Thursday, February 27, 2014

Virus Like Particles Applications for Bio-Defence Vaccine Delivery





Virus Like particles (VLPs) are a promising vaccine platform due to safety and efficiency. When we consider specific issues related to bio-defence vaccine development and of course non-bio-defence related vaccine R and D, VLP's would appear to be a highly promising technology. Bio-defence vaccines, particularly those which are perceived as less likely agents for development, testing and use for bio-terrorism and/or warfare,  have typically faced funding issues. BioShield and BARDA have attempted to level the playing field, but there remain significant concerns related to such investment. Its difficult to develop vaccines for diseases or therepeutic counter-measures for diseases which in some instances, have never occurred on a large scale or at all. As a bio-defence specialist and risk analyst, in my view, these vaccines and medical counter-measures are a critical aspect of public health protection. Threat reduction, on the security policy side is the perview of social sciences and the intelligence community, and although useful in countering the threat of bio-terrorism, it is not a replacement for the continued need to ensure public health security at the therapeutic level. The bio-defence community would be remiss in their responsibilities if they did not do this. Vaccines are costly and investment, partiuclarly in early stage or by the time a company is entering the "Valley of Death", is vital to production and ultimately public health security. 

Aside from reducing safety issues related to some vaccines and medical counter measures, VLPs may make investments in bio-defence vaccines and orphan drug research and development, a far more secure bet in terms of efficacy, safety, manufacturing costs, and timeline delivery. Investment in diseases which occurr naturally and have the potential to result in pandemic outbreaks with severe consequences to public health security, have typically been of more economic and investment interest than diseases which may or may not ever occur. It is the later group of vaccines, which is likely to benefit considerably from advanced VLP technology. One can imagine that even a product like VIG (Vaccinia Immune Globulin) for example, developed in the 1960's to treat those counter-indicated for smallpox vaccine, might be an interesting candidate. Still other neglected diseases could well benefit from advances in VLP research and development.

Monday, February 24, 2014

DARPA's 7-Day Bio-Defence and the Future of Synthetic Vaccines

US military personnel don special biohazard gear during a training exercise designed to simulate a biological weapon attack. The Department of Defense and other agencies routinely hold training sessions throughout the country as part of a domestic bioterrorism preparedness program. 'The challenge is to integrate these forces to mount an effective response under various attack scenarios," says Prof. Steven Block. Courtsey: US Navy 

Author's note: In 2007, I attended a bio-defence briefing delivered by a DARPA scientist. Since then I have always thought of DARPA as the 'bugs on the wall folks' and indeed they have successfully produced robotic bugs, but more significantly, they discussed advanced bio-defence technologies which we were not allowed to take notes on or photograph the slides. It was extremely exciting and I left with a sense of awe which DARPA tends to inspire. Six years later, they have successfully manufactured 10 million doses of vaccine within in a month. For pharmaceutical companies who invest up and over a billion per drug and which including research and development takes about ten years to bring onto the market, this was a phenomenal feat. Here we are on the threshold of major breakthroughs in vaccine research, development and production, even manufacturing technologies and the future of bio-defence couldn't look brighter. While industry of course continues to work on live attenuated vaccine production is the future a synthetic one?

Photo: www.csc.com
Current vaccine production based on inactivated  viruses (live attenuated vaccines) has been successful in reducing significant disease burden associated with major epidemics of the 19th and 20th centuries. However, a major draw back has been the lengthy research and development phase, the significant investment costs and the inability to respond rapidly to changing strains. Synthetic vaccines may overcome many of the more traditionally based production obstacles. In 2012 DARPA announced it's Blue Angel Program. On their site they lay out the problem quite concisely with regard to responding to pandemics of the future. Such pandemics may or may not be natural and may or may not be caused by natural pathogens, viruses and toxins. Consider fighting a synthetically produced outbreak. The DARPA site states:

"The 2009 Army Posture Statement, cites a World Health Organization estimate of between 20 and 50 percent of the world's population being effected if a pandemic were to emerge. WHO forecasts 'it may be six to nine months before a vaccine for a pandemic virus strain becomes available." In a separate report on pandemic influenza, the WHO described several challenges to producing sufficient volumes of vaccine using current, egg based protein-production technology, including the likihood that two doses per person could be required due to the absence of pre-existing immunity. In short, the potential for a pandemic exists and current technological limitations on defensive measures put the health and readiness of U.S. military forces at risk. A technological solution to increase the speed and adaptability of vaccine production is urgently needed to match the broad biological threat. DARPA's Blue Angel Program seeks to demonstrate a flexible and agile capability for the Department of Defence to rapidly react to and neutralize any natural or intentional pandemic disease. Building on a previous DARPA program, Accelerated Manufacture of Pharmaceuticals, Blue Angel targets new ways of producing large amounts of high quality, vaccine grade protein in less than three months in response to emerging and novel biological threats. One of the research avenues explores plant made proteins for candidate vaccine production. "Vaccinating susceptible populations during the initial stage of a pandemic is critical to containment," said Dr. Alan Magill, DARPA program manager. "We're looking at plant based solutions to vaccine production as a more rapid and efficient alternative to the standard egg-based technologies, and the research is very promising." In a recent milestone development under Blue Angel, researchers at Medicago Inc. produced more than 10 million doses (as defined in an animal model) of an H1N1 influenza vaccine candidate based on virus-like particles (VLP) in one month. Production adhered to Phase 1 appropriate current good manufacturing practices. The work was part of a 'rapid fire' test that ran from March 25, 2012 to April 24, 2012, at a facility in Durham, NC. A third party laboratory tested the production lots to confirm the immunogenicity of the vaccine candidate. Testing confirmed that a single dose of the H1N1 VLP influenza vaccine candidate induced protective levels of hemagglutinin antibodies in an animal model when combined with a standard aluminum adjuvant. The equivalent dose required to protect humans from natural disease can only be determined by future, prospective clinical trials. 

