Dual-Use Examples Lecture No. 15

1. Dual-Use ExamplesLecture No. 15

2. 1. Outline Contentious Research Slides 2 - 8 Mousepox Slides 9 - 14 Synthesis of Polio Virus Slides 15 – 18 Influenza Virus Slide 19 Virulence in Smallpox Slides 20-21

3. 2. Contentious Research (i) Fink Chapter 1 The Life Sciences Today The “Dual use” Dilemma A Brief History of Biological Warfare* U.S. Policy and the Creation of the Biological and Toxin Weapons Convention *Annex: Biological Warfare in History

4. 3. Contentious Research (ii) Fink Chapter 1 (continued) The New Threat Recent Examples of “Contentious Research” in the Life Sciences The Response of the Life Science Community to Previous Challenges Committee Charge and Process

5. 4. Contentious Research (iii) The Life Sciences Today “The biological sciences have experienced enormous growth over the last century…” “The ever-expanding research activity has resulted in numerous new…products that are transforming medicine…” “Biotechnology research is now a truly global enterprise…” “In addition to the dispersed research enterprise, publications and personnel are also widely spread…”

6. 5. Contentious Research (iv) The Life Sciences Today (continued) “The rapid spread of scientific knowledge and applications owes much to a research culture in which knowledge and biological materials are shared among scientists and people move freely between universities, government agencies, and private industry. Large numbers of foreign graduate students and postdoctoral associates have been an essential ingredient of the success of the biological research enterprise. The scientific workforce is increasingly international…”

7. 6. Contentious Research (v) The Dual Use Dilemma “The regulation of dual use biotechnology research is a highly contentious technical, political, and societal issue. In the language of arms control and disarmament, dual use refers to technologies intended for civilian application that can also be used for military purposes…” “…The key issue is whether the risks associated with misuse can be reduced while still enabling critical research to go forward.”

8. 7. Contentious Research (vi) The New Threat “Every major technology - metallurgy, explosives, internal combustion, aviation, electronics, nuclear energy - has been intensively exploited, not only for peaceful purposes but also for hostile ones. Must this also happen with biotechnology, certain to be a dominant technology of the coming century…?”

9. 8. Contentious Research (vii) The New Threat (continued) “…During the century just begun, as our ability to modify fundamental life processes continues its rapid advance, we will be able not only to devise additional ways to destroy life but…also…to manipulate it - including the processes of cognition, development, reproduction, and inheritance. A world in which these capabilities are widely employed for hostile purposes would be a world in which the very nature of conflict has radically changed. Therein could lie unprecedented opportunities for violence, coercion, repression, or subjugation.”

10. 9. Mousepox (i) “…Probably the most celebrated recent case involving the dissemination of research with the potential for bioterrorist uses was the report of an unexpected effect of the bioengineering of a strain of ectromelia virus (mousepox) that was intended to help eradicate mice in Australia…. Some have felt that the publication of this paper provides a blueprint or roadmap for terrorist to engineer a more virulent strain of smallpox that could overwhelm the human immune system in even well-vaccinated individuals…”

11. 10. Mousepox (ii) “The authors of the paper had originally set out to make an infectious immunocontraceptive for wild mice by incorporating a gene encoding an antigen from fertilized mouse eggs into the genome of ectromelia virus. Since the expression of this egg antigen of the virus did not result in infertility, the authors attempted to increase the virulence of ectromelia with the hope that this would increase the immune response…”

12. 11. Mousepox (iii) “They drew on previously published work…in which it had been shown that incorporating the gene for…IL-4 into the viral genome and thus overexpressing it in vivo enhanced the virulence of vaccinia virus in mice. The increased virulence is probably due to suppression of the antiviral immune response mediated through competing cytokines like IL-2…which work by stimulating immune effector cells to kill virus-infected cells and thus control the virus infection.”

