Botulinum Neurotoxin Subtype A4 Originating from Nontoxigenic Clostridium botulinum

EDITORIAL

In this issue of Applied and Environmental Microbiology (AEM), we publish a paper by Marite Bradshaw, William H. Tepp, Regina C. M. Whitemarsh, Sabine Pellett, and Eric A. Johnson from the Department of Bacteriology, University of Wisconsin—Madison (UW—M), reporting on the purification and characterization of subtype A4 botulinum neurotoxin (1). The authors describe the purification of recombinant Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain, Hall A-hyper. Previous research has indicated that the reduced stability and activity of the BoNT/A4 light chain (LC) compared to BoNT/A1 LC is due to a single-amino-acid substitution, I264R. A second mutation predicted to increase solubility (L260F) was also produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of recombinant BoNT/A4 (rBoNT/A4) and rBoNT/A4–L260F I264R showed 1,000-fold-lower activity versus BoNT/A1 in both mutated and nonmutated BoNT/A4, indicating that these mutations do not alter the activity of BoNT/A4 toxin.

We think this article makes important contributions. This endogenous clostridial expression system will be valuable for determination of the regulation of BoNT expression in dual-toxin producers, for elucidation of the steps in toxin complex formation, and for demonstration of new subtypes and serotypes. The number of botulinum neurotoxins has significantly expanded recently, in large measure because of advances in molecular biology (2–4). There are seven classic serotypes, designated by the letters A to G, and at least 40 subtypes (2). Recently, a new serotype, “H,” has been proposed (5, 6), but because this toxin is the minor toxin in a dual-toxin-producing strain, a clear characterization of the purified toxin has not been possible to date. Accurate characterization is essential for confirmation of new serotypes and for a clear understanding of the biochemical, toxicological, and immunological properties of such toxins. The only means to cleanly isolate a minor toxin from dual-toxin producers appears to be to express it recombinantly. Expression of recombinant full-length BoNTs has to date only been accomplished in nonclostridial hosts (7–11); however, the complex series of events that take place during toxin production (expression, folding, activation, secretion, etc.) have posed challenges in nonclostridial systems that can be mitigated by using an endogenous Clostridium host. Furthermore, we note that botulinum toxin has medical uses and that the findings in this study could potentially be useful to the pharmaceutical industry for improving production of this drug.

Botulinum neurotoxins are among the most poisonous substances known. BoNTs cause a severe neurological paralytic illness in humans and animals commonly known as botulism. Because of their remarkable strength and their serious and long-lasting consequences, there is significant concern about their potential undesirable use as bioterrorism agents (12, 13). To mitigate the risk associated with this type of research, the authors have made three specific accommodations. First, they used a nonsporulating and nontoxigenic strain as the recombinant host to avoid the possible dissemination of spores and native BoNT/A1. Second, they used BoNT/A4, which has approximately 1,000-fold-less specific toxicity than other type A strains and toxins from other serotypes that have been characterized. Finally, they removed some details of the recombinant expression system while maintaining scientific validity by providing a general description of the expression system.

The University of Wisconsin—Madison has developed a process for reviewing research that could potentially be considered dual use research of concern (DURC) at the grant level with additional review at the protocol, experimental, and publication levels. As described in the United States Government policy for oversight of life sciences (13), DURC is defined as “Life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misapplied to pose a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, materiel, or national security.” The grant application used to support the research reported in the article was reviewed by the University of Wisconsin—Madison's Institutional Biosafety Committee (UW—M IBC) and Biosecurity Task Force (UW—M BTF) for DURC, as requested by the National Institute of Allergy and Infectious Diseases (NIAID) in May 2012. Both UW—M committees felt the work described in the grant was not DURC because the research would not enhance the harmful consequences of botulinum neurotoxin (BoNT) type A4. This was because the specific aim of the grant was to develop a system to study BoNTs that are naturally produced in small quantities, and the data suggest that these types of BoNTs are significantly less toxic than currently known types of BoNTs. This research would allow type A4 and other BoNTs to be studied in a native background. The review was sent to NIAID, and NIAID respectfully disagreed with the UW—M assessment because they felt that the experiments could potentially increase the dissemination of the toxin. The UW—M risk mitigation plan was submitted and was reviewed for consistency with NIAID policy. The UW—M risk mitigation plan was approved by the NIAID in a letter dated 30 April 2013.

After review of the original version of the manuscript, the UW—M BTF and IBC determined the manuscript should be published and continued to respectfully disagree with NIAID that the research contained in the manuscript is DURC. Both UW—M committees felt that the experiments could not increase the dissemination of the toxin. The principle investigator, Eric Johnson, kept the UW—M responsible official (Timothy Yoshino) and UW—M alternate responsible official (Rebecca Moritz) informed of the results of the research as the experiments progressed, as requested by the IBC and BTF. The UW—M responsible officials noted in a letter dated 19 May 2014 that the risk mitigation plan had been followed appropriately. They also stated all research was done at the appropriate biosafety level, including the use of a C. botulinum strain that is incapable of forming spores and the use of a dedicated biosafety level 3 (BSL3) laboratory to grow that strain and subsequently isolate the toxin. The same letter indicated that the investigators followed all of the required biosecurity parameters that are listed in the select agent regulations. The UW—M IBC and BTF support the full publication of the manuscript because they concluded the expression system described in the manuscript will provide a greater understanding of BoNTs in their native environment. They also concluded that full publication will make the properties of the A4 toxin available to the BoNT research community, and the benefits of this information being disseminated to the public far outweigh the potential risks associated with publication.

In correspondence on 21 April 2014, Ryan Ranallo (NIAID) provided an evaluation of the original version of the manuscript. NIAID determined that the research did involve dual use research of concern (DURC), as defined in the document United States Government Policy for Oversight of Life Sciences Dual Use Research of Concern, published on 29 March 2012 (13). Despite the disagreement over DURC classification with UW—M, NIAID agreed with the lead author and UW—M IBC regarding publication of the manuscript. NIAID did recommend that upon submission, the authors include an accompanying letter outlining the procedures undertaken by UW—M to assess DURC, the results of the assessment, how the benefits of communication outweigh the risks, and the risk mitigation strategies employed (i.e., the three specific accommodations summarized above). Ranallo reiterated NIAID's belief that it is important to consider the impact on the efficacy of existing medical countermeasures, e.g., heptavalent botulinum antitoxin (HBAT), when communicating DURC. In response to this concern, the authors added data that supports BoNT/A4 neutralization by HBAT in the original manuscript.

The paper was considered for publication by AEM like all others submitted for evaluation. Reviewers were asked to evaluate the paper for scientific rigor and significance and to consider whether the research represented DURC. The manuscript also was evaluated for DURC by senior editors and the chair of the Journals Board at the American Society for Microbiology, which publishes AEM, to determine whether any potential risks of publishing the paper would outweigh the benefits. Based on the peer reviews, the authors' response to the reviews, the support for publication of the revised version by UW—M and NIAID, and our own internal review, we have decided to move forward with publication. The development of this expression system will allow researchers to study the native expression, folding, activation, secretion, and solubility of BoNTs that are naturally produced in small amounts or in a dual-toxin-producing strain. This expression system will also allow these types of BoNTs to be studied for their immunological features, cell entry characteristics, and pharmacological aspects. This research is fundamental to our ability to understand the properties of individual types and subtypes of BoNTs and to find new therapeutic interventions and medical uses as well as establish new countermeasures.