DARPA's 7 Day Bio-Defence 

Photo: DARPA

In May, 2013 Medicago Inc. announced it had successfully produced a VLP vaccine candidate for the H7N9 virus responsible for an influenza outbreak in China. Medicago's future in VLP vaccines couldn't look brighter especially given their success with rapid vaccine production and their work with DARPA. 
An additional, although slightly different project which I believe will make significant strides in bio-defence counter-measures is DARPA's 7-Day Bio-Defence project, again, worth considering in terms of how future vaccines will be manufactured and how investment in this technology could well shift. As stated on their site:

"Military readiness and national security depends on the health and well being of military service members. The Department of Defence's (DoD) cumulative investment in personnel comprises the second largest share of the total defence budget. As such DoD seeks advances in health care to ensure war-fighters can operate at peak performance. Research into natural and synthetic pathogens, and treatments against them is one plank of ensuring military readiness in the face of accidental and offensive biological threats to both war-fighters and the supply chain supporting them. In this context, the 7-Day Biodefence program will seek to develop novel technologies focused on preventing infection by any emerging pathogen, sustaining survival once infected, and building immunity. In recent years, global surveillance networks have determined an increase in the frequency and diversity with which new infectious micro-organisms are emerging. While this increase is due in part to improved reporting, multiple examples demonstrate it is also promulgated by changes in natural systems and possibly human activity. The potential biological threat breaks down into two primary categories: 

1. Exposure to natural pathogens that are the result of: increased human-animal interface; increased population densities and co-location of vulnerable species with pathogen reservoirs; climate change, particularly affecting migration and spread of vectors; and narrowing of genetic diversity among food-animal stocks. 

2. Exposure to synthetic and highly diverse pathogens that have become easier to produce as bio-medical and genetic-engineering technologies proliferate internationally; such that pathogens could be used by adversaries for offensive purposes in a direct attack on war-fighters for covert sabotage of the agricultural industry that supports war fighters. 

Together, these emerging threat agents challenge current medical countermeasures. Today's research and development cycle for countermeasures is ill-equipped for rapid response to emerging biological threats. In response to the unspecified potential threat from emerging pathogens, the goal of the 7-Day Biodefence program is to develop innovative approaches to counter pathogens without regard to their exact nature. The methods being explored do not require prior knowledge of the pathogen and are broadly applicable to multiple, unrelated infectious agents. The program consists of four technical areas invstigating novel technologies to: 1. prevent infection; 2. sustain survival; 3. provide transient immunity; and 4. create persistent immunity. See: http://www.darpa.mil/Our_Work/DSO/Programs/7-Day_Biodefense.aspx

Advances in synthetic vaccine manufacturing, VLP's and even 3D bio-printing will significantly change our concept of bio-defence and the manufacturing of counter-measures. The incorporation of these counter-measures will additionally change how we approach threat reduction and possibly remove many of the traditional concerns at the technical level. This could increase interest in areas which have typically been an after thought.
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Sunday, February 23, 2014

Manufacturing of Recombinant Botulinum Neurotoxin Vaccine

PHOTO: http://iai.asm.org/

As the  Medical Countermeasure System Joint Vaccine Acquisition Program (MCS/JVAP), the advanced developer for the Department of Defence (DoD) responsible  for developing, producing and stockpiling FDA licensed vaccines to protect the Warfighter from biological agents puts out a Request for Information a requirement to develop and license a pre-exposure prophylaxis recombinant botulim neurotoxin serotype A and B vaccine, scientists, Jason Barash and Stephen Arnon at California Department of Public Health announced they had discovered the first new form of botulinum toxin in over forty years.  Moreover, the scientist published two reports describing their work online in the Journal of Infectious Diseases. As with previous study which could provide a recipe for would be bio-weaponeers, the genetic sequences were withheld.See: http://www.npr.org/blogs/health/2013/10/11/230957188/why-scientists-held-back-details-on-a-unique-botulinum-toxin

The recent discovery of Botulinum Type H, the most lethal of botulinum neurotoxins, with no known antidote is likely to concern bio-defence specialists. The previously known seven serotypes: A,B,C1,C2,D,E,F and G produced by the bacterium clostridium botilinum, block acetylcholine, a neurotransmitter which then leads to muscle paralysis. According to New Scientist, this is the first time a genetic sequence has been withheld from publication over security concerns. Patients are usually treated with monoclonal antibodies, immune proteins which react to the specific toxin type. In a March, 2013 report,  Medscape notes: 
In an abstract (Botulinum neurotoxin vaccines: past present, and future),  published by PubMed back in 2007, Smith, LA and Rusnak JM detail that 'In the early 1930's, a formalin-inactivated toxoid against botulium neurotoxin was first tested in humans. In 1965, a pentavalent botulinum toxiod (PBT) received Investigational New Drug (IND) status under the Centers for Disease Control's IND 161 (for at risk workers), and in 1991 under the United States Army's Office of the Surgeon General IND 3723 (for military deployment). This PBT vaccine has been shown to be safe with over 20,000 injections given to date, and continues to be used in at risk individuals. During the past decade, recombinant DNA technology has been employed to develop second generation vaccines to prevent botulism. Recombinant subunit vaccines utilizing the receptor-binding domains of botulinum neurtoxin (BoNT) have been shown to be safe and efficacious in protecting animal models against BoNT serotypes A, B, C1, D, E and F. In 2004, the first recombinant subunit vaccine [rBV A/B(Pichia pastoris) vaccine] was tested in humans during a phase 1 clinical trial. Results from that study demonstrated that the recombinant bivalent vaccine was safe and well tolerated at all dosage levels tested and stimulated serotype specific neutralizing antibodies among the majority of vaccine recipients. See: http://www.ncbi.nlm.nih.gov/pubmed/18197811

PHIL Image 12052
Clostridium innocuum bacteria
"The US Food and Drug Administration (FDA) last Friday approved the first botulism antitoxin to neutralize all 7 known botulinum nerve serotypes valuable versatility for a drug in the nation's emergency medicine cabinet against a bioterrorist attack. The heptavalent botulism antitoxin (BAT,Cangene) had been available on an investigational basis from the Centers for Disease Control and Prevention (CDC). Cangene began supplying doses of BAT to the US Strategic National Stockpile in 2007 under a 427 million contract with the Department of Health and Human Services, according to a company press release. The CDC will distribute the stockpile antitoxin." See: http://www.medscape.com/viewarticle/781389

I would agree with Cangene's statement to the effect that "Currently, no specific, licensed therapies are available to treat all seven known serotypes" and "Given the inability to rapidly detect and identify botulinum neurotoxin (BoNT) serotypes in emergency situations, there is a critical need for a single effective treatment against all BoNT serotypes." See: http://www.raps.org/focus-online/news/news-article-view/article/2849/could-cangenes-botulism-antitoxin-become-the-seventh-product-approved-under-the.aspx

