (Wannemacher & Anderson, 2005).

**3.3.2 Amount of toxin administered** 

0.8 μg/kg (i.m.) (Millard & LeClaire, 2007).

2006a).

**3.3.3 Animal species** 

**3.4 Human toxicity** 

**3.5 Transmission** 

Yehezkelli, 2005; Mirarchi, 2010).

#### **3.2 Pathogenesis**

Ricin is a toxalbumin, a biological toxin whose mechanism of action is inhibition of protein synthesis in eukaryotic cells which results in cell death (CDC, 2006a). The dimeric A-B chain structure is crucial to cellular internalization and subsequent toxicity. Cell entry by ricin involves a series of steps, summarized as follows: 1) the RTB portion of the ricin molecule binds to cell surface glycolipids or glycoproteins possessing 1,4-linked galactose residues; 2) once bound to the cell surface, the toxin is internalized by endocytosis and routed to the cytosol. The presence of the B-chain facilitates transport of the A-chain into the cytosol; 3) toxin entry into early endosomes; 4) ricin vesicular transport from early endosomes to the trans-Golgi network; 5) retrograde vesicular transport through the Golgi network to reach the endoplasmic reticulum; 6) reduction of the disulfide bond connecting RTA and RTB; 7) partial unfolding of the RTA to render it translocationally competent to cross the endoplasmic reticulum (Endo et al., 1987; Olsnes & Koslov, 2001; Lord et al., 1994; 2003). Transport to the cytosol is the rate-limiting step during the decline in protein synthesis (Hudson and Neville, 1987).

Once transported from the ER to the cytoplasm, the RTA can interact with the ribosome, which acts as a suicidal chaperone stimulating proper refolding and resumption of catalytic activity (Lord et al., 2003). Ricin has a Michaelis constant (*K*M) of 0.1 μmol/L for ribosomes and an enzymatic constant (*K*cat) of 1,500/min. It depurinates a specific adenosine residue (A4324) near the 3' end of 28S ribosomal RNA subunit in the 60S ribosome subunit (Robertus, 1991). This halts the binding of elongation factor-2, which then results in the inhibition of protein synthesis in eukaryotic cells (Endo et al., 1987). Catalytic studies showed that single ricin molecule in the cytosol can inactivate over 1500 ribosomes per minute and eventually kills the cell (Olsnes et al.,1975; Cawley and Houston, 1979).

Ricin is more active against animal than plant or bacterial ribosomes (Cawley and Houston, 1979). Ribosomes which lack the specific 28S subunit containing the GAGA tetranucleotide sequence are generally not susceptible to the toxin.

#### **3.3 Toxicity**

Ricin's toxicity is dependent on a number of factors including route of exposure (inhalation, parenteral (injection), ingestion, dermal contact, or ocular contact), amount of toxin administered, and animal species.

#### **3.3.1 Route of administration**

Ricin is extremely toxic by inhalation, and least potent by the oral route. In mice, the approximate dose that is lethal to 50% of the exposed population (LD50) and time to death are, respectively, 3-5 μg/kg (Franz and Jaax, 1997) and 60 hours by inhalation (Kortepeter et al., 2001a), 20 mg/kg and 85 hours by ingestion (Franz and Jaax, 1997), 5 μg/kg and 90 hours by intravenous injection (Franz and Jaax, 1997), and 24 μg/kg and 100 hours by subcutaneous injection (Franz and Jaax, 1997). Low oral toxicity is possibly due to poor toxin absorption and partial degradation in the gut. Higher toxicities by other routes may be related to accessibility of target-cell populations and the availability of toxin receptors

Ricin is a toxalbumin, a biological toxin whose mechanism of action is inhibition of protein synthesis in eukaryotic cells which results in cell death (CDC, 2006a). The dimeric A-B chain structure is crucial to cellular internalization and subsequent toxicity. Cell entry by ricin involves a series of steps, summarized as follows: 1) the RTB portion of the ricin molecule binds to cell surface glycolipids or glycoproteins possessing 1,4-linked galactose residues; 2) once bound to the cell surface, the toxin is internalized by endocytosis and routed to the cytosol. The presence of the B-chain facilitates transport of the A-chain into the cytosol; 3) toxin entry into early endosomes; 4) ricin vesicular transport from early endosomes to the trans-Golgi network; 5) retrograde vesicular transport through the Golgi network to reach the endoplasmic reticulum; 6) reduction of the disulfide bond connecting RTA and RTB; 7) partial unfolding of the RTA to render it translocationally competent to cross the endoplasmic reticulum (Endo et al., 1987; Olsnes & Koslov, 2001; Lord et al., 1994; 2003). Transport to the cytosol is the rate-limiting step during the decline in protein synthesis

Once transported from the ER to the cytoplasm, the RTA can interact with the ribosome, which acts as a suicidal chaperone stimulating proper refolding and resumption of catalytic activity (Lord et al., 2003). Ricin has a Michaelis constant (*K*M) of 0.1 μmol/L for ribosomes and an enzymatic constant (*K*cat) of 1,500/min. It depurinates a specific adenosine residue (A4324) near the 3' end of 28S ribosomal RNA subunit in the 60S ribosome subunit (Robertus, 1991). This halts the binding of elongation factor-2, which then results in the inhibition of protein synthesis in eukaryotic cells (Endo et al., 1987). Catalytic studies showed that single ricin molecule in the cytosol can inactivate over 1500 ribosomes per

Ricin is more active against animal than plant or bacterial ribosomes (Cawley and Houston, 1979). Ribosomes which lack the specific 28S subunit containing the GAGA tetranucleotide

Ricin's toxicity is dependent on a number of factors including route of exposure (inhalation, parenteral (injection), ingestion, dermal contact, or ocular contact), amount of toxin

Ricin is extremely toxic by inhalation, and least potent by the oral route. In mice, the approximate dose that is lethal to 50% of the exposed population (LD50) and time to death are, respectively, 3-5 μg/kg (Franz and Jaax, 1997) and 60 hours by inhalation (Kortepeter et al., 2001a), 20 mg/kg and 85 hours by ingestion (Franz and Jaax, 1997), 5 μg/kg and 90 hours by intravenous injection (Franz and Jaax, 1997), and 24 μg/kg and 100 hours by subcutaneous injection (Franz and Jaax, 1997). Low oral toxicity is possibly due to poor toxin absorption and partial degradation in the gut. Higher toxicities by other routes may be related to accessibility of target-cell populations and the availability of toxin receptors

minute and eventually kills the cell (Olsnes et al.,1975; Cawley and Houston, 1979).

sequence are generally not susceptible to the toxin.

administered, and animal species.

