Smith et al., J Bioterr Biodef 2012, S7
http://dx.doi.org/10.4172/2157-2526.S7-003
Bioterrorism & Biodefense
Review Article
Open Access
Botulinum Neurotoxins as Biothreat Agents
Theresa J Smith1, Virginia I Roxas-Duncan1 and Leonard A Smith2*
1
2
Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702
Office of the Chief Scientist, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702
Abstract
Botulinum neurotoxins (BoNTs), a family of proteins produced by the bacterium Clostridium botulinum and other
clostridial species, are considered to be the most potent toxin known, and a potential biological weapon. BoNTs
cause botulism, a serious and life-threatening illness in humans and animals. There are seven immunologically
distinct serotypes of BoNTs, each with multiple unique subtypes/genetic variants. BoNT potency and diversity present
challenges for detection, diagnosis, treatment, and development of countermeasures. This article provides an overview
of the range, diversity, and potency of BoNTs, and how these features might affect their use as biothreat agents.
Keywords: Botulinum neurotoxins; Biothreat; Diversity; Assays;
Antitoxins
Abbreviations: BoNT: Botulinum Neurotoxins; PCR: Polymerase
Chain Reaction; SNARE: Soluble N-ethylmaleimide-sensitive factor
Attachment Protein Receptor; FRET: Fluorescence Resonance Energy
Transfer; PFGE: Pulsed Field Gel Electrophoresis; AFLP: Amplified
Fragment Length Polymorphism; MLST: Multi-Locus Sequence
Typing; MLVA: Multiple-Locus Variable number tandem repeat
Analysis; VNTR: Variable Number Tandem Repeat; SNP: Single
Nucleotide Polymorphism
Introduction
Botulinum neurotoxins (BoNTs) are extremely potent protein
toxins produced by the bacterium Clostridium botulinum and related
clostridial species. The bacteria are anaerobic spore-formers that are
relatively easy to culture, and the toxins that are produced are stable in
complexed form, but somewhat heat labile and easily destroyed during
autoclaving or cooking.
The toxins cause a neuroparalytic syndrome characterized by a
descending, bilateral flaccid paralysis that can be fatal due to respiratory
arrest. Naturally occurring intoxications can arise within hours of
ingestion of preformed toxin (food borne botulism). They may also
be due to the elaboration of toxins after colonization of contaminated
wounds and abscesses with BoNT-producing clostridia (wound
botulism), or from organisms that have colonized the intestinal areas of
susceptible infants (infant botulism) or adults (adult toxicoinfections).
Intoxications may also occur after deliberate introduction by injection
or inhalation.
BoNTs are considered to be category A select agents because
they have great potential for adverse public health impact with mass
casualties, they require broad-based public health preparedness efforts,
and they have moderate to high potential for large-scale dissemination
[1]. These factors also make botulinum neurotoxins primary candidates
as bioterrorism or biothreat agents.
History
Botulism has probably occurred sporadically since food storage
methods involving drying, salting, pickling, or smoking were introduced.
However, accurate descriptions of intoxications were not available
until early in the 19th century [2]. Justinus Kerner’s monographs on
“sausage poison” in 1820 and 1822 are generally considered the first
accurate and complete descriptions of the disease [2]. The connection
of botulism with a biological organism occurred with the isolation of
J Bioterr Biodef
a gram positive bacillus from ham which produced a substance that
was toxic in animals [3]. The first descriptions of botulism that were
not associated with meat or fish were published in 1904, after an
outbreak in Darmstadt, Germany that was due to canned white beans
[4]. Subsequently it was noted that, while most botulism outbreaks in
Europe were traced to meat or fish products, most food borne cases
in the US were due to vegetables. Several severe outbreaks of botulism
occurred in the US from late 1919 to early 1920, associated with the
consumption of commercially canned ripe olives. These incidents were
particularly deadly, with an overall fatality rate of 78%. The severity
and widespread nature of these cases prompted the establishment of
strict guidelines concerning commercial food canning and intensified
research in this area [5]. These highly publicized outbreaks, plus others
occurring in Europe over the following decade [6], brought attention
to the deadly nature of this toxin, which may have prompted some
governments to pursue this toxin as a biological weapon.
