Download STAC RESTRUCTURE

Biotechnology and Biodefense:
Security Challenges Presented by
Genetically Modified Organisms
Bioterrorism and Homeland Security Course
WMD Course Series
Uniformed Services University of the Health Sciences
25 April 2006
Objectives
• Review the properties of traditional biological warfare (BW)
agents and consider factors that may drive pursuit of
enhanced, emerging and advanced agents.
• Provide an overview of some relevant life science
technologies with potential for misapplication by those with
hostile intent.
• Discuss the potential for life science discovery to enhance
challenges posed by existing BW agents and enable evolution
of the BW threat.
Traditional BW Agents
Traditional Biological Agents
• Pathogens:
– Bacteria
– Viruses
• Toxins:
– Proteins
– Non-Proteins
Traditional BW Paradigm
• Agents developed by selecting a harmful pathogen, toxin,
or other biological compound, then “weaponizing” the agent
• The process is focused on the naturally-occurring agent,
not the target organism
• Historically, intrinsic limitations thought to hamper BW utility
for military employment to selective scenarios
Why Might an Adversary Seek to Move
Beyond Traditional Agents?
• Limiting the “sandbox” to traditional agents favors the defense
over the long term:
– Need to overcome medical/physical countermeasures
– Application of technology may provide best “bang for the buck”
• Concerns over attribution following use may drive
development or use of agents not formerly associated with
BW.
• Situations requiring covert application of BW for strategic
purposes may necessitate use of unique agents.
Enhanced BW Agents
These are traditional BW organisms that have been selected
or engineered to defeat countermeasures:
• Antibiotic-resistant
• Capable of circumventing detection
• Evading vaccine-mediated protection
How does one skin the cat?
• Selected from natural/clinical isolates
• Selective pressure of growth environment
• Random mutagenesis
• Recombinant DNA engineering
Emerging BW Agents
• Newly identified naturally-occurring agents of disease - also
occasionally referred to as Emerging Infectious Diseases (EIDs)
that demonstrate virulence, stability and production capacity
consistent with their potential for intentional dissemination.
• Infectious diseases whose incidence has recently increased or
threatens to increase in the near future and include:
– New infections resulting from changes or evolution of existing
organisms.
– Known infections spreading to new geographic areas or
populations.
– Previously unrecognized infections appearing in areas undergoing
ecologic transformation.
– Old infections reemerging as a result of antimicrobial resistance in
known agents or breakdowns in public health measures.
Advanced BW Agents
• Life science advances are likely to enable those with hostile
intent to identify critical physiologic nodes and rationally
engineer agents to exploit vulnerabilities.
• Advanced BW agents would be novel and would not exist in
nature.
• A variety of underlying technologies may support engineering
of a vast range of such pathogens.
Anticipating the Evolution of Biological
Threats
Traditional
Enhanced
Emerging
Advanced
Naturally occurring
microorganisms/
toxin products
Selected/modified to
circumvent
countermeasures
Naturally occurring
organisms/EIDs
(i.e. SARS, AI)
Artificial agents
engineered in
laboratories
Finite number of
agents
Finite yet related
number
Unknown number;
likely finite for BW
Unknown but
potentially infinite
Known signature
Identifiable signature
Identifiable signature
Signature unknown
Knowledge gaps
exist re: properties,
availability, stability
Knowledge gaps
exist re: feasibility,
stability, vulnerability
Knowledge gaps
driven by public
health community
Almost complete
gaps in knowledge