**3.3.1 Route of administration** 

**3.2 Pathogenesis** 

(Hudson and Neville, 1987).

**3.3 Toxicity** 

among cell types. Skin testing in mice showed no dermal toxicity, indicating poor absorption across the skin (Wannemacher & Anderson, 2005).

#### **3.3.2 Amount of toxin administered**

Ingestion and mastication of 3-6 castor beans is the estimated fatal dose in adults; the fatal dose in children is not known, but is likely less (CDC, 2006a). Most cases of castor bean ingestion do not result in poisoning, because: a) it is difficult for ricin to be released from ingested castor beans; b) ricin release requires mastication, and the degree of mastication is likely to be important in determining the extent of poisoning; and c) ricin is not as well absorbed through the gastrointestinal tract when compared to injection or inhalation (CDC, 2006a).

#### **3.3.3 Animal species**

The lethality of ricin by different routes in several animal species was summarized by Millard & LeClaire (2007). By ingestion, the hens were the least sensitive to the toxin (LD50 = 14 g/kg), and the horses were the most sensitive (LD50 = 0.1 g/kg). By injection, rabbits had an LD50 = 0.1 μg/kg (i.m.), and 0.5 μg/kg (i.v.), while guinea pigs had <1.1 μg/kg (i.v.) and 0.8 μg/kg (i.m.) (Millard & LeClaire, 2007).

#### **3.4 Human toxicity**

Limited information is available regarding human toxicity. Based on animal experiments and accidental human exposures, the approximate LD50 and time to death for humans are, respectively, 3 μg/kg and 36-72 hours by inhalation, 30 μg/kg and 6-8 days by ingestion, 3 μg/kg and 36-72 hours by intravenous injection, and 500 μg and 3-6 days by subcutaneous injection (based on Georgi Markov's assassination) (Franz & Jaax, 1997; Maman & Yehezkelli, 2005; Mirarchi, 2010).

The vulnerability of certain populations (e.g., children, pregnant women, the elderly, those with immunosuppression, or underlying respiratory or gastrointestinal tract disease) to the health effects of ricin exposure is unknown; however, persons with pre-existing tissue irritation or damage may sustain further injury upon ricin exposure (CDC, 2006a).

#### **3.5 Transmission**

Ricin may adhere to skin, nonetheless, person-to-person transmission through casual contact has not been reported. Ricin is transmitted by the airborne route through release of the toxin in the form of a powder, or a mist, or reaerosolization of ricin into the air from disturbed surfaces (CDC, 2006a). However, to be effective, the toxin would need to be dispersed in particles smaller than 5 microns (CDC, 2006a).

#### **4. Clinical symptoms, signs and pathology**

The clinical signs, symptoms, and pathological manifestations of ricin toxicity vary with the dose and route of exposure, as detailed below. For symptomatic patients, the clinical course presents with the rapid onset of nausea, vomiting, and abdominal pain. Gastrointestinal

deglycosylated RTA (RFT5.dgA and Ki-4.dgA) administered i.v. to Hodgkins' lymphoma patients revealed maximum tolerated doses (MTDs) of 15 and 5 mg/m2 of estimated body

Ricin is less toxic by oral ingestion than by other routes (Rauber & Heard, 1985), probably due to poor absorption of the toxin and possibly partial enzymatic degradation in the digestive tract. The effects of oral intoxication vary among individuals, are dose dependent, and have different signs and symptoms. Rauber & Heard (1985) reviewed 751 cases of castor bean ingestion and reported 14 fatalities (1.9% death rate). The number of beans ingested by patients who died greatly varied. For instance, of the two lethal cases of oral intoxication documented since 1930, one involved a 24-year-old man who ate 15 to 20 beans, and the other was a 15-year-old boy who had 10 to 12 beans. All of the described serious, or fatal cases of castor bean ingestion have the same general clinical history: rapid (less than a few hours) onset of nausea, vomiting, and abdominal pain followed by diarrhea, hemorrhage from the anus, anuria, cramps, dilation of the pupils, fever, thirst, sore throat, headache, vascular collapse, and shock. Death occurred on the third day or later. The most common autopsy findings in oral intoxication were multifocal ulcerations and hemorrhages of gastric and small-intestinal mucosa, which may be quite severe; lymphoid necrosis in the mesenteric lymph nodes, gut-associated lymphoid tissue (GALT), and spleen; Kupffer cell and liver necrosis; diffuse nephritis; and diffuse splenitis (Rauber & Heard, 1985; Bradberry

There are no documented cases of aerosol exposure to ricin in humans. Lesions induced by oral and parenteral exposure are consistent with those from animal studies, suggesting that the same would hold true for aerosol exposures. An allergic syndrome has been reported in workers exposed to castor bean dust in or around castor oil-processing plants (Brugsch, 1960). The clinical picture is characterized by the sudden onset of congestion of the nose and throat, itchiness of the eyes, urticaria, and tightness of the chest. In more severe cases, wheezing can last for several hours, and may lead to bronchial asthma. Affected individuals

Ricin poisoning can be diagnosed based on clinical and epidemiological information, e.g., ingestion of castor beans, or occurrence of multiple cases during a short period, suggesting a common-source etiology (Wortmann, 2004). Ricin intoxication should be suspected if clinicians are presented with a number of patients having acute lung injury. A covert dispersion of aerosolized ricin is expected to be diagnosed, post factum, only after clinical symptoms occur (Kortepeter et al., 2001b). One common problem encountered in patients treated with ricin immunotoxins is the VLS, in which fluids leak from blood vessels leading to hypoalbumina, weight gain and pulmonary edema (Ghetie & Vitetta, 1994a). In patients who may be targets of an assassination attempt, ricin injection should be considered if there

respond to symptomatic therapy and removal from the exposure source.

surface area for RFT5.dgA and Ki-4.dgA, respectively (Schnell et al., 2003).

