Team:Wageningen UR/Safety/Biosecurity

Xylencer

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Biosecurity

Although Biosafety and Biosecurity are often used interchangeably, there is a clear difference between the two. In contrast to Biosafety, Biosecurity deals with the technologies and practices that are implemented to prevent intentional exposure to pathogens and toxins, or the intentional release and misuse of biological agents and knowledge [1]. In other words, bioterrorism and biological warfare.

Biological warfare has a long history. One of the first accounts of biological warfare was the intentional introduction of the plague in the European city of Caffa in 1345 [2]. Since then, infectious organisms have been used in several wars throughout history [3]. Although there is no consensus about the specific risk involved, over half of the scientists participating in a Delphi study believed that there will be a bioterrorism attack in the coming 10 years [4].

Dual Use and Information Hazard

Nowadays, the threat of biological warfare has become even more severe due to advances in easy-to-use genetic engineering (GE) tools and the custom synthesis of DNA fragments. Additionally, the community of biohackers, do-it-yourself biologists who experiment without appropriate training and laboratory facilities, is growing. These advances also provide persons with bad intentions with the tools to create hazardous organisms [5]. This is true for tools and resources, but also for information itself. Research is intended to benefit the population, but no matter the primarily positive intention, gained knowledge and methodology can have the potential to be misused. Information, technologies and resources with the potential to be abused and posing a threat to society, even though their primary intention may be beneficial, are referred to as dual-use research of concern (DURC). Among DURC, there is also the specific case of information hazard which describes the “risks that arise from the dissemination or the potential dissemination of true information that may cause harm or enable some agent to cause harm”, according to Bostrom, professor of Philosophy at the University of Oxford [6]. However, the restriction of spread of information collides with the transparency in science which allows peers to replicate research to validate findings. Even though transparency is a cornerstone of the scientific world, the publication of research, which is classified as DURC, could allow people with malicious intentions to misuse this knowledge.

In cases of DURC, it is important to consider whether the expected benefits outweigh the potential damages from the research [7]. Ideally, this assessment would be performed prior to the start of a research project. Some factors that should be taken into account in this analysis include the extent of the posed threat, the probability of damages and whether extensive knowledge on techniques and complex machinery is required to apply the research. This analysis should be done to fit both the worst case and the best-case scenario. If the potential damages outweigh the potential benefits, the research should not be started or continued. The risk assessment of Xylencer can be found under biosafety.

Examples of Dual Use Research of Concern

Synthetic Horsepox Virus

One example of DURC is the construction of a complete horsepox virus from synthetically synthesized DNA fragments [8]. Horsepox virus is relatively similar to the modern vaccinia virus. Currently, there is a debate about the safety of the use of this vaccinia virus in vaccines [9]. According to the authors, horsepox virus could be safer vaccine treatment compared to the modern vaccinia virus, used to vaccinate against smallpox [10]. The techniques described in this paper could be applied to revive variola, the responsible agent for smallpox, as this is a highly similar virus. The techniques are however advanced techniques that require a high level of training. The release of the variola virus could have disastrous consequences. Publicly available sequences, combined with techniques described in this paper, could allow people with malicious intent to form these structures. This example also shows how advancements in synthetic biology, in this case, synthesizing large fragments of DNA, could increase the threat of bioterrorism.

Engineering influenza

Another example is the research on the avian influenza virus strain H5N1 in 2011 [11]. In this study, an H5N1 strain was modified by a few mutations to create a virus that was able to spread via air and infect ferrets. Modifying an avian virus to be able to spread via mammals, so also humans, caused commotion among a broad audience, scientific and non-scientific. American institutions such as the U.S. National Science Advisory Board for Biosecurity (NSABB) and the U.S. Department of Health and Human Services (HHS) advised to not publish methods that would allow other researchers to replicate the experiments. This case gained even more attention when the Dutch government obliged the publishers to apply for an export license before sending a revised version. This export license is based upon legislation used to prevent the use of nuclear, chemical and biological weapons. But now, for the first time, it was used to regulate the publication of a scientific research paper [12].

Mentioned cases and their attention among the scientific and non-scientific community caused biosecurity to become a more prominent topic. As a result, biosecurity aspects are implemented more intensively into research, as seen for example among the increasing number of iGEM teams concerned with biosafety & biosecurity in their projects [13].

