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Kevin Song

Dr. Milana Trounce

Publpol 222: Biosecurity and Bioterrorism Response

5 March 2017

Rational Design of Anti-Botulinum Neurotoxin Oral Vaccines

ABSTRACT

It has been well-documented that botulinum neurotoxin (BoNT) is one of mankinds most

potent weaponizable neurotoxins. However, compared to conventional terrorism/bioterrorism

agents (e.g., weaponized anthrax and nuclear weapons in the hands of non-state actors), BoNT is

noted for its relatively remarkable ease of synthesis and obtainment through legal and illicit

supply chains. Here, we argue for the need for development of an anti-BoNT oral vaccine, and

present a research methodology that would lead to the rational design of such a vaccine.

Key words: botulinum, botulinum neurotoxin, BoNT, oral vaccine, vaccine development,
bioterrorism

INTRODUCTION

A terrorist weapons potential to inflict casualties is typically positively correlated with

the difficulty with which it can be obtained or produced. Whereas nuclear weapons are capable

of inflicting millions of casualties, their development typically requires funding by nation-states,

is difficult to hide, and often requires extremely sophisticated technology (e.g., uranium

centrifuges). On the other hand, guns are readily available through legal and illicit means in

many developed countries, though they are capable of inflicting few casualties by comparison.

Coleman, et al. have previously devised a quantitative matrix to assess various terrorist

threats that encompass biological, chemical, nuclear, cyber, and conventional (e.g., guns and

IEDs) means. Their published matrix1 reveals that in general, agents with greater casualty
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potential (e.g., nuclear weapons and weaponized smallpox) are more difficult to procure than

lower-casualty agents. Nevertheless, the matrix indicated that a few agents, most notably

botulinum neurotoxin (BoNT), were capable of inflicting mass casualties (even equivalent to

those caused by weaponized smallpox), while being relatively easy to produce and procure. In

their discussion, Coleman et al. mention that it remains imperative for biosecurity authorities to

restrict the proliferation of such Quadrant I threats as BoNT, which constitute high-impact and

high-probability threats that could easily be weaponized to harm a nations population (e.g., by

contaminating the milk supply with just a few milligrams of BoNT).1

BoNT stands out among various other terrorist agents not only due to its lethal potency

and relative ease of synthesis/procurement, but also due to its widespread usage and global

demand for its consumption in illicit and sanctioned medical/cosmetic procedures. Apart from

the seven currently licensed producers of Botox, there are many more non-licensed, illicit

producers of botulinum toxin who currently supply the global legal and black markets. Coleman,

et al. have noted that in the recent decades, advances in biotechnology have eased BoNTs

difficulty of production by suppliers, while online distribution services have eased BoNTs

difficult of procurement by consumers and potential bioterrorists.1

Arguments for an oral anti-BoNT vaccine

An anti-BoNT vaccine could potentially eliminate and absolve BoNT as a critical and

urgent biosecurity threat. Such a vaccine could ideally initially produce antibodies in human

subjects that ligate to epitopes found on functional regions of active BoNT protein. In the case of

a subsequent BoNT attack, a vaccinated subjects body, due to immunological memory, would

immediately and autonomously produce the needed antibodies to functionally silence the BoNT

neurotoxin.
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The current treatment model of botulism requires administration of antibody antitoxins

following development of post-exposure symptoms. However, for both first-responders and the

general population alike, vaccinated individuals would not require such delayed treatment by

medical staff, as their bodies could ideally produce enough anti-BoNT antibodies to neutralize

the toxin on their own.

If an anti-BoNT is successfully developed and tested for toxicity, there are few negative

consequences that could arise from its administration to first-responders and medical staff (if not

the general population at large). In such a scenario, the Botox market would most likely suffer

or collapse, as illicit/legal and weaponized/sanctioned BoNT would be rendered ineffective for

vaccinated populations. On the flipside, basic research into the structural biology of BoNT

during vaccine development could expedite the development of

pharmaceutical/biopharmaceutical-based BoNT alternatives that exhibit less toxicity than BoNT

itself.

The reasons for choosing an oral formulation of a potential anti-BoNT vaccine are

multifaceted. Tablet-based oral vaccines, such as those produced by biotechnology company

Vaxart, do not require refrigeration, and thus can be easily stockpiled and self-administered by

individuals with no medical training.2 Furthermore, the Vaxart vaccine platform (which this

proposal will be partially based on) has the ability of producing vaccines for arguably any known

recombinant antigen. In addition, along with the fact that botulism typically occurs through

consumption of spoiled food, it has been shown in Vaxart clinical trials3 that gut-associated

lymphoid response to oral vaccines is greater than systemic response to conventional vaccines.
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RESEARCH METHODOLOGY

Mechanism of action of BoNT

BoNT is produced and exported by the anaerobic bacterium Clostridium botulinum. It

comes in seven known serotypes: A, B, C1, D, E, F, G, whereas human illness is only caused by

types A, B, E, and (rarely) F.4,5 BoNT is exported by C. botulinum as a single-chain 150 kDa

protein that is later cleaved by tissue proteases to form a 100 kDa heavy chain and a 50 kDa light

chain joined by a disulfide bond.5

BoNT is hypothesized to induce muscular paralysis by binding to presynaptic cholinergic

recognition sites and blocking the release of acetylcholine-containing vesicles.5 The light chain

of BoNT functions as a protease near its N-terminal end, whereas the heavy chain is responsible

for the toxins specificity for binding to presynaptic cholinergic receptors.5

In serotypes A and E, BoNT functions as a protease of SNAP-25s, which are needed for

vesicular fusion of acetylcholine-containing vesicles to the presynaptic membrane.5 In serotypes

B and F, BoNT is a protease of vesicle-associated membrane proteins (VAMPs, or

synaptobrevins) that are responsible for analogous fusion of acetylcholine-containing presynaptic

vesicles.5

Approach: identify and block proteolytically active binding sites of BoNT light chains

In the development of an anti-BoNT vaccine, one must realize that different BoNT

serotypes may require distinct vaccine formulations (or an overall combinative vaccine). Here

we, assume focus on the development of a vaccine for serotype A.