State-sponsored programs for the development of BoNTs as
biological weapons have been instituted in Canada, Germany, Iraq,
Japan, the UK, the US, and the USSR [7,8]. However, to our knowledge
only Japan has actually tested such weapons on humans. General Shiro
Ishii, head of the infamous Japanese Unit 731, admitted to testing
botulinum toxin as a weapon in the 1930s by feeding Manchurian
prisoners botulinum toxin-laced food [9].
In early 1944, Allied intelligence uncovered plans indicating that
Germany was planning to use BoNT as a weapon against invading
forces. This led to the initiation of a US program that included
researching the use of BoNTs as weapons [8]. While the US program
was abolished in 1969, the Soviet Union continued pursuing BoNT
as a biological weapon, despite their signing the Biological and Toxin
*Corresponding author: Leonard A Smith, Senior Research Scientist (ST) for
Medical Countermeasures Technology, Office of the Chief Scientist, United States
Army Medical Research Institute of Infectious Diseases, 1425 Porter Street,
Fort Detrick, MD 21702; Tel: (301) 619-4238; Fax: (301) 619-2348; E-mail:
leonard.smith@amedd.army.mil
Received November 24, 2011; Accepted January 20, 2012; Published January
24, 2012
Citation: Smith TJ, Roxas-Duncan VI, Smith LA (2012) Botulinum Neurotoxins as
Biothreat Agents. J Bioterr Biodef S7:003. doi:10.4172/2157-2526.S7-003
Copyright: © 2012 Smith TJ, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Bioterrorism: Toxins
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Citation: Smith TJ, Roxas-Duncan VI, Smith LA (2012) Botulinum Neurotoxins as Biothreat Agents. J Bioterr Biodef S7:003. doi:10.4172/2157-2526.
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Weapons Convention agreement in 1972. This research was continued
until the fall of the Soviet Union in the early 1990s.
BoNT subtypes
% identity
A1-A5
85.0-97.1
In 1992, Iraq admitted to the United Nations that they had
produced approximately 19,000 liters of concentrated BoNT; some of
this material had been loaded into military weapons [7].
B1-B6
92.7-98.4
C-C/D
75.8-76.5
D-D/C
72.7-76.4
State-sponsored programs were not the only sources of concern.
In the 1990s, the Japanese cult Aum Shinrikyo cultured C. botulinum
strains to produce toxin, which they disseminated on at least three
occasions in Tokyo using aerosol release. The toxin was produced by
a clostridial strain that was reportedly isolated from northern Japan.
These attempts were not successful, apparently due in part to a lack of
toxin production in the cultures [10].
E1-E8
94.7-99.0
F1-F7
63.8-97.0
Table 1 Amino acid residue identities among BoNT subtypes.
BoNT A1 BoNT B1 BoNT C1 BoNT D BoNT E3 BoNT F1 BoNT G
BoNT A1
BoNT B1
BoNT C1
Diversity
BoNT D
Clostridium botulinum organisms produce a series of neurotoxins
that are closely related in structure and function, but surprisingly
diverse in genetic and amino acid sequences. There are seven toxin
serotypes (A-G). Four of these, BoNT/A, /B, /E, and /F, are considered
to be human pathogens; the other three serotypes are not generally
thought to be human pathogens, even though there have been a few
human cases associated with these toxin types.
BoNT F1
BoNT serotypes differ in their host range, potency, and course of
intoxication. Host susceptibility differences that are related to toxin
differences are known. For example, humans are very susceptible to
BoNT/A and BoNT/B, but appear to be somewhat resistant to BoNT/C,
/D, and /G. Rats and chickens are susceptible to BoNT/A, but resistant
to BoNT/B [11]. Paralytic effects due to serotype A can last for many
months, but effects due to BoNT/B or /E diminish after a few weeks.
Infant botulism cases caused by BoNT/F can occur in very young
babies (less than one week old); in contrast to BoNT/A or /B, these
intoxications tend to resolve rapidly, especially after treatment with
specific antitoxin.