The Insect Allies Project

During our human practices, we received feedback that our research shows similarity to the Insect Allies Project. Insect Allies is a project by the Defense Advanced Research Projects Agency (DARPA), a governmental body in the USA. The project aims at delivering gene therapy to plants on-field using viruses in order to engineer traits such as drought or pest resistance. The viruses delivering this gene therapy would be spread using insects as vectors. This research has been criticized by European researchers and has been flagged as DURC [14]. The authors doubted that this project would live up to its promises to protect US agriculture against potential crop losses. Besides this, it was stated that there is a lack of discussion regarding legislative issues the project would face, giving the impression that it could never actually be put into practice for US agriculture. Therefore, the authors conclude that expected benefits do not outcompete potential risks of the Insect Allies project. Also, doubts about the efficiency and legal feasibility led to the perception that the project had no intentional beneficial application in the first place but was an effort to develop biological agents for use of bioweapons. Due to a statement on the similarity between Xylencer and the Insect Allies project, we delved into these similarities and the DURC potential of Xylencer.

Similarities between Insect Allies and Xylencer?

Xylencer is similar to Insect Allies in that we use genetically modified viruses that are spread by insects to combat plant pests (or also draught in the case of DARPA). However, there are large differences in the approach. We believe these differences address most dual-use concerns brought up for Insect Allies.

The first main difference is that Xylencer does not apply gene therapy to plants or other organisms outside laboratory environment. Our therapy consists of spreading bacteriophages via insects, we do not alter the genome of the plants and the insects as intermediates in our therapy. The only direct interaction between Xylencer and plants is the release of Microbe Associated Molecular Patterns (MAMPs) which activate the natural plant immune responses. However, activation of the plant’s own immune system is an established method in agriculture to increase the crop’s robustness towards pests. Xylencer makes use of genetically engineered bacteriophages specific to X. fastidiosa and Xanthomonas, an X. fastidiosa related plant pathogen [15]. This reduces the effect of our therapy on non-target organisms as compared to Insect Allies using less specific plant viruses as a delivery platform for gene therapy.

Dual Use and Information Hazard in Xylencer?

We performed an analysis of the dual-use potential within Xylencer considering it would be a fully developed product ready to be put on the market. One concern we came across was that our method of spreading specific bacteriophages could be used to target other bacteria within the microbiome of plants that have a beneficial effect on plant health and crop yield. Alternatively, phages could be used as a carrier for toxin encoding genes setting free toxins in the plant’s microbiome. However, the microbiome of plants is extensive though, so wiping out one of the bacterial species will not impact the plants extensively, as discussed with Britt Koskella, assistant professor at Berkeley University and expert on the effects of phages on the microbiome.

Another problem could be that the binding to the phage could be mimicked with viruses targeting eukaryotic cells. These viruses are evolutionary not closely related [16]. The techniques used for bacteriophages will therefore be different for viruses infecting eukaryotes. The research of the Insect Allies project is much closer related to this potential misuse.

Lastly, our Molecular timer kill switch might potentially be misused. Linking the molecular timer to a toxin could lead to late and programmed activation of a toxin. As it gives rise to less expression of the toxin, it is most likely not beneficial to use this system with malicious intent though.

In parallel, we performed an analysis of the information hazard of the research we have performed over the last six months and planned to perform. The short time period of iGEM is not enough to completely research the ideas behind Xylencer. We have concluded that the parts of our research that have been flagged as potentially applicable for malicious intent are based on previously published research. Chitin binding proteins have been characterized extensively, including their influence on virulence [17]. Capsid fusions of adhesion proteins have also already been used for bacteriophages [18, 19]. As these papers are already publicly accessible, there is no additional information hazard in the publication of our research approaches. Besides basing these parts of our research on prior publications, we also use model organisms rather than pathogenic bacterial strains or viruses. This means that we do not provide information on genetic engineering of plant pathogens, but only of these model organisms. The use of model organisms diminishes the potential of using our project for malicious intent and allows us to follow biosafety rules. We concluded that Xylencer does not add to the information hazard.

We have analyzed the potential risks and benefits of Xylencer as we were advised to do by experts. In conclusion, we believe that the benefits of eradicating X. fastidiosa outweigh the risks for malicious purposes of Xylencer. As discussed above, there is some potential for the use of our research for malicious parties. Both the monetary losses in the agricultural sector and the loss due to the destruction of cultural heritage in the form of more than a century old trees would outweigh the slight risk of misuse. The analysis of the potential risks and benefits of the Insect Allies project in the critical paper had a tendency to show that the risks were much higher than the benefits. While Xylencer saves plants that would otherwise be destroyed, Insect Allies prevents pests that have not occurred yet. For Xylencer, we believe that the potential benefits outweigh the risks of potential misuse.