13. 12. Mousepox (iv) “…They then demonstrated that this engineered mousepox virus was much more virulent than the parent virus and killed 60% of infected mice, even if the mice were from a genetically resistant strain. Even more unexpected was their observation that mice that had been vaccinated and were completely resistant to the parent virus…were now killed by the IL-4 gene-expressing virus.”

14. 13. Mousepox (v) “Some have felt that the publication of this paper provides a blueprint or roadmap for terrorists to engineer a more virulent strain of smallpox that could overwhelm the human immune system in even well-vaccinated individuals…. It has been suggested that either the paper should not have been published, or at the very least the ‘materials and methods’ section…should have been altered or omitted entirely from the published article…”

15. 14. Mousepox (vi) Reasons for publication? “…First, knowledge of these experiments allows the scientific community to explore how to overcome such engineered viruses…” “…Second, it suggests that we should be prepared to treat infections caused by such an engineered virus with antibodies that inactivate the relevant cytokine, with gamma interferon that would counter the effect of IL-4, or with both…”

16. 15. Synthesis of Polio Virus (i) “Wimmer and colleagues reported that they had reconstructed poliovirus from chemically synthesized oligonucleotides that were linked together and then transfected into cells. The report attracted considerable attention in the news media and concern in some segments of the public….This…raised public concern about bioterrorism because it suggested that the Wimmer experiment provided a recipe for terrorists to manufacture the virus…”

17. 16. Synthesis of Polio Virus (ii) “Many scientists concluded that the Wimmer experiment was neither a novel discovery nor a potential threat. The general principle that one could make live poliovirus from a DNA template was already known in 1981, when Baltimore and colleagues reported that a DNA copy of the positive strand RNA genome of poliovirus could be taken up into living cells under appropriate conditions and result in the generation of encapsulated, infectious virus…”

18. 17. Synthesis of Polio Virus (iii) “We have improved upon the methodology and dramatically shortened the time required for the accurate assembly of a 5- to 6-kb segments of DNA from synthetic oligonucleotides. As a test of this methodology, we have established conditions for the rapid (14-day) assembly of the complete infectious genome of the bacteriophage  X174 (5,386 bp) from a single pool of chemically synthesized oligonucleotides…”

19. 18. Influenza Virus (iv) “Influenza A virus has been responsible for widespread human epidemics because it readily transmits form humans to humans by aerosol. Recent events have highlighted the potential of influenza A virus as a bioterrorist weapon: the high virulence of influenza A virus that infected people in Hong Kong in 1997: and the development of laboratory methods to generate influenza A viruses by transfection of DNAs without a helper virus…”

20. 19. Virulence in Smallpox (i) “Variola major virus causes smallpox, which has a 30 - 40% mortality rate,whereas vaccinia virus, which is used to vaccinate humans against smallpox, causes no disease in immunocompetent humans….Both viruses have an inhibitor of immune response enzymes - vaccinia virus complement control protein (VCP) and smallpox inhibitor of complement enzymes (SPICE). The authors focused on a comparison of the genes encoding this inhibitor…”

21. 20. Virulence in Smallpox (ii) “…As live variola is not available for study, they used standard techniques to synthesize the SPICE gene. They found that variola spice has a greater degree of specificity for human complement and is nearly a hundredfold more active than VCP in inactivating this component of the human immune system (human complement component C3b)…”

22. Sample Questions • Do you agree with Meselson’s view that there is a grave danger that the modern life sciences will be used in a major way for hostile purposes? If you agree how do you think this might be prevented? If you disagree set out your reasons and discuss one of them in detail. 2. Outline the mousepox experiment. Should this work have been reported in the scientific literature? Give the reasons for your consideration. 3. “Wimmer’s synthesis of polio virus was not on example of dual-use research of concern”. Discuss. 4. Could influenza virus be used as a serious bioterrorism/biowarfare agent?