As a defence specialist, I would suggest that it is wise to recall in 1995, Iraq admitted it had produced 19,000 liters of botulinum toxin (See: http://www.fas.org/spp/starwars/crs/98042705_npo.html). Botulinum is listed by CDC and WHO as a Category A agent. It can and has been weaponized and poses a risk as bio-warfare agent.  In a 2005 Proceedings of the National Academy of Sciences (PNAS) announced it would publish a paper that presented a mathematical model of the possible consequences of deliberate botulinum toxin contamination of the US milk supply. The paper was authored by Dr. Lawrence Wein of Stanford University Graduate School of Business and Yifan Liu a graduate student. An overview is available here: http://www.nytimes.com/2005/05/30/opinion/30wein.html?pagewanted=all 
While I sat in on the ethics meetings to decide if Wein and Liu's study should be published in full, the first time such a meeting convened due to security concerns, botulinum and its potential use a weapon came to the foreground. Protection against such toxins is a vital aspect of national security. 
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Jill Bellamy is an internationally recognized expert on biological warfare and defence. She has formerly advised NATO and for the past seventeen years has represented a number of bio-pharmaceutical and government clients working on procurement strategy between NATO MS and Washington DC. Her articles have appeared in the National Review, The Wall Street Journal, The Washington Post, The Sunday Times of London, Le Temps, Le Monde and the Jerusalem Post among other publications. She is a CBRN SME with the U.S. Department of Defence, Chemical, Biological, Radiological and Nuclear Defence Information Analysis Center and CEO of Warfare Technology Analytics, a private consultancy based in the Netherlands. She is an Associate Fellow with the Henry Jackson Society, UK.

up-coming: Merging Social Network Analysis with DARPA's Bio-Intelligence Chips (BIC)

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Emerging Technologies: Bio-hacking and the future of bio-terrorism




For some time concern has been raised over 3D and 4D technologies, (with synthetic biology the emerging technological forerunner of these concerns and the NSABB playing watchdog), with regard to how inherent de-skilling may reduce the technical threshold which inhibits most would-be weaponeers from developing and deploying a weaponized biological agent capable of mass destruction. At the somewhat more extreme end, bio-hacking could reduce barriers which are perhaps better left in place.  Bio-hacking  was put on the map in 2013 when molecular biologist Ellen Jorgensen delivered a TED talk about Genespace, the DIY science lab she opened in New York in late 2010. See: http://new.ted.com/talks/ellen_jorgensen_biohacking_you_can_do_it_too  


The lab Jorgensen oversees is one of approximately 45 DIY international science groups,of  more than twenty in the US. While some of these labs are rather extreme in their goals, emerging technology such as 3D bioprinting could theoretically reduce the knowledge needed to develop synthetic weapons. So far several of the bio-hacking groups seem to be content with using themselves in experiments and implanting magnets but coding life for the masses and or the non-scientific community, could become a lot easier in a relatively short period of time. 

"But we don't smuggle plutonium. We don't supply chemical weapons. We don't build rockets. Instead we have a hobby that the FBI believes could be so dangerous that they have come up with a special programme to make sense of it. That hobby is to play with genes, proteins and bacteria in our spare time in a homemade lab we constructed from scratch. We are part of a rapidly growing community of amateur geneticists, who are often labelled biopunks, or outlaw biologists. Or, better still, in an analogy to the computer programming enthusiasts of a generation ago, some call us bio-hackers. But instead of software code, we try to tinker with DNA, the code of life. The FBI has set up the Biological Countermeasures Unit ( ) one of their goals in preventing acts of terrorism is to reach out to leading names in the field to quiz them about what they do." See: http://www.bbc.com/future/story/20130122-how-we-became-biohackers-part-1

This surely must be cutting edge bio-security, however, how close are bio-hackers to actually crossing what was considered the technological threshold to creating what might even be considered synthetic biological weapons? After 911 and the US anthrax attacks, I advised governments that mass casualty bio-terrorism was not as simple as it was being touted. In fact I, and several other scientists, focused on state warfare laboratories, considering bio-terrorism not of real world interest. Emerging technology which results in de-skilling however, may make the life of the would be bioweaponeer far easier and reduce what was always considered to be rather insurmountable technical barriers, certainly in the deployment of a mass casualty weapon. 

biohackspace.org 

What is the current view of life sciences deskilling, given the increase in DIY science? Johnathan B.Tucker, a former long time colleague, presented an excellent analysis of the issue in his paper, "Could Terrorists Exploit Synthetic Biology? published in The New Atlantis, see: http://www.thenewatlantis.com/publications/could-terrorists-exploit-synthetic-biology, although notably before bio-hacker movement emerged more openly into the media with a cohesive defined goal and group structure. Tucker, in his analysis states: 

"Member of this second school point to a contradictory trend in biotechnological development that they claim will ultimately prove stronger. They note that the evolution of many emerging technologies involves a process of de-skilling that, over time, reduces the amount of tacit knowledge required for their use. Chris Chyba of Princeton, for example, contends that as whole-genome synthesis is automated, commercialized, and 'black-boxed,' it will become more accessible to individuals with only basic scientific skills, including terrorists and other malicious actors (16).De-skilling has already occurred in several genetic-engineering techniques that have been around for more than twenty years, including gene cloning (copying foreign genes in bacteria), transfection (introducing foreign genetic material into a cell), ligation (stitching fragments of DNA together), and the polymerase chain reaction, or PCR (which makes it possible to copy any particular DNA sequence several million fold). Although one must have access to natural genetic material to use these techniques, the associated skill sets have diffused widely across the international scientific community. In fact, a few standard genetic-engineering techniques have been de-skilled to the point that they are now accessible to undergraduates and even advanced high school students, and could therefore be appropriated fairly easily by terrorist groups." See:http://www.thenewatlantis.com/publications/could-terrorists-exploit-synthetic-biology   

Gerald Epstein, of the Center for Science, Technology and Security Policy, write that whole-genome synthesis 'appears to be following a trajectory familiar to other useful techniques: Originally accessible only to a handful of top research groups working at state of the art facilities, synthesis techniques are becoming more widely available as they are refined, simplified, and improved by skilled technicians and craftsmen. Indeed, they are increasingly becoming 'commoditized,' as kits, processes, reagents, and services become available for individuals with basic lab training." (17). In 2007 Epstein and three co-authors predicted that 'ten years from now, it may be easier to synthesize almost any pathogenic virus than to obtain it through other means," although they did not imply that individuals with only basic scientific training will be among the first to acquire this capability.(18)" See:http://www.thenewatlantis.com/publications/could-terrorists-exploit-synthetic-biology 