Each serotype can be divided into two to eight subtypes/genetic
variants that differ by as little as 1% or as much as 36% in amino acid
sequence (Table 1). The toxins are expressed in one of six different
clostridial species (C. botulinum groups I-III, C. argentinense, C. baratii,
and C. butyricum) [12].
C. botulinum group I organisms may produce any one of three toxins
that are usually associated with human botulism (serotypes A, B and F).
In addition, members of this group may concurrently produce multiple
toxins, as in the case of BoNT/Ab, /Ba, or /Bf. The toxins produced by
Group I species are generally of higher yields and toxicity in culture
than those produced by other species. One of these strains, BoNT/
A1 Hall, has been reported to yield toxin concentrations of 1,000,000
mouse i.p. LD50/ml from crude cultures under optimal conditions
[13]. Toxins produced by Group I strains also show the greatest
subtype diversity. BoNT/A subtypes differ by as much as 15% at the
amino acid level; BoNT/F subtypes are even more diverse. Amino acid
differences of 26% are seen among the four BoNT/F sses produced by
Group I clostridia (BoNT/F2-F5) and there is 36% difference between
BoNT/F5 and BoNT/F7, which is produced in C. baratii strains (Table
1). This level of diversity is similar to that seen between the BoNT/E
and BoNT/F serotypes (Table 2). Some subtype differences also result
in host range differences. Mammals, especially cattle, are extremely
susceptible to BoNT/C1 but resistant to BoNT C/D. The reverse is true
in birds. Catastrophic die-offs in waterfowl due to BoNT C/D have been
seen in waterfowl and other migratory birds [14]. Subtype variation has
also impacted the effectiveness of BoNT countermeasures [15-17].
J Bioterr Biodef
BoNT E3
BoNT G
-----
37.5
30.4
31.3
37.7
38.7
38.0
-----
30.8
32.6
35.9
37.4
57.1
-----
51.5
30.9
30.9
32.5
-----
31.7
32.7
34.0
-----
62.8
36.4
-----
36.8
-----
Table 2: Percent identity of amino acid residue among BoNT serotypes. The most
conserved serotypes are in bold type.
C. botulinum group II organisms produce nonproteolytic BoNT/B,
/E and /F. Toxin yields and specific activities are much lower than with
the Group I organisms. However, these organisms have the capability to
grow and express toxin at low temperatures (3.3-10˚C) [18-20], making
vacuum-packaged foods stored at refrigerated temperatures vulnerable
if contaminated with botulinum spores.
Group III organisms produce BoNT/C and BoNT/D toxins, which
are not generally considered to be human botulism threats. Information
on toxin yields for these serotypes is not readily available. However,
recent calculations of BoNT/C and /D specific activity per mg of toxin
indicate that the two BoNT/C subtypes were not as potent in mice
as other toxin serotypes, but that BoNT/D subtypes may be the most
potent of all toxin types in mice [21]. Group IV organisms produce
BoNT/G, which is not considered to be a human pathogen. Culture
yields may vary, depending on the strain used, culture conditions, and
number of culture passages [22].
Range
Environmental samples from Argentina [23], Brazil [24], China
[25], Europe [26], Greenland [27], Hawaii [25], Indonesia [28], the US
[29], and Venezuela [30] have all yielded BoNT-producing clostridial
strains. BoNT-producing organisms have been found on every
continent with the exception of Antarctica, including remote places
such as Korea (unpublished – NCBI accession no. EF506573), Mauritius
[31], northern Scotland [6], Tasmania [32], and Tierra del Fuego [33].
With such a widespread geographic range, it should not be difficult to
isolate BoNT producing clostridia from most areas of the earth.
There have been 1,835 cases of naturally occurring botulism in the
US from 1950 to 1996. Of these, 921 were associated with BoNT/A, 818
were associated with BoNT/B, 67 cases were associated with BoNT/E,
and 23 were associated with BoNT/F [34]. Approximately 95% of all
naturally occurring cases in the US can be attributed to either BoNT/A
or BoNT/B. This predominance of serotypes A or B as causative toxins
in botulism appears to be a widespread phenomenon; for example, the
major serotype associated with food borne cases in Argentina [33],
France [35], Poland [36], the UK [37], and the country of Georgia [38]
has been either type A or B. Worldwide, 98% of reported infant botulism
cases have also been associated with either BoNT/A or /B [39].