regarding hazards
Potential for exponential expansion of threat highlights the need to
separate fact from fantasy in setting priorities
Need to
Inform Novel
Defensive
Strategies/
Architecture
Life Science Technologies May Impact
Adversary Capabilities Throughout the
“BW Cycle”
• Acquisition, Research and
Development
• Production
• Formulation
• Delivery
• Dissemination
Engineering Countermeasures Evasion:
Lessons Learned from Basic Science
JOURNAL OF VIROLOGY, Jan. 2004, p.999-1005
0022-538X/04/$08.00+0 DOI: 10.1128/JVI.78.2.999-1005.2004
Copyright @ 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 2
Production of Novel Ebola Virus-Like Particles from cDNAs: an
Alternative to Ebola Virus Generation by Reverse Genetics
Shinji Watanabe, Tokiko Watanabe, Takeshi Noda, Ayato Takada, Heinz Feldmann,
1
2,3
2,4
2,3
5
Luke D. Jasenosky,1 and Yoshihiro Kawaoka1, 2,3
15018-15023 | PNAS | December 23,2003 | Vol. 100 | no.26
www.pnas.org/cgi/dol/10.1073/pnas2433882100
Hypervirulent mutant of Mycobacterium tuberculosis
resulting from disruption of the mce1 operon
Nobuyuki Shimono, Lisa Morici, Nicola Casali, Sally Cantrell, Ben SIdders, Sabine Ehrt and Lee W. Riley
Issues equally pertinent to foreign and “domestic” publications
Vaccine, Vol. 15, No. 17/18, pp. 1846-1850, 1997
@ 1997 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0264-410X/97 S17+0 00
PH: S0264-410X(97)00132-1
Expression of cereolysine AB genes in
Bacillus anthracis vaccine strain
BIOCHEMISTRY AND BIOPHYSICS
ensures protection against
Additive Synthesis of Regulatory Peptide in Vivo: the
experimental hemolytic anthrax
Introduction of the Vaccine Strain of Francisella tularensis infection
Producing - Endorphin
A.P. Pomerantsev *, N.A. Staritsin, Yu. V. Mockov and L.L Marinin
V.M. Borzenkov, A.P. Pomerantsev, and I.P Ashmarin
UDC 615.371:579.841.95].012.6.07
Reverse Genetics: Creating Viruses from
Scratch
Plasmids
Activator
Plasmid
Containing
Virus
Genome
Plasmid
Virus
Proteins
Target Cell
Virus RNA
Virus
Assembly
Infectious Virus
(Thousands per Cell)
Containing Viral
Proteins
Practical Application: Targeted Mutation
of Ebola Viruses
Reverse
Genetic
Transcription Manipulation
Ebola
Virus
Ebola
RNA
Ebola
cDNA
Cell Culture
Genetically Tranfection Cells Genetically
Modified
Into
Produce Modified
Ebola
Cultured
Ebola
Ebola
cDNA
Mammalian Virus
Virus
CellsC
Volchkov, et al.Science 291:1965-9 (2001)
DNA Shuffling
DNA Shuffling
• Accelerated evolution
in a test tube could:
• Optimize a gene or
genes encoding
virulence factors
• Evolve a simple
viral genome
toward enhanced
virulence
• Create chimeric
organisms with
radically altered
properties
Fragmentation
DNA from 2 Related Organisms
(Green Genes Encourage
Desired Trait, Red Genes
Suppress Desired Trait)
Pool of Random DNA Fragments
Reassembly
Large Library of Recombinants
Screening for
Desired Trait
Recombinants Possessing
Desired Trait
DNA Shuffling: In practice, a variety of DNA sources and
fragments would be employed
Tropism Alterations
HIV
Ebola
HIV Pseudotyped with
Ebola Coat Proteins
Nucleus
Nucleus
Cell Receptors
T-Cell
(Highly specific for
(Virus not Infectious via Aerosol) Viruses)
Lung Epithelial Cell
(Virus Infectious via Aerosol
Kobinger, et al Nat Biotechnol 19:225-30 (2001)
RNA Interference: RNAi
Plasmid:
siRNA
shRNA
siRNA
• Rapidly degraded
• Limited expression
• Low association
shRNA
• Hairpin structure
prevents degradation
• Greater expression
• More efficient folding
Coding
Direction:
Transcript:
Product:
shRNA Can Impair Cell Function and
Survival
Delivery
Vehicle
shRNA
Plasmid
Expression
RISC (inactive)
shRNA
shRNA not degraded
RISC remains active
Activates RISC
Shuts off target gene
Nucleus
Prevents ‘reading’
Cytoplasm
of target mRNA
Induces mRNA
Degradation
Critical Proteins not made
Cell Death!