"To date, the de-skilling of synthetic genomics has affected only a few elements of what is actually a complex, multi-step process. Practitioners of de novo viral synthesis note that the most challenging steps do not involve the synthesis of DNA fragments, which can be ordered from commercial suppliers, but the assembly of these fragments into a functional genome and the expression of the viral proteins. According to a report by the U.S. National Science Advisory Board for Biosecurity, a federal advisory committee, "The technology for synthesizing DNA is rapidly accessible, straightforward and a fundamental tool used in current biological research. In contrast, the science of constructing and expressing viruses in the laboratory is more complex and somewhat of an art. It is the laboratory procedures downstream from the actual synthesis of DNA that are the limiting steps in recovering viruses from genetic material." (19)" See: http://www.thenewatlantis.com/publications/could-terrorists-exploit-synthetic-biology 

As technology emerges which contributes to deskilling and with the advent of DIY science, we may witness rather rapid advancements which overcome the long time presumed threshold. The bio-hacking community has emerged because techniques used in molecular biology have been de-skilled and the cost has dropped. 




"A couple of decades ago, it took three years to learn how to clone and sequence a gene, and you earned a PhD in the process. Now, thanks to ready made kits you can do the same in less than three days. Specialized materials and second hand equipment are much more affordable, not to mention more available. Machines for amplifying DNA can now be purchased online, whilst enzymes and chemicals for creating, manipulating and sticking together DNA an be ordered off the shelf. The cost of sequencing DNA has plummeted , from about 100,000 for reading a million letters or base pairs, of DNA code in 2001, to around 10 cents today. See:http://www.bbc.com/future/story/20130122-how-we-became-biohackers-part-1 full review: Warfare Technology Analytics
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Monday, February 17, 2014

How 3D Bioprinting would change a Wargame: Atlantic Storm




   Influenza virus Credit: Dr Paul Digard, Department of Pathology

Author's note: In 2007 I was invited to speak at a NATO instillation near Warsaw. I delivered a talk on variola major the causative agent of smallpox and highly pathogenic disease containment. At that time, the US Strategic National Stockpile was rapidly increasing its procurement of a number of vaccines and medical counter measures, including vaccine to prevent smallpox. The US strategic stockpile was comprised of vaccine from Acambis and Bavarian Nordic. As I noted in my discussion, NATO held only a virtual stockpile and one had wonder how a real response could be mounted by NATO, not just allocated to the Member States, holding their national stockpiles, but by NATO command, when they only had a virtual stockpile. Atlantic Storm, following on from Dark Winter, was in my view a conservative and realistic table top exercise. Below is the basic introduction to Atlantic Storm,  but what would happen if instead of strategic stockpiles we could use centrally located 3Dbioprinters? What if NATO had  3D bioprinters for example or even the World Health Organization? How would that change how we counter highly pathogenic and possibly deliberate outbreaks of disease? I do make a distinction between deliberate and  natural outbreaks, as a deliberate outbreak is likely to increase mortality rates. So here are the basics of Atlantic Storm:  
Atlantic Storm was a simulated bioterrorist attack which demonstrated the weakness of international public health and security systems when dealing with a sudden outbreak of highly infectious diseases. On 14 January 2005, ten heads of government from Europe and North America and the Director General of the World Health Organization (WHO; Geneva, Switzerland) were scheduled to meet for a ‘Transatlantic Security Summit' in Washington, DC, USA, to discuss the threat of international terrorism. On the eve of the meeting, news broke that citizens from several European countries appeared to have become ill with smallpox; shortly thereafter suspected smallpox cases appeared in the USA. Although the assembled leaders did not know it at the time, a radical terrorist group had obtained seed strains of Variola major—the virus causing smallpox—and deliberately released the virus in a number of main transport hubs and sites of commerce throughout Europe and North America. On 14 January, the heads of states who gathered in Washington were confronted with one of the worst nightmares imaginable: the use of contagious and deadly disease as a weapon. Full citation see:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1369238/

Just as Atlantic Storm began, the scientific journal Nature published a paper describing a new technology that allows the rapid and accurate synthesis of long DNA segments using standard laboratory chemicals (). In October 2005, scientists at the Centers for Disease Control and Prevention (Atlanta, GA, USA) published the reconstruction of the ‘Spanish' influenza virus, which killed at least 25 million people during the winter of 1918/19 (). These and other discoveries provide researchers with better tools and knowledge to develop new medicines and vaccines against infectious diseases. However, they also make the synthesis and modification of viruses and bacteria for criminal purposes more likely. Hundreds of biological laboratories around the world already have the technical capacity to synthesize or manipulate small viruses such as polio or flu. Smallpox, with its genome of approximately 200 kb, is technically more challenging, but within the next few years, technology will undoubtedly advance to the stage where the synthesis of Variola major—based on sequence information freely available on the worldwide web—will be possible. The age of engineered biological weapons is neither science fiction nor suspense thriller—it is here today ().
For decades, NATO and other security alliances have planned their response to all kinds of military crises. Planning with that degree of rigor and strategic and operational detail is also needed to cope with potential biological threats of international consequence. Such transatlantic cooperation is also at the core of many non-proliferation programmes, such as the US Department of Defense's Cooperative Threat Reduction Program or the G8 Global Partnership. These programmes seek to reduce the threat posed by weapons of mass destruction (WMD) by detecting, deterring and interdicting illegal trafficking in such items; improving the physical security of facilities and WMD materials; destroying chemical weapons agents; preventing radiological contamination by decommissioned Russian nuclear submarines; and providing former WMD programme personnel with a decent living so they will not seek to profit from selling their knowledge to terrorist organizations or states trying to acquire WMD. However, biosecurity has often been an orphan of such programmes and must be given both higher priority and more resources commensurate to the challenge (; Dalgaard-Nielsen & Hamilton 2005). “We live in a time of new threats… What we now see is that health and security go together, so we have to combine them, and I think the lesson we should draw from this…is that we don't have the organizational structures to deal with the new threats,” commented Jan Eliasson, who acted as the Swedish Prime Minister in Atlantic Storm. Similarly, Sir Nigel Broomfield (Fig 3), who played the British Prime Minister, said after the exercise, “we have a globalized economy and globalized society, but we don't yet have globalized effective institutions to deal with the questions that come out of the globalization process.”http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1369238/

If we consider not only the time it takes to deploy a vaccine, and the US CDC estimates the time at about twelve hours, and we consider international travel, this time frame becomes critical. This photo below illustrates one aspect of what is involved in deploying medical countermeasures. If however, a state or institution such as NATO or WHO could manufacture vaccines on site, something akin to having fire hydrants strategically located throughout a city for putting out fires and easily accessible to all, containing disease would be far more efficient and effective. 