**4.2 Oral intoxication** 

et al., 2003).

**4.3 Inhalation** 

**5. Diagnosis** 

bleeding, anuria, diarrhea, cramps, and vascular collapse can also occur (Challoner & McCarron, 1990). Most symptoms develop less than 6 hours after ingestion, although the lag time from ingestion of castor seeds to onset of symptoms has ranged from 15 minutes to almost 10 hours. Progression to death occurs within 36 to 72 hours of exposure, depending on the route of exposure and the dose received (CDC, 2008).

#### **4.1 Injection**

In humans, subcutaneous or intramuscular injection of high doses of ricin results in severe local lymphoid necrosis, gastrointestinal hemorrhage, liver necrosis, diffuse nephritis, and diffuse splenitis. Ricin injection leads to necrosis at the injection site, which may predispose one to secondary infection (Passeron et al., 2004). Crompton & Gall (1980) summarized the clinical signs and symptoms for the Bulgarian dissident Georgi Markov, whose wellpublicized assassination was attributed to intramuscular injection of ricin (~500 μg), as follows: there was an immediate local pain, followed by general weakness within about 5 hours. This was followed by elevated temperature, nausea, and vomiting 15 to 24 hours later. Thirty-six hours after the incident, the patient was admitted to the hospital feeling very ill. He exhibited fever, tachycardia, normal blood pressure, swollen and sore lymph nodes in the affected groin, and and a 6-cm diameter area of induration and inflammation was observed at the injection site on his thigh. Over the next 48 hours, he became suddenly hypotensive and tachycardic; developed GI hemorrhage, hypovolemic shock, and renal failure. His white blood count was 26,300/mm3. Early on the third day after the attack, he became anuric and began vomiting blood. An electrocardiogram demonstrated complete atrioventricular conduction block. He died shortly thereafter; at the time of death, his white blood count was 33,200/mm3. The autopsy revealed pulmonary edema that was thought to have been secondary to Markov's cardiac failure, hemorrhagic necrosis of the small bowel, and hemorrhages in lymph nodes near the injection site, myocardium, testicles, and pancreas (Crompton and Gall, 1980).

A case of a 20-year-old male who allegedly committed suicide by injecting (s.c.) castor bean extract was reported to show severe weakness, nausea, dizziness, headache, chest, back, and abdominal pain 36 hours after the injection (Targosz et al., 2002). This patient subsequently developed a bleeding diathesis, liver failure, and renal failure. He succumbed to cardiac arrest. Postmortem examination revealed hemorrhagic foci in the brain, myocardium, and the pleura (Targosz et al., 2002).

A 36-year-old chemist who allegedly injected (i.m.) himself with an unknown amount of ricin prepared from homogenized castor seeds reportedly experienced headache and rigors 10 hours after exposure. He then developed anorexia, nausea, sinus tachycardia, lymphadenopathy at the injection sites, and erythematous areas around the puncture wounds (Fine et al., 1992).

In a clinical study involving cancer patients, intravenous administration of 40 low doses (18– 20 μg/m2 of estimated body surface area) of ricin was well tolerated (Fodstad et al., 1984). Flu-like symptoms with fatigue and muscular pain were common, and sometimes nausea and vomiting occurred. The symptoms began 4 to 6 hours after administration and lasted for 1 to 2 days. Phase I/II clinical trials of two experimental immunotoxins containing deglycosylated RTA (RFT5.dgA and Ki-4.dgA) administered i.v. to Hodgkins' lymphoma patients revealed maximum tolerated doses (MTDs) of 15 and 5 mg/m2 of estimated body surface area for RFT5.dgA and Ki-4.dgA, respectively (Schnell et al., 2003).

#### **4.2 Oral intoxication**

144 Bioterrorism

bleeding, anuria, diarrhea, cramps, and vascular collapse can also occur (Challoner & McCarron, 1990). Most symptoms develop less than 6 hours after ingestion, although the lag time from ingestion of castor seeds to onset of symptoms has ranged from 15 minutes to almost 10 hours. Progression to death occurs within 36 to 72 hours of exposure, depending

In humans, subcutaneous or intramuscular injection of high doses of ricin results in severe local lymphoid necrosis, gastrointestinal hemorrhage, liver necrosis, diffuse nephritis, and diffuse splenitis. Ricin injection leads to necrosis at the injection site, which may predispose one to secondary infection (Passeron et al., 2004). Crompton & Gall (1980) summarized the clinical signs and symptoms for the Bulgarian dissident Georgi Markov, whose wellpublicized assassination was attributed to intramuscular injection of ricin (~500 μg), as follows: there was an immediate local pain, followed by general weakness within about 5 hours. This was followed by elevated temperature, nausea, and vomiting 15 to 24 hours later. Thirty-six hours after the incident, the patient was admitted to the hospital feeling very ill. He exhibited fever, tachycardia, normal blood pressure, swollen and sore lymph nodes in the affected groin, and and a 6-cm diameter area of induration and inflammation was observed at the injection site on his thigh. Over the next 48 hours, he became suddenly hypotensive and tachycardic; developed GI hemorrhage, hypovolemic shock, and renal failure. His white blood count was 26,300/mm3. Early on the third day after the attack, he became anuric and began vomiting blood. An electrocardiogram demonstrated complete atrioventricular conduction block. He died shortly thereafter; at the time of death, his white blood count was 33,200/mm3. The autopsy revealed pulmonary edema that was thought to have been secondary to Markov's cardiac failure, hemorrhagic necrosis of the small bowel, and hemorrhages in lymph nodes near the injection site, myocardium, testicles, and

