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MICROBIOLOGY COMMENT |
Department of Biochemistry and Molecular Biology and Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA
Correspondence
M. Zouhair Atassi
(matassi{at}bcm.edu)
In a recent article in this journal, Lee et al. (2005)
reported that immunization of mice with formaldehyde-treated botulinum neurotoxin B (BoNT/B) in the presence of haemagglutinin subcomponents HA1 and HA3b, normally present in the 16S toxin, produced higher levels of anti-BoNT/B antibodies (Abs) than immunization with BoNT/B alone. Because of the paper's potential clinical therapeutic implications, this comment discusses three major issues in the paper: quality of the antigen, dose and frequency of immunization. I show that these were quite different from those used in therapeutic applications of active BoNT/B (or any BoNT) complex.
BoNTs (mostly types A and B) are used to treat a variety of clinical conditions associated with involuntary muscle spasm and contractions as well as in cosmetic and other therapeutic applications (Jankovic, 2002, 2004; Atassi & Oshima, 1999; Brashear et al., 2004; Dressler & Bigalke, 2005). The therapeutic benefits are of limited duration and toxin injections need to be repeated every 3–6 months. In a very small percentage of patients (less with toxin A than with toxin B) the treatment elicits blocking Ab responses against the correlate toxin, which reduce or completely terminate the patient's responsiveness to further treatment (Göschel et al., 1997; Atassi & Oshima, 1999; Atassi, 2002, 2004; Jankovic, 2002, 2004; Jankovic et al., 2005; Dressler & Bigalke, 2005). It would be highly desirable to eliminate or substantially reduce the appearance of blocking antitoxin Abs in therapy of dystonia and other patients who become unresponsive to treatment.
In a recent article in this journal, Lee et al. (2005), following-up their earlier work on a neurotoxin product free of accessory proteins (Arimitsu et al., 2003), set out to determine if the haemagglutinins (HAs) in the products contributed to enhancement of the immune response. They noted that ‘type A and B PTXs (progenitor toxins) have been used for treating patients with many forms of dystonia. In both toxin types, PTXs are used because they are more stable than NTXs (neurotoxins). The treatment is very effective, but has serious side effects for some patients in whom anti-PTX, including anti-NTX Abs, is produced after several injections. ' They also reported ‘Although we have no quantitative data, there is some evidence that high levels of anti-NTX Ab are produced by immunization of rabbits with PTX alone compared with NTX alone.’ Thus, the purpose of the work was to determine if immunization of mice with BoNT/B in the presence of two haemagglutinin subcomponents, HA1 and HA3b, normally present in the 16S toxin, produced higher levels of anti-BoNT/B Abs than immunization with BoNT/B alone. In the Methods section the authors inform the reader that the ‘immunization schedule was based on the assumed treatment of patients with dystonia, i.e. recurrent injection of a low dose of antigen (toxin)’.
The reader could, therefore, reasonably conclude that the research was designed to understand the immunological factors responsible for neutralizing Ab formation in patients and to investigate the possibility of decreasing the antigenicity of BoNT. In view of the value of BoNT/A and BoNT/B in various therapies, including dystonias, an effective strategy that could extend their usefulness to immunoresistant patients would be most desirable.
Several factors can also influence the immune response to the toxin. These include the quality of the antigen and its dose, duration of treatment and frequency of immunization (Atassi, 2002, 2004). It has been well established that the immune responses to a protein are determined to a great extent by its form and by the presence of other proteins in the immunogen (Atassi, 2002, 2004). Furthermore, the immune response to the whole toxin is under genetic control, and the response to each epitope is under separate genetic control. The appearance of blocking Abs in treatment might be controlled by the major histocompatibility complex of the host (Atassi & Oshima, 1999; Atassi, 2002, 2004).
I wish to address here the main issues relevant to the paper of Lee et al. (2005): antigen quality, dose and frequency of immunization.
Antigen
Lee et al., (2005) treated the three forms of toxins, BoNT/B, and the 12S and 16S progenitor toxins (associated with non-toxic components) with formaldehyde to render them non-toxic. They were reportedly dialysed for 7 days at 37 °C against 0.10 M sodium phosphate buffer at pH 8.0, containing 0.6 % formaldehyde. Lee and colleagues inactivated the toxin in their work to be able to administer the relatively massive toxin doses that would have otherwise been lethal had the toxin not been inactivated. However, the consequences of formaldehyde treatment on these proteins preparations need to be considered.