Click photo for screen-resolution image


Air Force Senior Airman Jacob Lloyd, a member of the 153rd Logistics Readiness Squadron, moves simulated vaccines onto a C-130 Hercules as part of the Strategic National Stockpile exercise in Wyoming. May 14, 2012. Airmen assigned to the Wyoming Air National Guard worked with multiple state agencies to test state health officials' abilities to receive, deliver and distribute medical supplies to various parts of the state. (Air National Guard, photo by 1st Lt. Rusty Ridley)

While it is prudent to understand that such technology can potentially be used to manufacture biological warfare agents, I believe the benefits far outweigh this possibility. In terms of Atlantic Storm, 3D bioprinting would remove policy decision making on vaccination/containment strategy, logistical issues involved in deploying a stockpile, remove transnational issues related to supply and rapidly increase our ability to respond at the first instance. One of the issues particularly with regard to smallpox vaccine availability within EU/NATO states is inconsistent stockpiles. One can imagine if there were an outbreak of smallpox in Africa, given lengthy incubation periods,  flights arriving into Schipol or Zaventem would not raise any alarm until the first cases started emerging. By this time, Belgium which holds only slightly over a 15% coverage would see citizens possibly trying to cross the boarder to the Netherlands where they hold a 100% so 1:1 stockpile. 3D bioprinting could theoretically remove all these issues possibly making bio-terrorism and warfare a less attractive option. 
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For an interactive view see: http://www.atlanticstorm.org/flash/

Sunday, February 16, 2014

3D Bio-printing: Changing how we counter the threat of biological warfare and terrorism


As 3D bioprinting edges closer to revolutionizing the bio-pharmaceutical industry, battlefield medicine, public health infrastructures and a host of other fields, Organovo is by far the leader in 3D bioprinting, printing the first liver tissue for testing and creating strips of liver tissue  within a single plate. In 2012, Organoovo printed the first human blood vessel without the use of scaffolds. A deeper description is provided by Christopher Barnatt of Explainingthefuture.com: 

"Since 2008, Organovo has worked with a company called Invetech to create acommercial bioprinter called the NovoGen MMX. This is loaded with bioink spheroids that each contain an aggregate of tens of thousands of cells. To create its output, the NovoGen first lays down a single layer of a water-based bio-paper made from collagen, gelatin or other hydrogels. Bioink spheroids are then injected into this water-based material. As illustrated below, more layers are subsequently added to build up the final object. Amazingly, Nature then takes over and the bioink spheroids slowly fuse together. As this occurs, the biopaper dissolves away or is otherwise removed, thereby leaving a final bioprinted body part or tissue.

bioprinting stages

As Organovo have demonstrated, using their bioink printing process it is not necessary to print all of the details of an organ with a bioprinter, as once the relevant cells are placed in roughly the right place Nature completes the job. This point is powerfully illustrated by the fact that the cells contained in a bioink spheroid are capable of rearranging themselves after printing. For example, experimental blood vessels have been bioprinted using bioink spheroids comprised of an aggregate mix of endothelial, smooth muscle and fibroblast cells. Once placed in position by the bioprint head, and with no technological intervention, the endothelial cells migrate to the inside of the bioprinted blood vessel, the smooth muscle cells move to the middle, and the fibroblasts migrate to the outside.

In more complex bioprinted materials, intricate capillaries and other internal structures also naturally form after printing has taken place. The process may sound almost magical. However, as Professor Forgacs explains, it is no different to the cells in an embryo knowing how to configure into complicated organs. Nature has been evolving this amazing capability for millions of years. Once in the right places, appropriate cell types somehow just know what to do.

In December 2010, Organovo create the first blood vessels to be bioprinted using cells cultured from a single person. The company has also successfully implanted bioprinted nerve grafts into rats, and anticipates human trials of bioprinted tissues by 2015. However, it also expects that the first commercial application of its bioprinters will be to produce simple human tissue structures for toxicology tests. These will enable medical researchers to test drugs on bioprinted models of the liver and other organs, thereby reducing the need for animal tests.

In time, and once human trials are complete, Organovo hopes that its bioprinters will be used to produce blood vessel grafts for use in heart bypass surgery. The intention is then to develop a wider range of tissue-on-demand and organs-on-demand technologies. To this end, researchers are now working on tiny mechanical devices that can artificially exercise and hence strengthen bioprinted muscle tissue before it is implanted into a patient." See:http://www.explainingthefuture.com/bioprinting.html  

Researchers at Human Methodist Research Institute have developed a more efficient way to create cells called Block Cell Printing. This process allows 100 percent of the cells to live instead of the current 50 to 80 percent which normally survive during the current process. http://www.techrepulblic.com/article/10-industries-3d-printing-will -disrupt-or-decimate. While this technology is exciting from a number of perspectives, it could also prove highly efficacious in countering bio-terrorism and or bio-warfare agents. If a highly pathogenic disease were to be released, the ability to swiftly print vaccine or medical counter measures on site would certainly have a higher chance of successful containment and at a far earlier stage, than currently exists today with logistic challenges. From a bio-defence perspective, it's the pace not the space that makes the difference. 3D bio-printing will very likely change our concept of disease prevention and containment and is particularly well suited to counter bio-terrorism or bio-warfare where response time is critical to countering a planned, multi target strike. In more discreet ways bio-printing is likely to change how we conduct issues such as epidemiological tracebacks and will effect crisis management and  policy decisions.

Sunday, February 9, 2014

Assessing a BW Laboratory: Technical Methods Part Two

Author's note: All material below is drawn from open source literature and is available on line. While it is tempting to consider that the type of laboratory for which identification is critical would be at a BSL 3 or 4 level, this is not generally the case. Typically clandestine programs use lower level lab security to work on highly pathogenic agents. In some instances, a BSL 3 can utilize BSL 3+ in decontamination processess and even with regard to bio-safety standards typically found in Western laboratories. The photos provided of BSL 3 and 4 facilities are not intended to represent necessarily the type of facility that would typically be involved in work on highly pathogenic or Category A agents in a clandestine weapon lab or infrastructure. In fact BSL 4 in particular are rather rare (some will argue that the numbers have increased dramatically over the past two decades, but proportionately they are the exception). A BSL 4 usually stands out even in an attempt to conceal weapon research. Unless its burried it will incure some speculation so the type of laboratory infrastructure which this paper addresses and which I wrote back in 2009, is intended to review more mundane laboratories BSL 1, 2 and 3.

