Balancing the benefits and risks of a botulinum toxin vaccine

Botulinum toxin is one of the more extraordinary molecules encountered in the medical sciences. There are a host of reasons for the rather unique status of this molecule, but three are especially well known. First, botulinum toxin is generally viewed as the most potent of all biological substances [1]. Second, the toxin must proceed through a long and elaborate sequence of events to produce its pathologic effects [2]; and third, botulinum toxin is encountered in an unusually wide breadth of clinical settings, some of which are detrimental to patients and others are beneficial [3]. It is this multifaceted clinical presentation of the toxin that creates a challenge for investigators interested in vaccine development.

Botulinum toxin is the etiologic agent that causes the disease botulism [4,5]. This disease presents as muscle weakness or paralysis, which may be accompanied by an array of autonomic problems. In extreme cases, patients experience paralysis of the muscles of respiration and this may require weeks or even months of respiratory intensive care management.

Botulism is a naturally occurring disease but its incidence is so low that it has fostered no perceived need to develop a vaccine. Unfortunately, botulism can also occur under ‘unnatural’ circumstances. Owing mainly to its extraordinary potency, the toxin is viewed as a potential bioweapon [1]. As such, it could be used to attack either civilian (bioterrorism) or military (biological warfare) populations. In either mode, the toxin could be disseminated in food or air to produce mass illness or death. This troubling prospect has fostered intense efforts to develop a vaccine.

In striking contrast to its ability to produce natural or unnatural disease, botulinum toxin is also a medicinal agent that is being evaluated in an ever expanding number of neurologic and other disorders [3]. The toxin was originally introduced as a therapeutic agent for relief of neuro–opthalmologic problems, such as blepharospasm and strabismus. It has subsequently been tested for problems as diverse as hemifacial spasm, cervical dystonia, laryngeal dystonia, spasticity, tics and tremor. There are other disorders for which the toxin also appears to be efficacious, such as certain types of pain [6,7], particularly, headache pain [8].

The potential role of botulinum toxin as a weapon of bioterrorism and biological warfare, when viewed beside its emerging role as a therapeutic agent for treatment of an ever expanding number of clinical problems, brings to light an obvious and troubling quandary. Any medical countermeasure that can prevent the actions of botulinum toxin as a bioweapon would also diminish or abolish its actions as a therapeutic agent.

There is relatively little experimental work that specifically addresses ways to balance the desired versus the undesired consequences of vaccination. However, an examination of the literature that describes the mechanism of botulinum toxin action, when combined with the literature on the mechanism of antibody-induced protection against the toxin, may suggest experimental strategies worth pursuing. Fortunately, there is a substantial body of information in both of these areas.

In terms of the mechanism of action, there is now a reasonably clear picture of the steps through which the toxin must proceed to produce poisoning [2]. The majority of documented cases of botulism are due to oral exposure, so oral poisoning can be viewed as a model. Botulinum toxin is a dichain molecule (heavy chain: 100,000 Da; light chain: 50,000 Da) that is produced in bacteria as part of a noncovalent complex of several proteins, including three hemagglutinins (HA) and a so-called nontoxin nonhemagglutinin (NTNH). The role of the complex is to protect the toxin from metabolism in the stomach, thus ensuring delivery of the toxin to its site of absorption, which is the small intestine.

The process of absorption begins when the heavy chain of the toxin binds to receptors on the apical surface of gut epithelial cells [9,10]. The identity of these receptors is unknown, but their functional role has been established. They participate in receptor-mediated endocytosis that delivers the toxin to transport endosomes. The toxin is then carried across the epithelial cells and released into the general circulation.

There is only one site in the body where the toxin binds with high affinity and this is the membrane of cholinergic nerve endings [2,11]. The toxin binds exploitatively to receptors on the cell surface, after which, it is internalized by receptor-mediated endocytosis. In neuronal cells, unlike epithelial cells, the toxin escapes endosomes to reach the cytosol. The light chain of the toxin acts locally to cleave polypeptides that are essential for acetylcholine release. In the absence of transmitter release, patients experience muscle weakness or paralysis. When there is involvement of respiratory muscles, paralysis of transmission is potentially life threatening.

Remarkably, botulinum toxin is also a therapeutic agent. Its most-documented role is in the relief of problems characterized by excessive and involuntary efferent activity in motor nerves, causing excessive or uncontrolled muscle movement [3,12]. Physicians can treat these problems by injecting the toxin directly into the vicinity of overactive cholinergic nerve endings. The mechanism by which the toxin enters nerves and blocks transmitter release is the same as that when the toxin causes disease. The difference between disease and treatment is in the route taken by the toxin. In disease, the toxin is absorbed into the general circulation and can potentially be distributed to all peripheral cholinergic nerves, including those involved in respiration. In therapy, the toxin is administered in the vicinity of overactive nerves. Thus, it can diminish transmitter release in these nerves while not affecting other nerves in the body.