Only three outbreaks attributed to BoNT/C have been recorded
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worldwide. One case was in California, where type C was isolated from
the gastric contents of a person with fatal botulism [40]; the second
outbreak involved contaminated mushroom paste in France in 1955
[41], and the third was an infant case in Japan [42]. A lone foodborne
outbreak due to BoNT/D was reported in Chad in 1958 [43]. While
BoNT/G has been isolated from autopsy materials of several persons
that had experienced sudden unexplained deaths [44], no evidence was
found that supported botulism due to type G as the cause of death.
The ready availability of BoNT/A and BoNT/B strains worldwide,
coupled with their apparent potency as judged by their association with
botulism cases, make these the most likely two serotypes to be used as
biothreat agents.
There is also the problem of dissemination of standard BoNTproducing type strains that are used as controls in assay development
or assessment of food preparation conditions. For example, the ATCC
C. botulinum type strain, ATCC 27563, has been reported to reside
within strain collections in Australia and New Zealand [45], Brazil [46],
Finland [47], France [48], Iraq (US Dual-use Exports to Iraq and their
Impact on the Health of Persian Gulf War Veterans, Hearing before
the Committee on Banking, Housing, and Urban Affairs, United States
Senate, May 25, 1994), Sweden (Culture Collection of the University
of Goteborg), and the UK [48], as well as the states of Georgia [49],
Maryland [50], Ohio [51], Pennsylvania [52], and Virginia [53] within
the US. Tracing the origin of a possible bioterrorist event involving this
strain would be challenging.
A further concern is the manufacture of BoNTs as therapeutic
agents. A surge in manufacturing facilities that are outside of the US
and Europe might provide access to less secure stocks of BoNTs, which
could be used as biological weapons [54].
Potency
Botulinum neurotoxins are the most potent poisons known. Several
picograms of toxin can be lethal for small animals, and several human
deaths after tasting and spitting out contaminated food have been
recorded [55].
Judging by their predominance in naturally occurring botulism
cases, it would seem that BoNT/A and /B should be more potent than
other toxin serotypes. This appears to be the case, as reports of estimated
oral lethal doses in nonhuman primates have determined the minimum
lethal dose for serotypes A and B to be 7-16 ng/kg, while estimates for
other serotypes range from 50 ng/kg to 14 µg/kg (Table 3) [56].
However, the mode of introduction can be important. Estimated
human lethal doses for BoNT/A have been calculated as 0.09-0.15 µg (per
70 kilogram person) when injected, 0.7-0.9 µg when inhaled, and ~70
µg when ingested [9]. Additionally, while BoNT/C is rarely implicated
as the causative agent in foodborne botulism or toxicoinfections in
humans, it has been reported that BoNT/C, when injected, produces
an effect that is similar to BoNT/A in both intensity and longevity [57].
Thus, while intentional release of BoNT/C into the food supply would
not be very effective, an attack using injected toxin, analogous to the
assassination of the Bulgarian dissident Georgi Markov with ricin [58]
might prove to be very effective.
Economic and Agricultural Terrorism
As we have learned from the Amerithrax incident, the effects of a
bioterrorism incident are not limited to human illness and fatalities.
These incidents can cause severe emotional distress and cost millions
of dollars (it has been estimated that the clean-up costs following the
J Bioterr Biodef
serotype
estimated oral lethal dose in monkeys
BoNT/A
16 ng/kg
BoNT/B
7 ng/kg
BoNT/C
230 ng/kg
BoNT C/D
14 µg/kg
BoNT/E
50 ng/kg
BoNT/F
8 µg/kg
Table 3: The minimum estimated oral lethal dose of BoNT by serotype. Doses
were calculated from information in (Dolman and Murakami 1961) and calculated
mouse i.p. LD50 values obtained in our laboratory.
anthrax incidents were more than $1 billion) [59]. Deliberate exposure
of a civilian population to BoNT, while not having the issues of
continuing infectivity, would have a significant effect on the health care
delivery system. Costs after an aerosol exposure to BoNT in a major city
have been estimated at $1-$8 billion [60,61].