A case of a 20-year-old male who allegedly committed suicide by injecting (s.c.) castor bean extract was reported to show severe weakness, nausea, dizziness, headache, chest, back, and abdominal pain 36 hours after the injection (Targosz et al., 2002). This patient subsequently developed a bleeding diathesis, liver failure, and renal failure. He succumbed to cardiac arrest. Postmortem examination revealed hemorrhagic foci in the brain, myocardium, and the

A 36-year-old chemist who allegedly injected (i.m.) himself with an unknown amount of ricin prepared from homogenized castor seeds reportedly experienced headache and rigors 10 hours after exposure. He then developed anorexia, nausea, sinus tachycardia, lymphadenopathy at the injection sites, and erythematous areas around the puncture

In a clinical study involving cancer patients, intravenous administration of 40 low doses (18– 20 μg/m2 of estimated body surface area) of ricin was well tolerated (Fodstad et al., 1984). Flu-like symptoms with fatigue and muscular pain were common, and sometimes nausea and vomiting occurred. The symptoms began 4 to 6 hours after administration and lasted for 1 to 2 days. Phase I/II clinical trials of two experimental immunotoxins containing

on the route of exposure and the dose received (CDC, 2008).

**4.1 Injection** 

pancreas (Crompton and Gall, 1980).

pleura (Targosz et al., 2002).

wounds (Fine et al., 1992).

Ricin is less toxic by oral ingestion than by other routes (Rauber & Heard, 1985), probably due to poor absorption of the toxin and possibly partial enzymatic degradation in the digestive tract. The effects of oral intoxication vary among individuals, are dose dependent, and have different signs and symptoms. Rauber & Heard (1985) reviewed 751 cases of castor bean ingestion and reported 14 fatalities (1.9% death rate). The number of beans ingested by patients who died greatly varied. For instance, of the two lethal cases of oral intoxication documented since 1930, one involved a 24-year-old man who ate 15 to 20 beans, and the other was a 15-year-old boy who had 10 to 12 beans. All of the described serious, or fatal cases of castor bean ingestion have the same general clinical history: rapid (less than a few hours) onset of nausea, vomiting, and abdominal pain followed by diarrhea, hemorrhage from the anus, anuria, cramps, dilation of the pupils, fever, thirst, sore throat, headache, vascular collapse, and shock. Death occurred on the third day or later. The most common autopsy findings in oral intoxication were multifocal ulcerations and hemorrhages of gastric and small-intestinal mucosa, which may be quite severe; lymphoid necrosis in the mesenteric lymph nodes, gut-associated lymphoid tissue (GALT), and spleen; Kupffer cell and liver necrosis; diffuse nephritis; and diffuse splenitis (Rauber & Heard, 1985; Bradberry et al., 2003).

#### **4.3 Inhalation**

There are no documented cases of aerosol exposure to ricin in humans. Lesions induced by oral and parenteral exposure are consistent with those from animal studies, suggesting that the same would hold true for aerosol exposures. An allergic syndrome has been reported in workers exposed to castor bean dust in or around castor oil-processing plants (Brugsch, 1960). The clinical picture is characterized by the sudden onset of congestion of the nose and throat, itchiness of the eyes, urticaria, and tightness of the chest. In more severe cases, wheezing can last for several hours, and may lead to bronchial asthma. Affected individuals respond to symptomatic therapy and removal from the exposure source.

#### **5. Diagnosis**

Ricin poisoning can be diagnosed based on clinical and epidemiological information, e.g., ingestion of castor beans, or occurrence of multiple cases during a short period, suggesting a common-source etiology (Wortmann, 2004). Ricin intoxication should be suspected if clinicians are presented with a number of patients having acute lung injury. A covert dispersion of aerosolized ricin is expected to be diagnosed, post factum, only after clinical symptoms occur (Kortepeter et al., 2001b). One common problem encountered in patients treated with ricin immunotoxins is the VLS, in which fluids leak from blood vessels leading to hypoalbumina, weight gain and pulmonary edema (Ghetie & Vitetta, 1994a). In patients who may be targets of an assassination attempt, ricin injection should be considered if there

apparently produce lethal pulmonary damage, probably due to hypoxemia resulting from

Currently, there is no FDA-approved therapeutic for ricin exposure. Countermeasures that have demonstrated capability to disrupt the ricin intoxication process include vaccines and antibody therapy. Both rely on the ability of antibody to prevent the binding of ricin to cell receptors. To ensure maximum protection, the vaccine must be given before exposure, and sufficient antibody must be produced. Similarly, administration of preformed antibodies affords maximum protection if antibody is

Treatment is largely symptomatic and basically supportive to minimize the effects of the poisoning. Because ricin acts rapidly and irreversibly (directly on lung parenchyma after inhalation or is distributed rapidly to vital organs after parenteral exposure), postexposure therapy is more challenging than with slowly processed, peripherally acting agents that can

Inhalational exposure is best countered with active vaccination or prophylactic administration of aerosolized specific antibody (Franz and Jaax, 1997). However, there is currently no licensed vaccine available. Development of a ricin vaccine previously focused on either a deglycosylated ricin A chain or formalin-inactivated toxoid (Hewetson et al., 1996). Both vaccines confer protection against aerosolized ricin. Nevertheless, ricin is not completely inactivated by formalin and may retain some of its enzymatic activity (albeit approximately 1,000-fold lower than native ricin). Deglycosylated ricin A chain may lead to

Recent research has focused on developing recombinant RTA subunit vaccines devoid of cytotoxicity and other potential deleterious activities. Several ricin vaccines candidates that are based on engineered RTA molecules have shown protection with different animal models, but demonstration of their human protection proved to be more challenging (Vitetta et al., 2005). USAMRIID has engineered a recombinant ricin vaccine 1-33/44-198 (rRTA 1-33/44-198) (RV*Ec*), with increased protein stability over the parent RTA subunit and devoid of enzymatic (N-glycosidase) activity (Olson et al., 2004), lacking vascular leak activity (Porter et al., 2011) described in RTA-based immunotoxins, and fully protected vaccinated animals against supralethal aerosol challenges (McHugh et al., 2004; Carra et al., 2007). A cGLP pre-clinical toxicity study of RV*Ec* in New Zealand white rabbits demonstrated that no treatment-related or toxicologically significant effects were observed with RV*Ec* during this study (McClain et al., 2011). A phase I clinical study is ongoing at USAMRIID to evaluate the safety and immunogenicity of RV*Ec* in humans (USAMRIID, 13

A recombinant protein RTA vaccine, RiVax, has been developed based on mutations of both the enzymatic and a reported VLS-inducing site (Smallshaw et al., 2002). RiVax elicited

massive pulmonary edema and alveolar flooding (Poli et al., 2007).