Formaldehyde adds to the amino groups instantaneously, even at neutral pH and low temperature, to form highly electrophilic immonium cations (
), which could be reversible under mild conditions. However, in a prolonged reaction period (7 days) at a relatively high temperature and pH (37 °C and pH 8.0) the immonium intermediate would predominantly react with amino acid side chains within favourable distance to form stable methylene bridges (Means & Feeney, 1971). Aldehyde treatment under such conditions irreversibly cross-links amino groups to phenol (i.e. tyrosine), imidazole (i.e. histidine) and indole (i.e. tryptophan) side chains (Atassi, 1977). Formaldehyde would also react with thiol groups and even with the guanidinium side chain of arginine (Means & Feeney, 1971). The high non-selective reactivity of formaldehyde makes it totally unsuitable for employment in immunochemical studies of proteins (Atassi, 1977).
It has been well documented that treatment of proteins with formaldehyde gives products with enhanced antigenicity. For example, cytochrome c is a low-molecular-mass protein that is a very weak immunogen. The action of aldehyde causes the formation of intra- and intermolecular cross-links and produces oligomeric species of high molecular mass that exhibit strong immunogenic properties (Reichlin et al., 1970; Jemmerson & Margoliash, 1978, 1979). In the case of the study by Lee et al. (2005), treatment of the neurotoxin with formaldehyde at a relatively high temperature and pH (37 °C and pH 8.0) for a prolonged period of time (7 days) is bound to cause the formation of a heterogeneous mixture of oligomers of varying sizes due to intermolecular cross-links. Then to make matters even worse, the authors mixed these products with HA1, HA2 or HA3b without any attempt to remove the excess formaldehyde from the solution of the toxin preparation. Treatment of the 12S and the 16S progenitor toxin preparations with formaldehyde will be expected to generate complex homo- and hetero-oligomers and polymers in which the neurotoxin is covalently cross-linked to itself and/or to the non-toxic components. This means that the neurotoxin-HA complex is no longer formed by protein–protein association, but rather by covalent cross-links within and among the correlate proteins. This would lead to the obliteration of some epitopes and the creation of new epitopes that are not present on the corresponding native proteins. The authors made no effort to isolate and identify any products of this crude treatment.
It is important to point out that patients are not treated with formaldehyde-treated toxin preparations, but with an active toxin B (or toxin A) complex preparation that is not detoxified with any chemical agent and is as close as is possible to a native preparation.
For a control, Lee et al. (2005) injected human serum albumin (HSA) by itself or as a mixture with HA1, HA2 or HA3b and found that each of these mixtures gave higher anti-albumin Abs than immunization with HSA alone (Lee et al., 2005). This might be an acceptable control were it not for the fact that serum albumin is well known to be an insatiable binder of a plethora of proteins and small molecules (for examples of the vast literature, see: Lin et al., 2004; Coyle et al., 2006; Komatsu et al., 2005; Rozak et al., 2005; Chen et al., 2006) and readily undergoes aggregation under a variety of conditions and interactions (again, for examples of the vast literature, see: Vaiana et al., 2004; Militello et al., 2004; Oliva et al., 2003). Serum albumin also acts as a molecular chaperone and can be included in the extracellular chaperone family (Marini et al., 2005). The interactions of HSA with HA subcomponents of C. botulinum have not been studied, but with the known properties of HSA, it is not unreasonable to expect that it would associate with HA and/or form aggregates in the presence of these subcomponents. In either case, aggregates of HSA or HA-HSA species would be expected to evoke higher levels of anti-HSA Abs than monomeric HSA. For these reasons, HSA is not an appropriate control for these studies.
It is necessary to point out here other major advantages for the therapeutic application of the BoNT complex over pure neurotoxin. The non-toxin components present in the complex confer on the toxin protection against degradation and rapid clearance in vivo during therapeutic application, and its larger size helps in better targeting and keeping more of its activity in the afflicted, injected muscle by reducing its rate of diffusion and hence its spread to distant unaffected muscles (Borodic et al., 2001; Callaway et al., 2002).
Dose
Lee et al. (2005) injected detoxified BoNT/B at a concentration of 2 µg in 0.25 ml, mixed with NTNH-HA or different HA subcomponents (total 2 µg in 0.25 ml). The mean weight of 6- to 8-week-old mice is about 23 g. So, on the basis of body weight, the amount injected would be equivalent, in a 70 kg person, to a dose of 6.087 mg per injection.