Combinatorial Chemistry
• Encompasses a
broad range of
chemical techniques
and tools for creating
large libraries of
molecules
• Can also create
peptide libraries by
amino acid assembly
or cleaving existing
proteins
Step 1
+
+
Step 2
Split
Pool and
Wash
Step 3
Split
Pool and
Wash
Simplified Illustration of Split Synthesis on Solid Bead Supports
• Selection or screening processes identify those candidates displaying a specific
desired property
• Could be used to create and develop novel CBW agents (antipersonnel, antiagricultural, anti-material)
Stabilization and Delivery Means
Stabilization:
• Microencapsulation
• Biofilms
• Carrier Beads
Delivery Vectors:
• Viral
• DNA
• Transgenic
Delivery: Viral Vectors
Packaging
Genes
(From virus X)
Vector
(From virus Y can
include
Therapeutic
Gene)
Packaging Cell
Virus Vector
(Contains genetic material from Virus Y
coated with Surface proteins from virus X)
A variety of methodologies can potentially be applied to manipulate viral
systems not typically identified as biological warfare agents.
DNA Vaccines
Naked DNA inherently more
stable than protein
components of classical
vaccines:
• Could enhance aerosol
delivery of a vaccine.
• Also could enable
development and
delivery of DNA
constructs for hostile
purposes.
Relative Estimate of Evolving BW
Challenges
BW Agents
Advanced Agents
Enhanced Agents
Traditional Agents
Emerging Agents
2003
1980
1990
2000
2010
Notional Timeline
2020
2030
Implications: New BW Use Options
• Customizable aspects of ABW agent development could
increase confidence in results following agent release,
expanding the range of viable use scenarios.
• Agents could be tailored to target a specific population based
upon genetic or cultural traits.
• Creation of slow-acting or debilitating agents might permit
cover application of BW as a strategic weapon against a
target population for long term effects.
Implications: Continued
• Protection against CBW agents still necessary.
• New agents will continue to attempt to exploit weakness of
protective posture.
• Confirmation of use will be more difficult due to advent of new
agents.
Evolution of Biological Threats: Impact
upon Biodefense
By enabling development of novel agents, life sciences will
challenge efficacy of traditional approaches to biodefense:
 Counterproliferation
 Environmental detection
 Medical countermeasures
 Attribution
Counterproliferation
• Technology and methodology innovations coupled with
underlying life science community policies and practices will
likely pose significant proliferation challenges.
• Dissemination of life science information ubiquitous:
– Intrinsic barriers to regulation/restriction.
– Inhibition of knowledge transfer deleterious to biodefense and
public health.
• Focus on novel strategies for counterproliferation, monitoring
for indicators of “activities of concern.”
– Likely to necessitate successful partnership with life science
community.
Secondary Market Proliferation
• Sources of biological science laboratory and bioprocessing
equipment:
– Can be surplus, remanufactured, closeout, or used.
– Usually in good working order.
– Often late model or slightly outdated, but reliable.
• Typically Internet-based:
– Low overhead, little direct interaction with clients.
– Global shipping and delivery.
– Little concern regarding client identity provided reliable
payment.
• Small-scale or part of larger overall business:
– Small private reseller of overstock or labs that have closed.
– Subsection of larger auction category.
Digital Proliferation of Organisms
Challenges efficacy of strategies focused on physical transfer of specimens
Environmental Detection
• Currently, no “real time” detection systems for traditional
organisms of concern.
– Current efforts highly agent specific.
• Optimal systems would profile a variety of physical
(biological?) characteristics likely to be incorporated into
novel agents.
– Wide range pathogen detection could also encompass
traditional, enhanced and emerging agents.
Attribution
• Need to continue development and maturation of a robust
National capability for forensic analysis of biologicals.
– Genetic databases and post-incident molecular analyses a
component.
• However: alternate options focused on identifying the source
of genetic and supporting production material also critical.
– Post-incident attribution will remain a long-term goal.
Medical Countermeasures
• Current diagnostics, prophylaxis, and therapeutics:
– All standard approaches can potentially be circumvented by
enhanced or advanced agents.
• “Next Generation” approaches:
– Detection: Bioprofiling:
 Host response to infection.
 Low level nucleic acid indices of infection.
– Prophylaxis: Stimulating innate and “Midspectral” immunity:
 Generalized immunomodulation via pharmaceuticals.
 Targeted vaccination for generalized immune memory.
– Therapeutics: Will remain a unique challenge:
 High throughput approaches may aid drug discovery.
 “Gene trap” knockouts may highlight novel targets for therapeutics.
Conclusion
• Traditional BW Agents will continue to be the primary threat for
the immediate future.
– Enhanced and emerging agents likely to present near term
challenges as well.
• Advances in biotechnology and genetic engineering likely to
enable development of Advanced agents.
• Such agents would require fundamental shift in US biodefense
paradigms.