**6. Medical management** 

present before exposure.

local or systemic VLS.

April 2011; AP, 13 April 2011).

be treated with antibiotics (Franz & Jaax, 1997).

**6.1 Vaccination and passive protection** 

are signs of rapid onset of symptoms similar to VLS. Ricin ingestion should be suspected if patients with gastrointestinal hemorrhage and hypotension have eaten from the same food source.

Because ricin is immunogenic, acute as well as convalescent sera should be obtained from patients 2 weeks after exposure for measurement of antibody response (Franz & Jaax, 1997). Immunoassay (for blood or other body fluids) or immunohistochemistry techniques (for direct analysis of tissues) may be useful for confirming ricin intoxication (Poli et al., 2007). However, identification of the toxin in body fluids or tissues is challenging because ricin is bound very quickly, and is also metabolized before excretion (Ramsden et al., 1989).

Currently, two types of laboratory testing are available for suspected ricin exposures. For environmental cases (determined by the CDC for suspected exposures from the environment, or by the FDA for suspected exposures from food or medication), ricin can be detected qualitatively by time-resolved fluorescence immunoassay (TRFIA), and polymerase chain reaction (PCR) in specimens (e.g., filters, swabs, or wipes). For biologic samples, selected specimens can be assessed for urinary ricinine, a marker of ricin exposure, using HPLC-ESI-MS (CDC, 2006b).

The differential diagnoses of aerosol exposure to ricin include staphylococcal enterotoxin B (SEB), community-acquired pneumonia, inhalational anthrax, Q fever, tularemia, plague, and exposure to pyrolysis by-products of organofluorine polymers, or other chemical warfare agents such as phosgene (Eitzen et al., 1998). For ingested ricin, differential diagnoses include enteric pathogens, enterotoxins, and other toxins, including caustic agents, mushroom species, hydrocarbons, and pharmaceuticals such as salicylates and colchicine. Several factors discriminate ricin intoxication from other agents, such as: 1) clinical progression despite antibiotics (as opposed to infectious agents); 2) lack of mediastinitis (as seen with pulmonary anthrax); 3) progressive decline in clinical status (patients exposed to SEB tend to stabilize), and a slower progression than patients exposed to phosgene (Eitzen et al., 1998; Kortepeter et al., 2001a; 2001b).

#### **5.1 Prognosis and cause of death**

Death from ricin exposure could occur within 36 to 72 hours (CDC, 2008). If death has not occurred within 3-5 days, the patient usually survives. A mortality rate of 1.9% (14 of 751 patients) was reported after castor bean ingestion (Rauber & Heard, 1985). Even with little or no effective supportive care, the death rate in symptomatic patients has been approximately 6%. No information is available regarding human mortality rate after ricin inhalation.

The exact cause of death from ricin poisoning possibly varies with route of exposure. Ricin ingestion results in ulceration and hemorrhage of the stomach and small intestine mucosa, necrosis of the mesenteric lymphatics, liver necrosis, nephritis, and splenitis (Poli et al., 2007). Injection of the toxin may lead to severe local lymphoid necrosis, gastrointestinal hemorrhage, liver necrosis, diffuse nephritis, and diffuse splenitis. Intravenous administration of ricin in rats resulted in diffuse damage to Kupffer cells within 4 hours, followed by endothelial cell damage, formation of thrombi in the liver vasculature, and finally, hepatocellular necrosis (Bingen et al., 1987; Derenzini et al., 1976). In mice, rats, and primates, high doses by inhalation

are signs of rapid onset of symptoms similar to VLS. Ricin ingestion should be suspected if patients with gastrointestinal hemorrhage and hypotension have eaten from the same food

Because ricin is immunogenic, acute as well as convalescent sera should be obtained from patients 2 weeks after exposure for measurement of antibody response (Franz & Jaax, 1997). Immunoassay (for blood or other body fluids) or immunohistochemistry techniques (for direct analysis of tissues) may be useful for confirming ricin intoxication (Poli et al., 2007). However, identification of the toxin in body fluids or tissues is challenging because ricin is

Currently, two types of laboratory testing are available for suspected ricin exposures. For environmental cases (determined by the CDC for suspected exposures from the environment, or by the FDA for suspected exposures from food or medication), ricin can be detected qualitatively by time-resolved fluorescence immunoassay (TRFIA), and polymerase chain reaction (PCR) in specimens (e.g., filters, swabs, or wipes). For biologic samples, selected specimens can be assessed for urinary ricinine, a marker of ricin exposure, using

The differential diagnoses of aerosol exposure to ricin include staphylococcal enterotoxin B (SEB), community-acquired pneumonia, inhalational anthrax, Q fever, tularemia, plague, and exposure to pyrolysis by-products of organofluorine polymers, or other chemical warfare agents such as phosgene (Eitzen et al., 1998). For ingested ricin, differential diagnoses include enteric pathogens, enterotoxins, and other toxins, including caustic agents, mushroom species, hydrocarbons, and pharmaceuticals such as salicylates and colchicine. Several factors discriminate ricin intoxication from other agents, such as: 1) clinical progression despite antibiotics (as opposed to infectious agents); 2) lack of mediastinitis (as seen with pulmonary anthrax); 3) progressive decline in clinical status (patients exposed to SEB tend to stabilize), and a slower progression than patients exposed