The 2005 publication of a journal article predicting the possible
poisoning of hundreds of thousands of people from a single pointsource contamination of the milk supply with 10 g of BoNT [62] caused
alarm among the scientific community. While these predictions were
later found to be exaggerated [63], deliberate contamination of the milk
supply remains a concern [64].
Accidental contamination of central food stocks for fur-bearing
animals housed on small farms caused the catastrophic loss of over
50,000 animals [65]. Botulism outbreaks in cattle and other domestic
animals may cause the deaths of hundreds of animals over the course
of only a few days. Cattle are particularly susceptible to BoNTs. They
are more than ten times more sensitive to BoNT/C than mice on a
per kilogram weight basis [66]. Botulism outbreaks in cattle have
been increasing over the past decade, which has also raised concern
over possible outbreaks of human botulism due to contaminated dairy
products [67]. Domestic fowl are also targets for botulism; fatality rates
are similar to those seen in cattle [68,69]. Deliberate contamination of
feed with BoNTs could cause heavy economic losses, especially in areas
that are dependent on dairy farming and meat production.
Detection and Identification
The classical method of detection and serotype determination for
BoNT has always been through the use of in vivo mouse neutralization
assays [70,71]. While sensitive, this method requires a large amount
of sample and takes several days, making it somewhat impractical in
the age of rapid diagnosis/detection. Classic ELISA methods that use
colorimetric endpoints have also been used. They are rapid, but not
particularly sensitive [72]. Recently, several alternative methods have
been developed that are rapid, require small sample volumes, and are
equivalent in sensitivity to mouse neutralization assays.
Primary among these are PCR-based assays that are capable of
detecting and amplifying toxin-specific DNA that is associated with
the neurotoxins. Multiple assays have been developed that detect and
serotype BoNTs directly from food matrices and clinical samples [7378]. These assays can be completed in a matter of hours and, with
the advent of multiplexing, tests for multiple subtypes can be run
simultaneously using a single small sample. Limits of detection with
these assays may be as low as 10 gene copies per reaction [77].
In vitro protein-based detection assays are also being developed that
show great sensitivity and specificity. Summaries of various procedures
and their limits of detection can be found in Bagramyan et al. [79] and
Cai et al.[80]. ELISAs that utilize high-affinity antibodies and sensitive
detector systems, such as digoxigenin or chemiluminescent reagents,
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provide assays with sensitivities approaching that of mouse bioassays
(50-400 pg) [81, 82] in different matrices and food samples. However,
the sensitivity of these assays can vary depending on the choice of
capture and/or detector antibodies.
An interesting merger of DNA- and protein-based methods is the
immuno-PCR assay. The assay utilizes a capture antibody that binds
toxin, with a biotinylated antibody as detector. The detector antibody is
linked to a biotinylated reporter DNA fragment via streptavidin. PCR
reagents and specific primers are added to the mix, and the resulting
PCR products are reflective of the amount of toxin present in the
sample. This assay is very sensitive, with reported limits of detection
of 50 fg/ml [83]. A further development on this assay encapsulates the
DNA within liposomes. The liposomes protect the DNA from DNAses
during antibody binding and washing steps; release occurs during real
time PCR. Reported limits of detection with this liposome PCR assay
are a staggering 0.02 fg/ml BoNT/A [84], under ideal conditions using
purified toxin.
Additional sensitive protein assays have been developed that are
based on the enzymatic activity of the BoNTs. Each BoNT cleaves one
of three target SNARE proteins (SNAP-25; synaptotagmin, or VAMP;
and syntaxin) at a unique site. Multiple FRET assays are available that
use fluorogenic reporter reagents containing peptides with cleavage
sites for the toxin to be assayed [85-87]. Intact peptides are in a
quenched state; cleavage results in an increase in fluorescence. As this is
an enzymatic process, each toxin molecule may cleave many peptides,
resulting in enhancement of the fluorescent signal.