We spoke with several critics of known Dual-Use Research of Concern projects. More information on this can be found on our Integrated Human Practices page.

Dual Use and Information Hazard in iGEM

Many DURC projects receive extensive media attention. This indicates that communication is very important. With Xylencer, we have noticed that communication around iGEM projects, is key in avoiding unnecessary negative attention. Human practices are essential for clear communication. Current advances in the rapid development of the internet allow it to be very useful in propagating. But new sources of news (e.g. Facebook, Twitter, etc.) and the ability to go ‘viral’ within seconds have led to the need for a clear communication platform and strategy.

Regarding the awareness on DURC, the iGEM Bielefeld 2018 team held a nationwide survey in Germany. They found that 51% of the participants were not familiar with the term dual-use. Our team was also not aware of DURC at the start of the iGEM season. This low familiarity among students of DURC is worrying. We believe that we should start teaching students to perform benefit/damage analyses on their research early on. Students should also learn about safe-by-design in research during their studies. We believe that iGEM is the perfect time to educate future synthetic biologists on biosecurity. The field of synthetic biology is still very young, so new practices can still be taken up relatively easy.

What you can do about DURC

We have made a biosecurity timeline to help other teams consider biosecurity in their project, based on our experiences in this regard. This is an easy tool, including a checklist, for teams to use to help them integrate biosecurity into their projects. We hope this will make DURC more familiar among iGEM teams. For a very extensive report on dual-use, see iGEM Bielefeld 2015.

Continue to Lab Safety

References
1. National Academies of Sciences, Engineering, and Medicine. (2017). Dual use research of concern in the life sciences: current issues and controversies. National Academies Press.
2. Wheelis, M. (2002). Biological warfare at the 1346 siege of Caffa. Emerging infectious diseases, 8(9), 971.
3. Riedel, S. (2004, October). Biological warfare and bioterrorism: a historical review. In Baylor University Medical Center Proceedings (Vol. 17, No. 4, pp. 400-406). Taylor & Francis.
4. Boddie, C., Watson, M., Ackerman, G., & Gronvall, G. K. (2015). Assessing the bioweapons threat. Science, 349(6250), 792-793.
5. WHO. (2010, December). Scientific review of variola virus research, 1999–2010.
6. Bostrom, N. (2011). Information hazards: a typology of potential harms from knowledge. Review of Contemporary Philosophy, (10), 44-79.
7. Security and Defense Research–Working Group. (2012). Guidelines and Rules of the Max Planck Society On A Responsible Approach To Freedom Of Research And Research Risks.
8. Noyce, R. S., Lederman, S., & Evans, D. H. (2018). Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments. PloS one, 13(1), e0188453.
9. Fulginiti, V. A., Papier, A., Lane, J. M., Neff, J. M., Henderson, D. A., Henderson, D. A., ... & O'Toole, T. (2003). Smallpox vaccination: a review, part II. Adverse events. Clinical infectious diseases, 37(2), 251-271.
10. Noyce, R. S., & Evans, D. H. (2018). Synthetic horsepox viruses and the continuing debate about dual use research. PLoS pathogens, 14(10), e1007025.
11. Herfst, S., Schrauwen, E. J. A., Linster, M., Chutinimitkul, S., de Wit, E., Munster, V. J., … Fouchier, R. A. M. (2012). Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets. Science, 336(6088), 1534–1541. https://doi.org/10.1126/science.1213362.
12. Enserink, M. (2015). Dutch appeals court dodges decision on hotly debated H5N1 papers. Science. https://doi.org/10.1126/science.aac8867.
13. Guan, Z. et al. (2013) ‘Biosafety Considerations of Synthetic Biology in the International Genetically Engineered Machine (iGEM) Competition’, BioScience, 63(1), pp. 25–34. doi: 10.1525/bio.2013.63.1.7.
14. Reeves, R. G., Voeneky, S., Caetano-Anollés, D., Beck, F., & Boëte, C. (2018). Agricultural research, or a new bioweapon system?. Science, 362(6410), 35-37.
15. Ahern, S. J., Das, M., Bhowmick, T. S., Young, R., & Gonzalez, C. F. (2014). Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas. Journal of Bacteriology, 196(2), 459–471. https://doi.org/10.1128/JB.01080-13.
16. Koonin, E. V., Dolja, V. V., & Krupovic, M. (2015). Origins and evolution of viruses of eukaryotes: the ultimate modularity. Virology, 479, 2-25.
17. Killiny, N. and Almeida, R. P. P. (2009) ‘Xylella fastidiosa afimbrial adhesins mediate cell transmission to plants by leafhopper vectors.’, Applied and environmental microbiology. American Society for Microbiology, 75(2), pp. 521–8. doi: 10.1128/AEM.01921-08.
18. Hodyra, K., & Dąbrowska, K. (2015). Molecular and chemical engineering of bacteriophages for potential medical applications. Archivum immunologiae et therapiae experimentalis, 63(2), 117-127.
19. Pavoni, E., Vaccaro, P., D’Alessio, V., De Santis, R., & Minenkova, O. (2013). Simultaneous display of two large proteins on the head and tail of bacteriophage lambda. BMC Biotechnology, 13(1), 79. https://doi.org/10.1186/1472-6750-13-79.