Death from ricin exposure could occur within 36 to 72 hours (CDC, 2008). If death has not occurred within 3-5 days, the patient usually survives. A mortality rate of 1.9% (14 of 751 patients) was reported after castor bean ingestion (Rauber & Heard, 1985). Even with little or no effective supportive care, the death rate in symptomatic patients has been approximately 6%. No information is available regarding human mortality rate after ricin

The exact cause of death from ricin poisoning possibly varies with route of exposure. Ricin ingestion results in ulceration and hemorrhage of the stomach and small intestine mucosa, necrosis of the mesenteric lymphatics, liver necrosis, nephritis, and splenitis (Poli et al., 2007). Injection of the toxin may lead to severe local lymphoid necrosis, gastrointestinal hemorrhage, liver necrosis, diffuse nephritis, and diffuse splenitis. Intravenous administration of ricin in rats resulted in diffuse damage to Kupffer cells within 4 hours, followed by endothelial cell damage, formation of thrombi in the liver vasculature, and finally, hepatocellular necrosis (Bingen et al., 1987; Derenzini et al., 1976). In mice, rats, and primates, high doses by inhalation

to phosgene (Eitzen et al., 1998; Kortepeter et al., 2001a; 2001b).

bound very quickly, and is also metabolized before excretion (Ramsden et al., 1989).

source.

HPLC-ESI-MS (CDC, 2006b).

**5.1 Prognosis and cause of death** 

inhalation.

apparently produce lethal pulmonary damage, probably due to hypoxemia resulting from massive pulmonary edema and alveolar flooding (Poli et al., 2007).

#### **6. Medical management**

Currently, there is no FDA-approved therapeutic for ricin exposure. Countermeasures that have demonstrated capability to disrupt the ricin intoxication process include vaccines and antibody therapy. Both rely on the ability of antibody to prevent the binding of ricin to cell receptors. To ensure maximum protection, the vaccine must be given before exposure, and sufficient antibody must be produced. Similarly, administration of preformed antibodies affords maximum protection if antibody is present before exposure.

Treatment is largely symptomatic and basically supportive to minimize the effects of the poisoning. Because ricin acts rapidly and irreversibly (directly on lung parenchyma after inhalation or is distributed rapidly to vital organs after parenteral exposure), postexposure therapy is more challenging than with slowly processed, peripherally acting agents that can be treated with antibiotics (Franz & Jaax, 1997).

#### **6.1 Vaccination and passive protection**

Inhalational exposure is best countered with active vaccination or prophylactic administration of aerosolized specific antibody (Franz and Jaax, 1997). However, there is currently no licensed vaccine available. Development of a ricin vaccine previously focused on either a deglycosylated ricin A chain or formalin-inactivated toxoid (Hewetson et al., 1996). Both vaccines confer protection against aerosolized ricin. Nevertheless, ricin is not completely inactivated by formalin and may retain some of its enzymatic activity (albeit approximately 1,000-fold lower than native ricin). Deglycosylated ricin A chain may lead to local or systemic VLS.

Recent research has focused on developing recombinant RTA subunit vaccines devoid of cytotoxicity and other potential deleterious activities. Several ricin vaccines candidates that are based on engineered RTA molecules have shown protection with different animal models, but demonstration of their human protection proved to be more challenging (Vitetta et al., 2005). USAMRIID has engineered a recombinant ricin vaccine 1-33/44-198 (rRTA 1-33/44-198) (RV*Ec*), with increased protein stability over the parent RTA subunit and devoid of enzymatic (N-glycosidase) activity (Olson et al., 2004), lacking vascular leak activity (Porter et al., 2011) described in RTA-based immunotoxins, and fully protected vaccinated animals against supralethal aerosol challenges (McHugh et al., 2004; Carra et al., 2007). A cGLP pre-clinical toxicity study of RV*Ec* in New Zealand white rabbits demonstrated that no treatment-related or toxicologically significant effects were observed with RV*Ec* during this study (McClain et al., 2011). A phase I clinical study is ongoing at USAMRIID to evaluate the safety and immunogenicity of RV*Ec* in humans (USAMRIID, 13 April 2011; AP, 13 April 2011).

A recombinant protein RTA vaccine, RiVax, has been developed based on mutations of both the enzymatic and a reported VLS-inducing site (Smallshaw et al., 2002). RiVax elicited

In addition to studies pertaining to the natural toxicity of the protein, ricin has also been used extensively in the design of therapeutic immunotoxins. In such, ricin, RTA, or a related toxin is chemically or genetically linked to a binding ligand such as an antibody or growth factor that recognizes cancer cells, then it may be taken up by the cancer cells and ultimately kill them (Frankel, 1988). Immunotoxins using RTA or blocked ricin, have been evaluated in phase I clinical trials for control of several cancers (Ghetie & Vitetta, 1994b; Lynch et al.,

Ricin is a potent toxin derived from the seeds of the castor plant, *R. communis*. Because of its potency, stability, worldwide availability, and relative ease of production, ricin is considered a significant biological warfare or terrorism threat. Ricin was developed as an aerosol biological weapon by the U.S. and its allies during WWII, although it was never used in battle. As a biological or chemical weapon, ricin has not been considered as very powerful in comparison with other agents such as botulinum neurotoxin or anthrax. However, its effectiveness as a discrete weapon of terror-targeted assassinations, biocrimes, or small-scale operations does raise potential concern. Ricin's popularity as well as its track record in actually being exploited by extremists groups and individuals highlight the need to be vigilant of its latent misuse. Clinical manifestations of ricin poisoning vary depending on the routes of exposure. Diagnosis is based upon both epidemiological and clinical parameters. Laboratory confirmation of clinical samples is possible by immunoassay but complicated by pharmacokinetic factors. Currently, there is no U.S. FDA-approved drug or vaccine against ricin poisoning. Treatment is purely supportive. Prophylaxis will be best accomplished by vaccination. Ricin vaccine candidates are currently in advanced

This work was supported by Defense Threat Reduction Agency, JSTO-CBD Project Numbers CBM.VAXBT.03.10.RD.P.011 and CBCALL12-VAXBT4-1-0385. We are grateful to Dr. Mark A. Olson for providing the three-dimensional images of ricin, and Lorraine

The opinions or assertions contained herein are the private views of the authors and are not

Amlot, P.L., Stone, M.J., Cunningham D, et al. (1993). A Phase I Study of an Anti-CD22-

deglycosylated Ricin A Chain Immunotoxin in the Treatment of B-cell Lymphomas Resistant to Conventional Therapy. *Blood*, Vol. 82, pp. 2624-2633, ISSN 0006-497 AP. (13 April 2011). Army Starts Clinical Trials on Ricin Vaccine, In: Army Times, 20072011.