23. References (Slide 2) National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from http://books.nap.edu/openbook.php?record_id=10827&page=15 (Slide 4) National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from http://books.nap.edu/openbook.php?record_id=10827&page=16 (Slide 5 and 6) National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from http://books.nap.edu/openbook.php?record_id=10827&page=18 (Slide 7) Meselson, M (2000) Averting the Hostile Exploitation of Biotechnology, The CBW Conventions Bulletin, June 48, 16-19. Available from http://www.sussex.ac.uk/Units/spru/hsp/pdfbulletin.html

24. (Slide 8) National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from http://books.nap.edu/openbook.php?record_id=10827&page=23 (Slide 9) National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from http://books.nap.edu/openbook.php?record_id=10827&page=25 Jackson, R. J., Ramsay, A. J., Christensen, C. D., Beaton, S., Hall, D. F., and Ramshoaw, I. A. (2001) Expression of Mouse Interleukin-4 by a Recombinant Ectromelia Virus Suppresses Cytolytic Lymphocyte Responses and Overcomes Genetic Resistance to Mousepox. Journal of Virology, 75(3), 1205–1210. Available from http://www.ncbi.nlm.nih.gov/pubmed/11152493 National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from http://books.nap.edu/openbook.php?record_id=10827&page=25

25. (Slide 12 and 13) National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from http://books.nap.edu/openbook.php?record_id=10827&page=26 (Slide 14) National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from http://books.nap.edu/openbook.php?record_id=10827&page=27 Bembridge, G. P., Lopez, J. A., Cook, R., Melero, J. A., and Taylor, G.. (1998) Recombinant Vaccinia Virus Coexpressing the F Protein of Respiratory Syncytial Virus (RSV) and Interleukin-4 (IL-4) Does Not Inhibit the Development of RSVSpecific Memory Cytotoxic T Lymphocytes, whereas Priming Is Diminished in the Presence of High Levels of IL-2 or Gamma Interferon, Journal of Virology 72(5):4080-7. Available from http://www.ncbi.nlm.nih.gov/pubmed/9557697 Parker, S., Touchette, E., Oberle, C., Almond, M., Robertson, A., Trost, L. C., Lampert, B., Painter, G., and Buller, R. M.(2008) Efficacy of Therapeutic Intervention with an Oral Etherlipid Analogue of Cidofovir (CMX001) in a Lethal Mousepox Model. Antiviral Research 77(1):39-49. Available from http://www.ncbi.nlm.nih.gov/pubmed/17904231

26. (Slide 15) National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from http://books.nap.edu/openbook.php?record_id=10827&page=27 Wimmer, E. (2007) The Test-Tube Synthesis of a Chemical Called Poliovirus, EMBO Reports Special Issue 7, 3-9. Available from http://www.nature.com/embor/journal/v7/n1s/full/7400728.html Cello, J., Paul, A. V., and Wimmer, E. (2002) Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template, Science 297(5583), 1016 – 1018. Available from http://www.sciencemag.org/cgi/content/abstract/1072266 (Slide 16) National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from http://books.nap.edu/openbook.php?record_id=10827&page=28

27. (Slide 17) Smith, H. O., Hutchison, C. A., Pfannkoch, C., and Venter, J. C. (2003) Generating a Synthetic Genome by Whole Genome Assembly: _X174 Bacteriophage from Synthetic Oligonucleotides, PNAS, 100(26), 15440–15445. Available from http://www.pnas.org/ Tian, J., Gong, H., Sheng, N., Zhou, X., Gulari, E., Gao, X., and Church, G.. (2004) Accurate multiplex gene synthesis from programmable DNA microchips, Nature, 432(23/30). Available from http://www.nature.com/nature/journal/v432/n7020/full/nature03151.html (Slide 18) Krug, M. R. (2003) The Potential Use of Influenza Virus as an Agent for Bioterrorism, Antiviral Research 57, 147-150 Available from http://www.elsevier.com/wps/find/journaldescription.cws_home/521852/description#description (Slide 19) Rosengard, A. M., Liu, Y., Nie, Z., and Jimenez, R. (2002) Variola virus immune evasion design: Expression of a highly efficient inhibitor of human complement, PNAS, 99(13), 8808–8813 Available from http://www.pnas.org/