As explained earlier, botulinum toxin is a potential bioweapon that can be used against civilian and military populations. The threat posed by the toxin as a bioweapon has intensified efforts to develop a modern vaccine. Most of this effort has focused on expression of nontoxic recombinant polypeptides that represent functional subunits within the holotoxin. The recombinant molecule that appears most likely to gain regulatory approval is a 50,000 Da polypeptide that represents the C-terminal half of the heavy chain [13,14]. This polypeptide was initially selected because it is known to be the most immunogenic portion of the toxin molecule. However, it has subsequently been discovered to retain the ability to bind and penetrate gut and airway epithelial barriers [15]. Thus, this polypeptide is not merely an immunogen; it is also a carrier device that allows the immunogen to be administered as a mucosal vaccine.

As part of the effort to develop modern vaccines against the botulinum toxin, investigators have also sought to localize the sites and identify the mechanisms by which antibodies can neutralize it. The underlying motive is to ensure that any candidate vaccine is capable of evoking as many layers of protection as possible. To date, there are three major layers of protection that have been characterized when the carboxyterminal portion of the heavy chain is administered as a mucosal antigen [16].

First, the vaccine evokes a local immune response at mucosal surfaces, including the lumen of the gut and airways. Antibodies (IgA) that are secreted at these surfaces coat the toxin in a way that blocks binding and transcytosis across epithelial barriers. This prevents the toxin from being absorbed into the general circulation.

Second, the vaccine triggers a systemic immune response (both IgG and IgA). Antibodies bind to toxin in the circulation, marking it for clearance by the liver and spleen. This in turn means that there is a dramatic reduction in the levels of free toxin available for distribution to vulnerable nerve endings.

Third, a fraction of the circulating antibodies recognize epitopes that are in, or near, the portion of the toxin molecule that binds to receptors. When antibodies decorate this region, they prevent the toxin from paralyzing neuromuscular transmission.

A knowledge of the sequence of events through which the toxin progresses to produce its pathologic effects, plus a knowledge of the sites at which antibodies can act to neutralize the toxin, suggest testable ways of addressing the quandary inherent in administration of a botulinum vaccine. At least three of these potential solutions are worthy of consideration.

An unobvious vaccine

Botulinum toxin is released from bacteria as part of a noncovalent complex with other proteins. All three serotypes typically associated with human illness (A, B and E) possess NTNH; two of these serotypes (A and B) also possess a family of HAs. The role of the auxiliary proteins is to protect the toxin from the harsh conditions of low pH and proteolytic enzymes in the gut.

If the complex of toxin and auxiliary protein does indeed remain intact until (or even after) binding to the epithelium, this would raise an interesting possibility for vaccination. Rather than using a subunit of the toxin as an antigen, one could use the auxiliary proteins as mucosal antigens. The bulky structure that arises when multiple antibodies become associated with the complex should prevent binding to epithelial cells. In essence, these antibodies would be expected to produce the same outcome as that obtained when the C-terminal half of the toxin heavy chain is used as antigen. Mucosal IgA would interact with antigen in a way that blocks absorption and, therefore, blocks poisoning.

This potentially attractive approach has received almost no serious attention and in the one instance in which the idea was tested, the results were not impressive. Mahmut et al. reported an attempt to achieve mucosal immunization with auxiliary proteins but the results indicated that the level of resistance to challenge with toxin was low [17]. It is not clear why the level of immunoresistance was poor, but the putative value of this approach suggests that further evaluation of the idea is warranted. Intuitively, the approach should work, especially if the complex remains intact in the gut. However, if the complex comes apart before binding to epithelial barriers, the strategy of using an unobvious vaccine would be of limited value.

An obvious vaccine with an unobvious mechanism

Most current approaches to the development of a botulinum vaccine have focused on recombinant polypeptides and, particularly, the C-terminal half of the heavy chain. The various mechanisms by which antibodies against this polypeptide produce neutralization have been described [16]. The first of the several mechanisms occurs in the lumen of the gut or airway, where the antibodies associate with the toxin and prevent its binding to epithelial cells. In the absence of binding, the toxin cannot be absorbed.

When the C-terminal polypeptide is administered as a vaccine, its actions are not limited to mucosal surfaces. Instead, this polypeptide can evoke systemic antibodies that:

•	Enhance clearance of toxin from the general circulation
•	Block toxin binding to the neuromuscular junction

The latter would mitigate the therapeutic benefits of the toxin.