Available from http://www.armytimes.com/news/2011/04/ap-army-medical-

necessarily the official views of the U.S. Army or the Department of Defense.

**7. Medical therapy of ricin** 

1997; Schnell et al., 2003; Vitetta, 2006).

development in laboratory and clinical trials.

**9. Acknowledgment and disclaimer** 

Farinick for assistance with graphics.

research-ricin-041311/

**10. References** 

**8. Summary** 

protective immunity in mice, and had sufficient pre-clinical safety data (Smallshaw et al., 2005). Results from the initial Phase I human trial showed that RiVax appeared to be immunological and well tolerated in humans (Vitetta et al., 2006). However, while such results were encouraging, vaccine formulation and stability remain problematic. Hence, a lyophilized formulation that retained immunogenicity when stored at 4ºC was developed (Smallshaw & Vitetta, 2010; Marconescu et al., 2010).

Passive protection with aerosolized anti-ricin immunoglobulin (IgG) has also been evaluated as prophylaxis before aerosol challenge. Administration of nebulized anti-ricin IgG effectively protected against lung lesions and lethality in mice when challenged with an aerosol exposure to ricin (Poli et al., 1996). Extrapolation of these data to clearance rates of IgG from the airways of rabbits suggests that anti-ricin–specific antibodies may provide protection for up to 2 to 3 days or longer. These findings imply that inhaling protective antibody from a portable nebulizer just before an attack might provide some protection in nonimmune individuals (Poli et al., 1996). However, the window of opportunity for treatment by intravenous administration or inhalation of specific antibody after exposure is probably minimal at best.

Recent pre-clinical studies have shown the powerful protection afforded by neutralizing monoclonal antibodies (administered singly or in combination) against a lethal dose challenge of ricin, demonstrating proof of concept for passive immunotherapy for the treatment of ricin poisoning or for preexposure prophylaxis (Neal, 2010; 2011; Prigent, 2011).

#### **6.2 Supportive and specific therapy**

Supportive medical care depends on the route of exposure and clinical manifestations. For oral intoxication, supportive therapy includes intravenous fluid, electrolyte replacement, monitoring of liver and renal functions, gastric emptying/lavage, syrup of ipecac, cathartics, and, activated charcoal (Ellenhorn, 1997; Franz & Jaax, 1997). Patients who have ingested beans and presented asymptomatic should remain under observation for 4-6 hours after ingestion. For inhalational intoxication, respiratory support is given as needed. Aerosolexposed patient may require the use of positive-pressure ventilator therapy, fluid and electrolyte replacement, antiinflammatory agents, and analgesics (Kortepeter et al., 2001b). Dermal exposures require supportive treatment. Percutaneous exposures would necessitate judicious use of intravenous fluids and monitoring for symptoms associated with VLS, including hypotension, edema, and pulmonary edema (Poli et al., 2007). Supportive care includes correction of coagulopathies, respiratory support, and monitoring for liver and renal failure .

Several research groups have engaged in the development of RTA active site inhibitors or RTB receptor antagonists as clinical antidotes against ricin poisoning, or as therapeutic adjuncts to vaccination. Small molecules that exhibited modest IC50 values (Bai et al., 2010; Wahome et al., 2010; Pang et al., 2011), including a compound that showed *in vivo* efficacy (Stechmann et al., 2010) have been described. An effective and essentially irreversible RTA inhibitor is thought to be practically useful as a pretreatment for military forces or civilian first-responders (Millard & LeClaire, 2007).

#### **7. Medical therapy of ricin**

In addition to studies pertaining to the natural toxicity of the protein, ricin has also been used extensively in the design of therapeutic immunotoxins. In such, ricin, RTA, or a related toxin is chemically or genetically linked to a binding ligand such as an antibody or growth factor that recognizes cancer cells, then it may be taken up by the cancer cells and ultimately kill them (Frankel, 1988). Immunotoxins using RTA or blocked ricin, have been evaluated in phase I clinical trials for control of several cancers (Ghetie & Vitetta, 1994b; Lynch et al., 1997; Schnell et al., 2003; Vitetta, 2006).

#### **8. Summary**

148 Bioterrorism

protective immunity in mice, and had sufficient pre-clinical safety data (Smallshaw et al., 2005). Results from the initial Phase I human trial showed that RiVax appeared to be immunological and well tolerated in humans (Vitetta et al., 2006). However, while such results were encouraging, vaccine formulation and stability remain problematic. Hence, a lyophilized formulation that retained immunogenicity when stored at 4ºC was developed

Passive protection with aerosolized anti-ricin immunoglobulin (IgG) has also been evaluated as prophylaxis before aerosol challenge. Administration of nebulized anti-ricin IgG effectively protected against lung lesions and lethality in mice when challenged with an aerosol exposure to ricin (Poli et al., 1996). Extrapolation of these data to clearance rates of IgG from the airways of rabbits suggests that anti-ricin–specific antibodies may provide protection for up to 2 to 3 days or longer. These findings imply that inhaling protective antibody from a portable nebulizer just before an attack might provide some protection in nonimmune individuals (Poli et al., 1996). However, the window of opportunity for treatment by intravenous administration or inhalation of specific antibody after exposure is