The logic of this vaccine development process is such: BoNT light chain without a

functional epitope is non-functional, whereas a partial BoNT construct that contains the intact

epitope and is minimally functional would be an ideal candidate for expression by oral vaccine.
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The first step in vaccine development is to identify the protein epitopes of BoNT light

chain that confer BoNT active protease functionality. For BoNT-A, these would be the protein

epitopes on the light chain that allow active BoNT to cleave SNAP-25 proteins. Once such

epitopes are identified and verified for necessarily conferring protease functionality, an

analogous protein that features the epitope (but not the heavy chain or the entirety of the light

chain), yet is minimally functional so as to reduce side effects, can be designed for expression by

a packaged, non-self-replicating, adenovirus-related oral vaccine.

To do this, one may create truncated versions of BoNT light chain, as well as constructs

for which amino acid substitutions have been introduced. Specifically, ProteinCCD6

bioinformatics software can be used to direct oligonucleotide design for truncated protein

synthesis by recombinant C. botulinum. After synthesizing and purifying these constructs from

genetically modified, recombinant C. botulinum, one may conduct a simple assay in which

individual construct types are incubated with SNARE-25s. In this assay, one may subsequently

perform a Western blot to later assess whether cleavage of SNARE-25 by the recombinant light

chain constructs occurred.

The goal of these first steps is to identify as many potential light chain epitopes as

possible that are necessary for facilitating SNARE-25 proteolysis. Once the described

experiment has been conducted, one may then use known protein sequences and structural

determination via crystallography and related methods to backtrack and determine approximately

which regions of the light chain are needed, such that if they are deleted or mutated, would

negate protease activity.

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After identification of such potential epitopes, one may design secondary light chain

protein constructs that contain the intact, properly folded versions of one or more of these

epitopes, yet contain as little of the remaining light chain as possible. Afterwards, one could

synthesize, purify, and test these secondary constructs using the above assay to confirm that they

have minimal SNARE-25 proteolytic activity. Then, one could have a third party produce

monoclonal antibodies against each of these secondary products. Each of these monoclonal

antibody varieties could be tagged, and tested for ligation to intact BoNT light chain in a wash-

down assay, and the secondary constructs corresponding to properly ligating antibody varieties

would then be candidates for oral vaccine packaging (e.g., by the Vaxart platform which uses

dsDNA and a gene sequence of interest packaged by a non-self-replicating adenovirus-related

vector).7

An important caveat exists, however, throughout the entirety of this specific vaccine

development process: these strategies may not be fruitful if desired functional epitopes of interest

do not happen to lie on the surface of BoNT light chain.

APPLICATIONS AND CONCLUSIONS

We have shown that the design of an anti-BoNT oral vaccine is not infeasible, and such a

vaccine may potentially rid society of BoNTs urgency and need to be addressed as one of the

worlds most deadly bioterrorist threats. The research methodology we present above

incorporates a simple Western blot-based cleavage assay, as well as a simple tagged-antibody

protein binding assay. Most of this developmental processs challenges arise from the structural

biological problems posed by identifying epitopes necessary for protease ability, as well as

designing secondary protein structures that bear these intact epitopes while also exhibiting

functionality and minimal potential side effects.

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If such a vaccine were to be developed, following clinical review, it would ideally be

dosed to all first-responders and relevant medical staff (e.g., ED workers and paramedics). As

such a vaccine would be orally formulated, it could then be widely distributed and stockpiled in

times of need, or administered as a required prophylactic vaccination for major populations.

In addition, using knowledge of functional BoNT light chain epitopes, one may

potentially investigate future drug targets and pharmaceuticals/biopharmaceuticals that can

mimic BoNTs mechanism of action, but with remarkably less lethality and potency, for the sake

of producing viable Botox alternatives (also likely to the detriment of the botulinum toxin

black market).
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REFERENCES

1. Coleman K, Ishisoko N, Trounce M, and Bernard K. Hitting a Moving Target: A

Strategic Tool for Analyzing Terrorist Threats. Health Security. December 2016, 14(6):
409-418. doi:10.1089/hs.2016.0062.
2. Vaxart :: Technology :: Advantages. Vaxart, Inc; c2016.
http://vaxart.com/TechAdvantages.html
3. Vaxart, Inc.; unpublished.
4. Wheeler A and Smith HS Botulinum toxins: Mechanisms of action, antinociception and
clinical applications. Toxicology. April 2013, 306: 124-146. doi:
10.1016/j.tox.2013.02.006
5. Kedlaya D. Botulinum toxin: Overview, History, Mechanism of Action. WebMD LLC:
c2016. http://emedicine.medscape.com/article/325451-overview
6. Mooij Wijnand TM, Mitsiki E, and Perrakis A. ProteinCCD: enabling the design of
protein truncation constructs for expression and crystallization experiments. Nucleic
Acids Research. April 2009, 37: W402-W405. doi:10.1093/nar/gkp256.
7. Vaxart: Platform Science. Vaxart, Inc; c2016.
http://vaxart.com/TechPlatformScience.html