Biosafety Level 4 (BSL-4) laboratories are among the most complex facilities to design, operate and maintain. See:http://www.absa.org/abj/abj2011v16n2.html


Let's take a look at methods used to identify underground facilities as this is problematic. both in terms of nuclear weapon programs but also very much in terms of biological weapon laboratories.  Assessing an above ground laboratory for BW traits posses fewer obsticals. A National Security Archive Electronic Briefing Book No. 372 entitled: Underground Facilities: Intelligence and Targeting Issues, provides background into concerns arising over the use of underground or hardened facilities. The archieve brief states:

"The concern about underground facilities (or 'hard and buried' targets) is evident  in the establishment of a number of components in various intelligence and defence agencies. The National Reconnaissance Office has a Hard and Buried Targets Working Group, while the National Geospatial Intelligence Agency, had (as of 2005) an Information and Underground Issues Division in its analysis directorate, and by 2008, the Defence Threat Reduction Agency had Hard Target Research and Analysis Center (4). But the most significant indication of the concern about underground facilities was the establishment, in 1997 of the Underground Facility Analysis Center (UFAC), which while subordinate to DIA also relies on participatoin from a number of other intelligence agencies. Exactly how many facilities around the world fall into each category is not clear, particularly not at the unclassified level. But a 2001 report to Congress (Document23) noted the Intelligence Community's suspicion that there were over 10,000 potential hard and buried targets and that number owuld increase over the next decade. 

U.S. intelligence requirements with respect to monitoring underground facilities can be divided into four basic categories. The first is verifying the existence of such a facility at a specific location, hints of which may come from intelligence sources or claims that may emanate from defectors. A second requirement is determining the facility's mission--whether it be leadership protection, weapons production, weapons storage or something else. The third requirement is the develoment of specific intelligence concerning the facility-including its physical layout and size, the number of personnel, the equipment present, and its capability and/or output-whether that be the number of troops that can pass through a tunnel, the number of weapons stored, or the facility's ability to produce enriched uranium or a biological agent. The intelligence developed concerning those requirements can be employed to assess a foreign capabilities, monitor treaty compliance, as well as plan or conduct military operations." 


Laboratory Identification


Considering the analysis required to determine if an underground laboratory exists is largely the pervue of the US intelligence community (IC). As a general rule, because most BW programs have tended to be embedded in legitimate research, civilian bio-pharma infrastructures (hiding in plane site approach), I will focus on above ground laboratory BW infrastructures first, then discuss military labs which have a higher probability of being constructed specifically to avoid identification. A number of characteristic are present in clandestine WMD programs, not solely related to BW research and development however, a hallmark of BW is compartmentalization. Nearly all clandestine biological weapon programs share this trait. This characteristic has historically diverted indentification of said infrastructures. It should also be noted that while most pharmaceutical firms are subordinate to the Ministry of Health or Education, institutes suspected of conducting BW research are often subordinate to the respective Ministry of Defence or state security services. Moreover, laboratory scientific staff may be drawn from sections of the military or have a military or security service background. The interface between military and civilian institutions, a sophisticated clandestine network covering tens or hundreds of facilities or any other military or security section is highly concerning. At the facility level there are often, but not always, specific indicators of a possible BW program.

John Pike detailed criteria for assesing a potential weapon lab at the technical level which often poses the most difficulty. I have argued for the use of network analysis in identifying would be weaponeers across a latent infrastructure but Pike's work is truely inspiring.  In his work entitled: Biological Warfare Agent Production, see: John, “Biological Warfare Agent Production”, Weapons of Mass Destruction, GlobalSecurity.Org.  http://www.globalsecurity.org/wmd/intro/bio_production.htm He offers an indepth analysis with rather precise indicators for assessing a biological weapon research and development laboratory and program:  . 

"The design of a production facility provides important information regarding whether the facility is intended to produce pharmaceutical grade products or biological weapon grade materials. Relevant design elements include containment, purification equipment, sterilization equipment, and ventilation and filtration systems.Relevant design elements include containment, purification equipment, sterilization equipment, and ventilation and filtration systems." 

The following overview provided by Pike in GlobalSecurity.Org., defines at the facility level and laboratory level, quite precise indicators for assessing a biological weapon research and development program: 


“The design of a biochemical processing plant is an important signal of covert biological agent production. Containment of the biological material during processing is of special interest. There is a clear distinction between processing materials for biological or toxin agent weaponization and processing protective agents to be used for countermeasures or personnel performance enhancement. For the production of biological agents for offensive military activities, the processing containment requirement is to protect the environment from the agent because of its infectious nature. For the production of biomaterials, such as vaccines, biological response modifiers, antibiotics, and anti-viral agents, for defensive military activities, the containment requirement is to protect the processed biomaterial from contaminating materials in the environment.”

“Effectiveness of countermeasures is enhanced by achieving high levels of purity and cleanliness in the product before it is administered to friendly personnel. By contrast, an unpurified biological agent that will be used in BW is generally more stable than the purified agent that is needed to produce vaccines and biological response modifiers (BRMs). Consequently, a proliferant does not require a high level of purity if production is for BW use only.”

“Generation of biological agents requires fermenters or single cell production capabilities including smooth, highly polished stainless steel surfaces, self-containment capability, and negative pressure conditions. The primary difference between the production requirements for biological weapons and non-military commercial purposes lies in containment and contamination. During biological agent production, efforts are generally made to avoid contaminating the environment with the organism. Less concern arises about the contamination of the product. Conversely, the pharmaceutical, brewing, and biotechnology industries are most concerned about protecting the purity and quality of the product. This concern is reflected in the nature of the sealing joints, positive or negative pressure chambers, and containment of venting systems. Utilities involving clean steam, sterile air, and inert gas supply are most critical for containment in the processing of biologically based materials for human use, which must meet good manufacturing practices (GMP). Clean steam, generated from a purified water supply, must be supplied to all processing equipment having direct contact with the product to ensure sterility and prevent the influx of environmental contaminants.”