Two different assays have adapted this method for testing clinical
and food samples. The ALISSA (assay with a large immunosorbent
surface area) utilizes a two-step procedure. The first step involves the
binding of toxin to antibody-coated beads which are then washed to
remove potential inhibitory substances, and the second step is a FRET
assay [79]. Limits of detection for BoNT/A with this method were 0.5
fg/ml using spiked human serum or liquid food samples.
A second activity-based method is the Endo-PEP assay, which also
involves an initial antibody/toxin-binding and wash step to concentrate
the toxin and remove inhibitory substances. However, enzymatic
activity is detected using mass spectrometry. Peptides containing the
target sites for each of the BoNTs have been manufactured. The sizes (or
masses) of the cleavage products from these peptides, which are unique
for each serotype, are measured using MALDI-TOF mass spectrometry.
The sensitivity and specificity of these activity-based assays make them
appealing, as they are capable of identifying the presence of toxins of
a specific serotype (or multiple serotypes in the case of bivalent toxin
producers) from a variety of food matrices and clinical samples in a
matter of hours with sensitivities that are below mouse bioassays [88].
More definitive identifications, such as strain or isolate level
comparisons, require larger sample sizes and more time, whether
using DNA-based or protein-based assays. Differentiation of BoNTproducing strains has been done using a variety of DNA-based methods
[27,50,89-91]. PFGE methods for strain comparisons are useful but
they require culture of the toxin-producing organisms [92]. AFLP and
some SNP analyses require a source of pure, concentrated DNA, but
MLST, MLVA (VNTR) and other types of SNP analyses can be done
using DNA that is naturally found in conjunction with the neurotoxin.
Toxin subtypes can be differentiated using mass spectrometry
methods; these identifications require nanogram amounts of toxin,
which may require culture of the organism [93,94].
J Bioterr Biodef
In naturally occurring cases of botulism, the causative organism
is often obtained from contaminated food or clinical samples, but this
might prove difficult in a scenario where toxin has been deliberately
released. Thus, the choice of screening assay and characterization
methods might depend heavily on the amount and type of sample
provided.
Treatment and Countermeasures
Current treatment for botulism consists of supportive care and
the administration of antitoxin. Case severity can range from minor
changes in sight or voice [95] to extended periods of near-total paralysis
with loss of respiratory and gastrointestinal function. Mild cases may
require minimal care, but more severe cases could require weeks in
intensive care or special hospital units. Occasional severe cases can
be handled with a minimum of disruption, but an influx of severely
intoxicated patients, such as might be seen after a deliberate release,
would overwhelm the health care systems of most major metropolitan
centers [60].
An investigational heptavalent botulinum antitoxin (HBAT) has
recently been made available under an Investigational New Drug
(IND) protocol for use in the treatment of non-infant botulism [96].
Pretesting for reactogenicity is required, and the product must be
diluted and slowly administered to avoid serious adverse reactions. This
product has been despeciated to reduce its reactogenicity; however, the
resulting Fab and F(ab’)2 fragments are cleared from the system more
rapidly than intact IgG, so that repeat dosing may be necessary [96].
BabyBig (Botulism Immune Globulin Intravenous (Human)), an
FDA approved human antibody product, is used for the treatment of
infant botulism [97]. As this product is made from human antisera, the
adverse side effects associated with administration of equine antitoxin
have been eliminated. The product is indicated for the treatment of
infant cases involving serotypes A and B (product insert, BabyBig,
revised September 2009). It may also be effective against serotypes C,
D, and E, but it is not effective against BoNT/F.
While these products make potent therapeutics, they are expensive
to produce and lot-to-lot variability is common. Human and humancompatible monoclonal and polyclonal anti-BoNT antibodies are
currently being manufactured to replace HBAT equine antibody
preparations and BabyBig [98-102]. Combinations of recombinant
human monoclonal antibodies specifically engineered for potent
protection against multiple toxin serotypes and subtypes (oligoclonal
antibody) have been developed [101,103]. These oligoclonal antibodies
are extremely potent, and they can be inexpensively produced and
custom-mixed, eliminating lot variability problems and adverse events
associated with the infusion of large amounts of heterologous protein.