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[1] National Academies of Sciences, Engineering, and Medicine. (2017). Dual use research of concern in the life sciences: current issues and controversies. National Academies Press.

[2] Wheelis, M. (2002). Biological warfare at the 1346 siege of Caffa. Emerging infectious diseases, 8(9), 971.

[3] Riedel, S. (2004, October). Biological warfare and bioterrorism: a historical review. In Baylor University Medical Center Proceedings (Vol. 17, No. 4, pp. 400-406). Taylor & Francis.

[4] Boddie, C., Watson, M., Ackerman, G., & Gronvall, G. K. (2015). Assessing the bioweapons threat. Science, 349(6250), 792-793.

[5] WHO. (2010, December). Scientific review of variola virus research, 1999–2010.

[6] Bostrom, N. (2011). Information hazards: a typology of potential harms from knowledge. Review of Contemporary Philosophy, (10), 44-79.

[7] Security and Defense Research–Working Group. (2012). Guidelines and Rules of the Max Planck Society On A Responsible Approach To Freedom Of Research And Research Risks.

[8] Noyce, R. S., Lederman, S., & Evans, D. H. (2018). Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments. PloS one, 13(1), e0188453.

[9] Fulginiti, V. A., Papier, A., Lane, J. M., Neff, J. M., Henderson, D. A., Henderson, D. A., ... & O'Toole, T. (2003). Smallpox vaccination: a review, part II. Adverse events. Clinical infectious diseases, 37(2), 251-271.

[10] Noyce, R. S., & Evans, D. H. (2018). Synthetic horsepox viruses and the continuing debate about dual use research. PLoS pathogens, 14(10), e1007025.

[11] Herfst, S., Schrauwen, E. J. A., Linster, M., Chutinimitkul, S., de Wit, E., Munster, V. J., … Fouchier, R. A. M. (2012). Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets. Science, 336(6088), 1534–1541. https://doi.org/10.1126/science.1213362.

[12] Enserink, M. (2015). Dutch appeals court dodges decision on hotly debated H5N1 papers. Science. https://doi.org/10.1126/science.aac8867.

[13] Guan, Z. et al. (2013) ‘Biosafety Considerations of Synthetic Biology in the International Genetically Engineered Machine (iGEM) Competition’, BioScience, 63(1), pp. 25–34. doi: 10.1525/bio.2013.63.1.7.

[14] Reeves, R. G., Voeneky, S., Caetano-Anollés, D., Beck, F., & Boëte, C. (2018). Agricultural research, or a new bioweapon system?. Science, 362(6410), 35-37.

[15] Ahern, S. J., Das, M., Bhowmick, T. S., Young, R., & Gonzalez, C. F. (2014). Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas. Journal of Bacteriology, 196(2), 459–471. https://doi.org/10.1128/JB.01080-13.

[16] Koonin, E. V., Dolja, V. V., & Krupovic, M. (2015). Origins and evolution of viruses of eukaryotes: the ultimate modularity. Virology, 479, 2-25.

[17] Killiny, N. and Almeida, R. P. P. (2009) ‘Xylella fastidiosa afimbrial adhesins mediate cell transmission to plants by leafhopper vectors.’, Applied and environmental microbiology. American Society for Microbiology, 75(2), pp. 521–8. doi: 10.1128/AEM.01921-08.

[18] Hodyra, K., & Dąbrowska, K. (2015). Molecular and chemical engineering of bacteriophages for potential medical applications. Archivum immunologiae et therapiae experimentalis, 63(2), 117-127.

[19] Pavoni, E., Vaccaro, P., D’Alessio, V., De Santis, R., & Minenkova, O. (2013). Simultaneous display of two large proteins on the head and tail of bacteriophage lambda. BMC Biotechnology, 13(1), 79. https://doi.org/10.1186/1472-6750-13-79.