“Steam sterilization is accomplished before product processing by direct supply to the equipment. Steam is supplied to the equipment seals (e.g., sample ports, agitator shafts, raw material addition ports) during processing as a primary barrier. Equally important is the removal of collapsed steam or condensate formed on the equipment. This prevents the formation of pockets of standing water, which promote bacterial growth, and maintains the high temperature necessary for sterilization. The collected contaminated condensate can be channelled to an area for final sterilization or inactivation before it is released into the environment. Efficient steam supply and condensate removal requires pressure regulators, pressure relief devices, venting, and the capability for free draining of all lines.”

“Supplying sterile, inert gases to processing equipment is a method of containment. This can protect oxygen-sensitive biomaterials and prevent aerosol generation of toxic products. Inert gases, such as nitrogen, helium, and argon, are usually supplied directly to processing equipment through sterile, in-line filters, maintaining a pressurized system or providing an inert blanket over the product in processing vessels.”

“To attain a higher level of containment, many bioprocessing industries have employed greater degrees of automation. Potential contamination of purified product, human exposure to toxic products or constituents, and the risk of human error are minimized. Processing facilities make use of state-of-the-art computerized distributed control systems (ABB, Modicon, Allen Bradley Corp.), which allow automatic control, control from remote locations, and automatic data logging and trending.”

“Another component in bio-processing is the design of ventilation within the primary and secondary barriers of a process area. Ventilation at primary barriers (i.e., barriers separating product from equipment operators and the rest of the processing area) is accomplished with dedicated, in-line air/gas membrane filters. Ventilation across secondary barriers requires more complicated air handling system design to allow for the maintenance of clean areas (rated by the number of particles per volume of air) and maintenance of positive or negative pressure between the processing area and the outside environment or between different processing areas in the same facility. Equipment used in these designs includes high efficiency fans and high efficiency particulate air (HEPA) filters.”

“The procedure used for the actual replication of an organism is a function of the organism itself. Techniques include cell culture, fermentation, viral replication, recombinant DNA, and powdering and milling. Cell culture is necessary for the reproduction of pathogenic viruses and Rickettsiae since they will not reproduce outside a living cell (e.g., chick embryo or tissue cultures). Single cell growth chambers, including fermentation, are used for the production of bacteria and bacterial toxins, although some bacteria (e.g., plague bacteria) can also be cultivated in living animals. Recombinant DNA techniques are a preferred method to produce rare animal toxins. Because of the complexity of this technique, the capability is not as widespread as the others. Powdering and milling is the technique generally used to produce BW and toxin weapons (TW) agent particles having diameters less than or equal to 10 mm, the size most effective for respiratory delivery.”

“Toxins and pathogens that affect animals, such as anthrax, brucella, plague, and tularemia, are widespread. Vaccines are widely produced and administered. The issue of the need for the same toxic agent for either BW/TW production or countermeasure vaccine production emphasizes the dual-use nature of the technologies. Indeed, initial processing of agents and processing of their associated vaccines only differ by a few steps (e.g., the degree of purification and the type of containment used).”

While we may be able to assess a lab above ground, I well imagine the criticizm will be 'what if the lab is underground?' The criteria above would not apply. After international criticism that the United States failed to find a biological weapon program in Iraq, which I would argue, after extensive talks with weapons inspectors and then heads of those inspection teams, existed but was never presented to the media or public in a way they could understand, intent on finding an 'obsolete' stockpile, not realizing that biological weapon programs simply were no longer constructed along the old Soviet model of stockpiling BW in vast quantities. In fact I would argue an agile program is far more efficient and deadly than a strategic stockpile, particularly if we are looking at profiles that involve state sponsored and highly trained terrorist organizations such as Hezbollah.


The BBC has a rather nice overview of the first round of inspections for interest please see: http://news.bbc.co.uk/2/hi/uk/2180237.stm
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Saturday, February 8, 2014

BW Infrastructure Analysis in a Stand Off Environment: Dispelling Political Myths First


Often times criticism is leveled at the bio-defence community versus the non-proliferation community, a significant distinction in approach, when it comes down to the public understanding of  how potential biological weapon facilities might be identified, or mistakenly identified as the criticism goes. A glance back at the UNSCOM and UNMOVIC inspections is rather disheartening but not for lack of identifying or finding BW. Not only did the public fail to understand what Iraq's biological weapon programs looked like, indeed they would not have had even general knowledge of what any biological weapon program would look like, but the media portrayal of antiquated Soviet stockpiles promoted the impression to the public that inspectors were out in the dessert searching for a secret cache of Scud-B's or R-400's tipped with anthrax or smallpox. Moreover, it didn't seem to matter if the stockpile was chemical or biological because this distinctions also goes by or is lumped together as 'bio-chem.' A stockpile is a stockpile. I imagine the vision that popped into the public's head was something akin to this:


Chemical weapon stockpile Mustard gas Syria


Instead of this:

Vials: A total of 97 vials-including those with labels consistent with the al Hakam cover stories of single-cell protein and biopesticides, as well as strains that could be used to produce BW agents-were recovered from a scientist's residence. See: Statement by David Kay on the Interim Progress Report on the Activities of the Iraq Survey Group (ISG) before the House Permanent Select committee on Intelligence and the House Committee on Appropriations Subcommittee on Defence, and the Senate Select Committee on Intelligence, Source: http://www.odci.gov/cia/public affairs/speeches/2003/david kay 10022003.html. 



or far worse this:


A nightmarish and realistic concept of what a modern BW program is likely to consist of, biological laboratories where vaccines and medical counter measures are in full production. This is what a biological warfare complex generally consists of today. It is not identifiable, there is no tidy stockpile to be discovered and generally such a program exists in both legitimate manufacturing, whilst retaining the capability to produce warfare agent usually at relatively short notice, depending on the type of laboratory.

The view that a stockpile of BW is the only acceptable criteria for identifying an active or latent BW program is negligent at best, complacent and possibly dangerous at worst. Clinging to the stockpile criteria and dismissing infrastructure analysis or lab analysis as an aspect of  how current programs are constructed, negates the need to develop stand off methods for detection. As with most mythical creatures the search for the elusively portrayed 'stockpile' and the crushing criticism following the public's disappointment that there was no 'stockpile' lead to significant misconceptions regarding what constitutes a BW program and how to identify this either in country or in a standoff environment which I contend is technically possible today.