Recent pre-clinical studies have shown the powerful protection afforded by neutralizing monoclonal antibodies (administered singly or in combination) against a lethal dose challenge of ricin, demonstrating proof of concept for passive immunotherapy for the treatment of ricin poisoning or for preexposure prophylaxis (Neal, 2010; 2011; Prigent,

Supportive medical care depends on the route of exposure and clinical manifestations. For oral intoxication, supportive therapy includes intravenous fluid, electrolyte replacement, monitoring of liver and renal functions, gastric emptying/lavage, syrup of ipecac, cathartics, and, activated charcoal (Ellenhorn, 1997; Franz & Jaax, 1997). Patients who have ingested beans and presented asymptomatic should remain under observation for 4-6 hours after ingestion. For inhalational intoxication, respiratory support is given as needed. Aerosolexposed patient may require the use of positive-pressure ventilator therapy, fluid and electrolyte replacement, antiinflammatory agents, and analgesics (Kortepeter et al., 2001b). Dermal exposures require supportive treatment. Percutaneous exposures would necessitate judicious use of intravenous fluids and monitoring for symptoms associated with VLS, including hypotension, edema, and pulmonary edema (Poli et al., 2007). Supportive care includes correction of coagulopathies, respiratory support, and monitoring for liver and

Several research groups have engaged in the development of RTA active site inhibitors or RTB receptor antagonists as clinical antidotes against ricin poisoning, or as therapeutic adjuncts to vaccination. Small molecules that exhibited modest IC50 values (Bai et al., 2010; Wahome et al., 2010; Pang et al., 2011), including a compound that showed *in vivo* efficacy (Stechmann et al., 2010) have been described. An effective and essentially irreversible RTA inhibitor is thought to be practically useful as a pretreatment for military forces or civilian

(Smallshaw & Vitetta, 2010; Marconescu et al., 2010).

probably minimal at best.

**6.2 Supportive and specific therapy** 

first-responders (Millard & LeClaire, 2007).

2011).

renal failure .

Ricin is a potent toxin derived from the seeds of the castor plant, *R. communis*. Because of its potency, stability, worldwide availability, and relative ease of production, ricin is considered a significant biological warfare or terrorism threat. Ricin was developed as an aerosol biological weapon by the U.S. and its allies during WWII, although it was never used in battle. As a biological or chemical weapon, ricin has not been considered as very powerful in comparison with other agents such as botulinum neurotoxin or anthrax. However, its effectiveness as a discrete weapon of terror-targeted assassinations, biocrimes, or small-scale operations does raise potential concern. Ricin's popularity as well as its track record in actually being exploited by extremists groups and individuals highlight the need to be vigilant of its latent misuse. Clinical manifestations of ricin poisoning vary depending on the routes of exposure. Diagnosis is based upon both epidemiological and clinical parameters. Laboratory confirmation of clinical samples is possible by immunoassay but complicated by pharmacokinetic factors. Currently, there is no U.S. FDA-approved drug or vaccine against ricin poisoning. Treatment is purely supportive. Prophylaxis will be best accomplished by vaccination. Ricin vaccine candidates are currently in advanced development in laboratory and clinical trials.

#### **9. Acknowledgment and disclaimer**

This work was supported by Defense Threat Reduction Agency, JSTO-CBD Project Numbers CBM.VAXBT.03.10.RD.P.011 and CBCALL12-VAXBT4-1-0385. We are grateful to Dr. Mark A. Olson for providing the three-dimensional images of ricin, and Lorraine Farinick for assistance with graphics.

The opinions or assertions contained herein are the private views of the authors and are not necessarily the official views of the U.S. Army or the Department of Defense.

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**1. Introduction** 

for detecting bio-terrorism events.

introduced strain early.

**8** 

*Australia* 

**Spatio-Temporal Disease Surveillance** 

Concern over bio-terrorism has led to a demand for automated methods for the surveillance of disease counts with the ability to rapidly detect outbreaks of disease. While traditional statistical process control methods such as control charts have been found to have early detection properties when monitoring univariate disease counts, these are often inadequate

There are two principal impediments in statistical process control methods for the detection of bio-terrorism events: firstly, these methods aggregate over space by examining total counts and thus ignore the spatial dimension of the task and secondly they fail to adjust for the usual (seasonal) behaviour of diseases (e.g., Steiner *et al.*, 2011 where the focus is early detection of the start of influenza outbreak). If disease outbreaks were expected to be spread relatively uniformly in space, then the former reason is unimportant. However, since bioterrorism attacks are likely to be introduced to specifically targeted locations, then the resulting disease instances are likely to cluster in space. Consequently, monitoring total counts is likely to reduce the signal-to-noise ratio of this outbreak by aggregating over regions where there is no outbreak. Exploiting the spatial clustering should be able to

There is much ongoing work in developing methods that are efficient at detecting outbreaks in a spatial context but these methods may still fail to deal with the latter fault mentioned above. When surveillance for bio-terrorism involves the monitoring of diseases or syndromes already present in the population then the detection of an attack may be delayed if it is introduced during a period of normal seasonal increased activity. For example, respiratory complaints are often much more frequent in the cooler months so detecting an intentional disease outbreak with respiratory symptomatology would need to differentiate between the usual and an unusual increase in cases. Therefore, it is often necessary to remove the influence of the expected behaviour of a disease to detect the signal of an

Before presenting the method we are proposing in this chapter to deal with both of the above concerns, we begin by outlining some of the existing spatial disease detection methods. The current benchmark in spatio-temporal surveillance is the spatial SCAN statistic (Tango, 1995, Kulldorff, 1995, 1997, 2001, 2005). This method is a spatio-temporal moving average plan that systematically scans the target space applying a test to all windows of data up to a given fixed size in time and space. This presents an intuitive

provide additional power and efficiency in detecting the outbreak.

Ross Sparks, Sarah Bolt and Chris Okugami *CSIRO Mathematics, Informatics and Statistics, Sydney,* 