At least conceptually, there may be a unobvious way to use this recombinant antigen in a way that will prevent poisoning, as in an act of bioterrorism, yet preserve clinical benefits, as in the treatment of dystonia. A host of endogenous factors have been described that can promote certain types of immune responses while diminishing others. For example, signal molecules have been identified that can stimulate a mucosal IgA response, and other signal molecules have been shown to inhibit a circulating IgG response. A combination of these signal molecules could lead to an immune outcome that is localized to the mucosal surfaces where the toxin is normally absorbed into the body [18].

One embodiment of this strategy would be to use an adenovirus delivery system that can introduce both the message for the antigen, as well as the message(s) for signal molecules that would elicit the desired immune response. The use of adenovirus to deliver the message for the C-terminal half of the heavy chain has just been described and this approach did elicit immunity to the parent toxin [19]. Efforts to deliver the antigen message by the mucosal route and to combine this with signal messages that will determine the nature of the immune response are currently underway.

An obvious vaccine with an obvious mechanism of action, but an unobvious duration of action

Seeking to limit an immune response to mucosal surfaces where the toxin is absorbed may prove to be a daunting task. Even if this can be accomplished in the long term, there may not be an adequate conceptual and empirical framework for meaningful progress in the near term. Unfortunately, it is not plausible to delay efforts at vaccine development until there is a perfect solution. Efforts to protect those who may be vulnerable to bioterrorism or biological warfare must progress in a timely manner. This suggests that it would be prudent to consider ways to diminish the impact that vaccination has on the therapeutic utility of the toxin, without necessarily abolishing that impact. The idea of evoking an immune response that is limited in its magnitude does not appear to be wise. Botulinum toxin is so potent that there can be little doubt about the need to evoke a robust magnitude of immunity. On the other hand, the idea of evoking an immune response that is limited in duration may be an acceptable alternative.

One of the basic tenets of vaccinology is that, in most instances, the longer the duration of an immune response the better. The tetanus vaccine, which is the most widely administered vaccine in the world, is an excellent example of this. There is universal recognition that it is desirable to afford patients lifelong immunity against tetanus toxin. There is no licensed vaccine that can achieve this goal, so patients are typically advised to maintain immunity by receiving boosters at regular intervals, which, for most individuals, would be of 10 years. This practice will continue until a clinical product is available that reliably affords longer protection. This is in recognition of the fact that tetanus is a constant and lifelong threat.

Botulism is, in many respects, a counterpoint to tetanus. It would be hyperbole to claim that this disease is a constant and lifelong threat to the entire population. With the exception of certain individuals, such as laboratory workers who do research on the toxin, the risk of botulism would have to be described as low and/or episodic. For instance, members of the military assigned to theaters where there is an authentic threat of biological warfare need a high level of immunity, but this need is not lifelong. Typical tours of duty in such theaters are 2–3 years or less. Thus, the real need is for episodic protection.

The mentality of those who are involved in vaccine development is so geared to promoting lengthy duration of action that the idea of promoting a term duration may seem disconcerting. Even so, this may be the most rational near-term solution to a vaccine that can be beneficial at times and detrimental at others. Fortunately, there is a way to make this approach palatable, even to those who may need extended protection. This approach involves two components:

•	An initial vaccination procedure to evoke an immune response of respectable magnitude but limited duration;
•	A booster procedure that is easy to administer (namely, a single oral dose) and that re-evokes something similar to the original immune response.

Considerable progress has already been made in achieving the latter of these two goals. A trivalent vaccine (serotypes A, B and E) that is active by the inhalation route has already been described [16]. The authors of this study are now engaged in efforts to develop an oral formulation. The prospects of success appear high because the antigen used in the inhalation and oral work contains the minimum essential domain from the toxin molecule that mediates binding and transport across gut and airway epithelial cells. Thus, it seems likely that this antigen will enter the body to evoke a systematic immune response. This would be in addition to its ability to act locally to evoke a mucosal immune response that can block toxin absorption.

The more vexing issue is duration of immunity and, more precisely, the mechanism for limiting duration. It may be that some innovative thinking will be required to cope with this issue; or alternatively, it may prove to be surprisingly simple. Virtually all work on botulinum vaccines entails the use of adjuvants. For example, both laboratory and human research on parental formulations involves the use of alum. This material is thought to play multiple roles, from acting as a backbone in microscopic depots to acting as a stimulant of immune cascades. Obviously, it is the latter that would be troublesome when the goal is to limit duration of action. This raises an interesting question: would administration of sufficient amounts of naked antigen evoke an immune response that is robust but limited? Given the immense body of data that have accumulated over decades of research on antigens with and without adjuvant, an investigator would not be unreasonable in hypothesizing that a naked antigen formulation could at least approximate the intended outcome of producing immunity of limited duration. No one can know with certainly how such experiments will turn out until they are performed. However, one can know with certainty that the experiments to test the hypothesis are straightforward and should be performed.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.