An anti-BoNT/A product is currently in clinical trials to support
licensure as an FDA-approved product; anti-BoNT/B and /E antibody
products are in advanced development. Because of their high potency,
these products can be administered as a bolus injection in much lower
doses than antitoxin. In addition, the half-life of human antibodies in
humans is much longer than heterologous antibody, so that it is possible
to protect against intoxication for months with a single application.
These characteristics could permit both prophylactic and therapeutic
use of these antibody preparations. Vaccination confers possible lifelong immunity that eliminates the option of treatment with therapeutic
BoNT products. On the other hand, annual or semi-annual injection
of antibodies, which protect for a specified time period only, could be
discontinued if no longer necessary, allowing individuals to benefit
from therapeutic BoNT use.
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A pentavalent toxoid vaccine was manufactured in the 1980s to
protect laboratory workers and others who might be exposed to large
amounts of botulinum neurotoxins. While the vaccine was shown
to be effective against BoNT/A-E [104], it has been declining in
immunogenicity and potency over the past decade. In addition, reports
of injection site-related adverse reactions have been increasing. Because
of these issues and the age of the product, the CDC has discontinued
providing this vaccine and no longer recommends its use [105].
Newer vaccines have been developed that are based on large subunit
fractions of the toxins. Second-generation vaccines based on LC-HN or
HC domains are potent and effective against a variety of toxin subtypes
[17,106,107]. Phase II clinical trials of a recombinant bivalent BoNT
A/B HC to support FDA licensure have recently been completed [108].
Third-generation vaccines that employ nontoxic, catalytically
inactive BoNT holotoxins are currently being developed [16,109,110].
These recombinant proteins have been genetically altered in their
active enzyme sites, rendering them catalytically inactive but fully
immunogenic. As the entire protein is represented, a larger number of
epitopes are presented for immune recognition. This results not only in
greater overall efficacy, but also improved protection against alternative
toxin subtypes [16].
There are currently a large number of disorders that can be
effectively treated using therapeutic BoNT preparations. However,
vaccination can effectively neutralize these toxins, rendering them
ineffective as therapeutics. To avoid this problem, a mucosal vaccine of
limited immunogenicity has been proposed [111].
Development of Alternative Countermeasures
The limitations of current therapies against botulism have prompted
research for alternative therapeutics that can be potentially used to treat
pre- and post-exposure BoNT intoxication. This includes studies on
molecules that antagonize nearly every aspect of BoNT pathogenesis,
such as binding, internalization, translocation, and enzymatic activity
[112-114], and compounds that bind directly to neuronal cell receptors
or receptor mimics [115,116].
Over the past several years, considerable research efforts have been
directed to the discovery and development of small molecular weight
inhibitors (SMWIs) that target the BoNT/A active site, due to the
serotype’s persistence and highly toxic nature. A number of BoNT/A
SMWIs, identified using conventional and novel approaches have been
reported [117-130]; most of these molecules have been discussed in a
recent review [112].
Other regions of the BoNT LC, e.g., exosites (substrate-binding
sites remote from the catalytic pocket) [131], have been also targeted
for inhibitor development. Merino et al. [132] has focused on a family
of bis-imidazole BoNT/A inhibitors that inhibit the endopeptidase
activity via a two-site binding mode. Silhar et al. [133] reported on
D-chicoric acid, a natural product isolated from Echinacea, that binds
to an exosite, and showed a synergistic effect when combined with an
active site inhibitor. Recently, chicoric acid was reported to be a crossover inhibitor of BoNT/B LC [134]. In addition to SMWIs, a llama
antibody has been reported to bind to the α-exosite binding region
of BoNT/A LC [135]. A recent publication details a noncompetitive
inhibitor that appears to block the BoNT/A enzymatic activity by
covalently binding to a cysteine residue (Cys1065, also known as the γ
exosite) adjacent to the active enzyme site [136].
J Bioterr Biodef
Some natural products have also been reported to inhibit BoNT.