In treatment of dystonia patients with Myobloc (a BoNT/B complex; Solstice Neurosciences Inc.), the recommended dose range is 2500–10 000 U, which corresponds to 25–100 ng per treatment session (Brashear et al., 1999; Myobloc package insert rev. 11/04). This means that the 2 µg per mouse injected by Lee et al. (2005) is about 105 times higher than the dose range given to the patient.
The mean dystonia dose of Botox (a BoNT/A complex; Allergan Inc.) in the USA is approximately 236 U or 11.8 ng BoNT/A complex per session (Aoki, 2002; Botox package insert 71390US12.J). So the dose used by Lee et al. (2005) was 5.16x105 higher than is used in dystonia therapeutic applications. In a recent study of 929 dystonia and other patients (Yablon et al., 2005) using a current BoNT/A formulation (Botox), dystonia patients (n=326) were given 1–15 treatments of BoNT/A (mean per treatment: 187.3 U=9.365 ng; range per treatment: 20–500 U=1–25 ng; maximum total per patient: 4210 U=210.5 ng). Other patients received similar dose ranges. It was reported that neutralizing Ab appeared in 5 (less than 0.6 %) of the 929 patients.
The mean recommended dose of Dysport (a BoNT/A-complex product; Ipsen Ltd) for treatment of dystonia is about 500 units. Currently, the reported BoNT/A complex amount is 12.5 ng per 500-unit vial. Therefore, on normalized weights the dose administered by Lee et al. (2005) was 4.87x105 higher than that used in dystonia patients.
Both the Ab and T cell immune responses to a protein antigen are dose-dependent (Young & Atassi, 1982; Atassi, 1984, 2004). In the same manner, there is an optimum dose of BoNT for a maximum response by the host (Atassi et al., 1996; Rosenberg et al., 1997; Poewe et al., 1998). In BALB/c, the optimum dose of inactivated toxin has been reported to be 1 µg per mouse (Rosenberg et al., 1997). The dose of 2 µg per mouse used by Lee et al. (2005) was close to the optimal range for the Ab response. However, it is evident that the doses used in patient therapy are extremely suboptimal. Thus, the Ab response will increase with an increase in antigen dose and frequency of immunization. Therefore, increasing the BoNT dose by 105-fold would exert an enormous boost to the Ab titre. Studies of dystonia patients have shown that the Ab response is dependent on the toxin dose (Göschel et al., 1997).
On another level, the injection of humans with a dose equivalent to 2 µg active neurotoxin per mouse would be perilous. It is equivalent to about 3x105-fold LD100 values (based on mouse LD100 values in our hands of BoNT/B=6.25 pg per mouse and BoNT/A=6.5 pg per mouse). Finally, it should be noted that that antigen dose could even influence the genetic control of the immune response (Young & Atassi, 1982).
Frequency of immunization
Lee et al. (2005) injected the mice once a week for 6 weeks. Dystonia patients receive a neurotoxin-complex injection once every 3–6 months, with variations being dependent on the patient. It is well established that the immune response is influenced by the frequency of immunization (Atassi, 2004). As with all antigens, the appearance of Ab responses against BoNT is closely linked to duration of treatment and frequency of immunization. The Ab response will increase with the increase in antigen dose and frequency of immunization. In dystonia patients treated with toxin complex, the appearance of blocking Abs seems to correlate with the cumulative toxin dose, the time interval between injections and the total protein load (Göschel et al., 1997; Inagi et al., 1997; Jankovic, 2002). In addition, Ab recognition and epitope recognition have been shown to improve with duration of treatment and frequency of immunization (Rosenberg et al., 1997; Dolimbek et al., 2005).
Type of antibody
It is important to point out that the Ab level, although suggestive, does not necessarily always equate with blocking activity. Blocking activity is only partially determined by Ab level, while Ab affinity, the epitopes recognized by these Abs, immunoglobulin class and isotype play major roles (Atassi, 2004; Atassi et al., 2005). Lee et al. (2005) do not consider these other factors. Also, they do not determine whether the differences observed in Ab levels are equivalent to differences in blocking activity.
In conclusion, the work of Lee et al. (2005) used a chemically altered antigen not used in therapy, and injected it at a much higher dose and more frequently. Unfortunately, the findings have little or no relevance to the therapeutic applications of active BoNT/B (or any BoNT) complex.
Acknowledgements
The author wishes to thank the Welch Foundation for the award of the Robert A. Welch Chair of Chemistry.
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