1. Dual-Use Examples Lecture No. 15

2. 1. Outline Contentious Research Mousepox Synthesis of Polio Virus
Slides 2 - 8
Mousepox
Slides
Synthesis of Polio Virus
Slides 15 – 18
Influenza Virus
Slide 19
Virulence in Smallpox
Slides 20-21
Notes: The aim of this lecture is to look in more detail at the Fink committee’s treatment of dual-use in the life sciences and, in particular to review the three examples they give of ‘contentious research’ in the life sciences. Links are provided to the original papers and to some other papers of interest in considering this issue. 2

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
Notes: The first half of the introductory chapter of the Fink Committee’s report sets the problem of the potential misuse of the modern life sciences in the kind of wider context that is developed in the earlier lectures in this series. The modern history of biological warfare, for example, is expanded in a short annex describing the pre-scientific history of biological warfare. 3

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
Note: Another point made in the introductory chapter to the Fink Committee’s report is that the scientific community responded quickly when concerns arose about the new techniques of gene splicing and the 1975 Asilomar Conference discussion on the safety of such experiments led to guidelines for such research being developed in the US and in many other countries. So the dual-use problem is not an entirely new challenge for life scientists. 4

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…”
Notes: The report enumerates the well-known characteristics of the modern life sciences and the draws a fundamental conclusion which is set out in the next slide. 5

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…”
Notes: So the great success of the modern life sciences in providing for human welfare is based on a system in which people and ideas move with considerable freedom. To close down that freedom of movement would not be sensible unless there are overwhelmingly powerful reasons to do so. That then is the context in which the committee set its discussion of the dual use dilemma. 6

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.”
Notes: Given their view of the need for openness in the scientific enterprise and their recognition of the possibility of misuse the committee formulated their key concern as “whether the risks associated with misuse can be reduced while still enabling critical research to go forward”. This introductory chapter then makes the risks clear in its sections on the history of biological warfare and briefly introduces the Biological and Toxin Weapons Convention before turning its attention to the new threat. 7

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…?”
Notes: The report uses this famous question from Professor Matthew Meselson to illustrate what is really the question that life scientists face today. What are the responsibilities of scientists in the prevention of the misuse of their work? Failure to prevent the intensive exploitation of the life sciences for hostile purposes could lead to dreadful consequences as Meselson made clear in the next sentences of this quotation (set out on the next slide). 8

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.”
Notes: So here is the big problem set out clearly. If the life sciences are applied in a major way to conflict the nature of conflict will change in ways that none of us would wish to see (recall the discussion of warfare in the modern world in lecture 6). This section of the introduction then leads into the committee’s review of three examples of what it calls ‘contentious research’. That is research which had been carried out for benign civil reasons but which had clearly caused concern about the possible misuse for hostile purposes.
p. 23 9

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…”
Notes: This paper is undoubtedly very well known amongst people concerned with security issues even though it is not very well known amongst practicing life scientists. The original academic paper was published in the Journal of Virology in It should be carefully studied by students. 10

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…”
Notes: The civil objective of the work was clear. In order to deal with rodent plagues a naturally infective pox virus was modified so that it produced an egg protein. This it was hoped would generate a sufficient immune response to lead to the rejection of the eggs by mice and thus prevent the build up of the plague of mice (which if you have seen film of such a plague is quite startling). 11

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.”
Notes: Thus the plan was to create a double modification of the mousepox virus. First the gene for an egg protein was inserted and the when that did not produce a sufficient immune response they sought to enhance the response by also adding the gene for IL-4. 12

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.”
Notes: What the scientists did not expect was that the doubly modified mousepox virus now killed mice which has been vaccinated against the original virus. Moreover they had used quite simple techniques that were described in the methods section of the paper. The Fink Committee’s report then goes on to discuss the pros and cons of publication in the academic literature (though it should be noted that prior to publication in the Journal of Virology there was an major article and editorial about the work in the popular science New Scientist which made the potential connection to a modified smallpox very clear). 13

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…”
Notes: The authors consulted about whether the paper should be submitted for publication and the editors of the journal also sought guidance. Eventually, however, the paper was published exactly as submitted. 14

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…”
Notes: Notwithstanding the fact that even vaccinated mice were killed by the doubly modified mousepox the report gives reasons to support the publication as shown in the quotations given in this slide. Of course, given the prominence of this experiment there is a large literature on the topic that students could easily explore on the internet. An example of earlier work of relevance is given in the first link below to another paper in the Journal of Virology in Later work can be followed in the link to the 2008 paper in Antiviral Research provided in the second link below.
Ref:
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 Ether-lipid Analogue of Cidofovir (CMX001) in a Lethal Mousepox Model. Antiviral Research 77(1): Available from 15

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…”
Notes: Wimmer published his work in 2002 and following the anthrax letters in the US it again attracted considerable attention and concern enough to provoke a resolution in congress.
Ref:
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 – Available from 16

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…”
Notes: The Fink committee’s report was rather unconvinced about the threat of bioterrorism arising from misuse of this work, particularly as the method used had been laborious and taken a long time. However, for many people not familiar with life scientist’s interest in matching their capabilities in genome sequencing with capabilities in genome synthesis this work came as something of a surprise. More surprises were to quickly follow as the next slide demonstrates. 17

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…”
Notes: In December 2003 Science carried another paper by Craig Venter and his colleagues that demonstrated a much faster method of synthesis and in 2004 Nature carried a paper by George Church and his colleagues that described gene synthesis using programmable chips. There could be no doubt that DNA synthesis capabilities were moving forward rapidly. 18

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…”
Notes: This quotation is taken from a careful examination of the potential for influenza to be used as a bioweapon by Robert Krug. Of course all of these concerns became very clear when the 1918 Spanish Influenza was recreated in 2005 (see the next lecture).
Ref:
Krug, M. R. (2003) The Potential Use of Influenza Virus as an Agent for Bioterrorism, Antiviral Research 57,
Available from 19

20. 19. Virulence in Smallpox (i)
“Variola major virus causes smallpox, which has a % 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…”
Notes: This third example used in the Fink report deals with a paper by Rosengard and colleagues which deals directly with an aspect of the virulence of smallpox - certainly a very dangerous potential bioweapons agent. 20

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)…”
Notes: Again the report finds reasons for agreeing that this work should have been carried out, but as we saw in the last lecture they also recommended that an oversight of research projects of this kind was needed. We shall examine oversight systems in more detail in lecture No. 18. 21

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
(Slide 4)
National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from
(Slide 5 and 6)
National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from
(Slide 7)
Meselson, M (2000) Averting the Hostile Exploitation of Biotechnology, The CBW Conventions Bulletin, June 48, Available from

24. (Slide 8)
National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from
(Slide 9)
National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from
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
National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from

25. (Slide 12 and 13)
National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from
(Slide 14)
National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from
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): Available from
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): Available from

26. (Slide 15)
National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from
Wimmer, E. (2007) The Test-Tube Synthesis of a Chemical Called Poliovirus, EMBO Reports Special Issue 7, 3-9. Available from
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 – Available from
(Slide 16)
National Research Council (2004) Biotechnology Research in an Age of Terrorism. Washington: National Academies Press. Available from

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– Available from
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
(Slide 18)
Krug, M. R. (2003) The Potential Use of Influenza Virus as an Agent for Bioterrorism, Antiviral Research 57, Available from
(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