Zou et al. [137] described the inhibitory effects of toosendanin, a
triterpenoid derivative from the bark of Melia toosendan, against
BoNT/A, /B and /E in monkeys. The ability of toosendanin and a more
potent tetrahydrofuran analog to selectively inhibit translocation of
BoNT/A and /E LCs with subnanomolar potency has been elucidated
[138]. A function-oriented synthesis (FOS) strategy has been applied to
toosendanin analogs [139,140]. Other reported natural product BoNT
inhibitors include the aqueous extract from stinging nettle (Urtica
dioica) leaf [141] that showed inhibitory effects against BoNT/A LC, and
the thearubigin fraction of black tea (Camellia sinensis) that reduced
the onset of paralysis in mice due to intoxication by BoNT/A, /B and /E
[142]. Lomofungin, a secondary metabolite that was first isolated from
the soil-dwelling gram-positive bacteria Streptomyces lomodensis, was
identified as a BoNT/A LC inhibitor from a high throughput screening
(HTS) of a drug library [143]. Lomofungin displays noncompetitive
inhibition kinetics against BoNT/A LC [133].
Despite intensive efforts on BoNT inhibitor development, to
date, no compounds have yet been identified that exhibit suitable
characteristics (safety, efficacy, solubility) and possess ADMET
(absorption, distribution, metabolism, excretion and toxicity) profiles to
make them effective pharmaceutical interventions. Part of the extreme
difficulty in the BoNT/A inhibitor development can be attributed to the
unusually large peptide substrate-enzyme interface [131] that requires a
small-molecule with high affinity to effectively disrupt protein-protein
interactions, i.e., block substrate binding [126]. Moreover, the BoNT
toxin and its domains show considerable conformational flexibility,
making design of effective inhibitors complicated [128].
Assays and model development are also critical to the BoNT
therapeutic effort. A number of in vitro, cell-based, tissue-based, and
in vivo assays have been developed (reviewed in Hakami et al. [144]).
Though advances in the development of high throughput screening
and cell-free biochemical assays have greatly enhanced the initial
identification and testing of candidate BoNT inhibitors, the noncorrelation between in vitro and in vivo results [127, 145] emphasized
the limitations of conventional drug development archetypes. Greater
research is vital toward the development of in vitro assays that will allow
prompt identification of BoNT inhibitors that are effective both in vitro
and in vivo. An added challenge is the demonstration of the in vivo
efficacy of candidate small molecule inhibitors. Targeting of drugs to the
nerve terminal, ensuring their access to the intracellular compartments
and increasing their bioavailability to match the duration of the toxin
will be aided enormously by the efforts and progress on delivery vehicle
research.
Summary
Botulinum neurotoxins are extremely potent toxins produced by a number of
clostridial species that are found worldwide. Their diversity poses challenges for
detection/diagnosis, treatment, and countermeasures. Deliberate introduction of
BoNTs may be hazardous to both humans and domestic animals, and could cause
widespread economic damage.
Recent developments in detection have produced extremely sensitive and
specific rapid assays that detect and serotype BoNTs, particularly those known
to be human pathogens. In addition, strain- or isolate-level identifications are now
possible.
New antitoxin treatments have been developed. A new equine heptavalent
antitoxin has been approved for use in adults, and a human antibody product
is available for the treatment of infant botulism. Recombinant human/humancompatible antibodies are being produced that effectively neutralize a range
of BoNT serotypes and subtypes. Research to develop drug therapies is also
underway.
Bioterrorism: Toxins
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Citation: Smith TJ, Roxas-Duncan VI, Smith LA (2012) Botulinum Neurotoxins as Biothreat Agents. J Bioterr Biodef S7:003. doi:10.4172/2157-2526.
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This work was partially supported by NIAID IAA 120-B18 and DTRA
projects CBM.VAXBT.03.10.RD.012 and 3.10037_07_RD_PP_B. The opinions,
interpretations and recommendations are those of the authors and do not
necessarily represent those of the US Army.
botulinum associated with bovine botulism in Japan. Vet Microbiol 140: 147154.
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Bioterrorism: Toxins
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Citation: Smith TJ, Roxas-Duncan VI, Smith LA (2012) Botulinum Neurotoxins as Biothreat Agents. J Bioterr Biodef S7:003. doi:10.4172/2157-2526.
S7-003
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