Searching for the Stockpile and believing that there was one or that lack of one equated to proof that there was no BW program in Iraq or anywhere else. Unfortunately the propagating of this myth that Iraq had no BW programs and the general criticism over the basis of going to war, has meant that nations who most likely do possess a BW program are perceived as not having one, due to public distaste for accusing a suspected state proliferator and the distaste of possibly going to war again on what is largely perceived to be false information. Believing BW are in nice neat stockpiles waiting to be discovered is like believing there are magical unicorns, then being disappointed when only horses are found. Of course it's easier to have a map that designates the sites, but in the case of most BW programs there is no map until after a country has announced it has a program and is willing to work with an inspection regime to decommission such weapons.



Political discussions aside whether or not identifying BW programs merits any deep consideration,  how do we identify a potential military BW lab or infrastructure in a standoff environment? Is it possible, beyond the dual use bio-tech infrastructure of most developed nations to identify a biological warfare complex? Is it possible within a field where dual-use is the rule, not the exception, to make such an identification, much like an assessment of a deep bunker nuclear weapon program?

Unlike nuclear weapon complex, with identifiable signatures, biological weapon facilities for the most part have been assessed as not possessing identifiable characteristics. Recently, Spenser Ackerman, published an article entitled: Beyond Radiation:   Pentagon Seeks Better Ways to Detect Nuclear Weapons.  For the full article see: http://www.wired.com/dangerroom/2013/02/nuclear-sensors/ If we compare how we assess a nuclear weapon program to that of a biological one in a standoff environment, it is comparing apples and oranges. It is worth understanding how nuclear programs are identified and then to take a look at current methods for assessing a BW program. See Assessing a BW Laboratory: Technical Methods Part Two

The BBC has a rather nice overview of the first round of inspections for interest please see: http://news.bbc.co.uk/2/hi/uk/2180237.stm
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Pakistan's Absent Biological Warfare Complex





Author's note: Its incredibly rare, in fact this is the first time, I've ever written about a state's non-existing biological warfare program. Although much has been written over the last couple decades with regard to Pakistan's nuclear program and proliferation by the AQ Khan network, its apparent lack of a BW program is notable. Open source literature is rather sparse on Pakistan's biological weapon programs, largely due to the fact that Pakistan does not, in my view, posses one. While an argument can certainly be made for most states in possession of a bio-pharmaceutical infrastructure that they maintain the capability to do so, it appears Pakistan has chosen not to pursue this path. 

"Pakistan signed the Biological and Toxin Weapons Convention (BTWC) in 1972, and ratified it in 1974.(1) Although it has a well developed biotechnology research and development infrastructure, there is no evidence of any Pakistani program to develop, produce, or stockpile biological weapons or agents." NTI See: http://www.nti.org/country-profiles/pakistan/biological/  
A Pakistani Think Tank: (Pakistan Defence:http://defence.pk/threads/pakistans-biological-and-chemical-weapons-capabilities.6106/) posted the statement below. While one might be tempted to debate the credibility of a Think Tank which promotes Pakistani defence issues and a specific agenda,  it is worth considering as it is in line with external, open and literary sources which I will review in relation to this statement below :

"Although allegations have been leveled against Pakistan for conducting research into biological warfare since the early 1990s, Pakistan is not suspected of either producing or stockpiling biological weapons (BW).[1] However, it is generally believed that Pakistan has a well developed bio-technology sector that is capable of supporting limited biological warfare-related research and development.[2] In 1996, the U.S. Department of Defense stated that Pakistan "had the resources and capabilities appropriate to conducting research and development relating to biological warfare," and "was conducting research and development with potential biological warfare applications."[3] But the U.S. government has not presented any evidence to corroborate its assertions.See: http//www.expressindia.com

The Pakistani government insists that it has never developed, produced, or stockpiled biological weapons or agents and that a bio-warfare program is not part of the country's defense matrix. Pakistan signed the Biological and Toxin Weapons Convention (BTWC) in 1972, which it ratified in 1974.[4] Since then, Pakistan has remained a vocal advocate for the success of the BTWC. During the various Review Conferences of the BTWC, Pakistani representatives have urged more robust participation from state signatories, invited new states to join the treaty, and as part of the non-aligned group of countries have made the case for guarantees for states' rights to engage in peaceful exchange of biological and toxin materials for purposes of scientific research.[5]

In the wake of Pakistan's May 1998 nuclear tests, the U.S. Department of Commerce imposed sanctions on a large number of government, quasi-government, and private sector entities suspected of participating in Pakistan's nuclear weapons and nuclear weapons-related delivery programs. In the process, the U.S. government also imposed sanctions on chemical and biological facilities on suspicion that they might be involved with chemical and bio-warfare programs. These four entities were: The Center for Advanced Molecular Biology, Lahore; Karachi CBW Research Institute; Karachi CW & BW Warfare R&D Laboratory; and the National Institute of Biotechnology and Genetic Engineering, Faisalabad.[6] Despite being sanctioned, there is no independent evidence to suggest that any of the above four institutes were or are engaged in offensive biological warfare programs.

Indeed, Pakistan's focus seems to be on preventing proliferation and protecting their national public health security from external BW threats. Although dual-use is a problem for any nation with a bio-technology infrastructure, the Pakistani government has worked to increase awareness of the threat of proliferation. NTI states:


"Pakistan's biotechnology sector has continued to expand in recent years, encompassing nearly 30 institutions dealing with biotechnology and genetic engineering.(8). Of greater concern than a dedicated BW program is the possibility that dangerous dual-use biological materials from these facilities could be inadvertently exported or fall into the hands of non-state actors as a result of possible weaknesses in Pakistan's export control and biological security systems. Since the mid-2000's Pakistan has increased its regulation of the biological industry, issuing a set of bio safety rules in 2005 which established a National Biosafety Committee to create guidelines, issue export licenses, and inspect facilities dealing with 'living modified organisms or genetically modified organisms." (9) Islamabad has also taken measures to improve its WMD-relevant export controls. See:http://www.nti.org/country-profiles/pakistan/biological/  It certainly is interesting to see a nation such as Pakistan which has entered the nuclear club, refrain from developing biological weapons which is a general track nations seeking Weapons of Mass Destruction tend to take. The Arms Control Association, a well known non-proliferation think tank state's:  No government has alleged Pakistan is violating its Biological Weapons Convention commitments. Islamabad has not filed a voluntary BWC confidence building declaration." However I will note that a large number of countries have failed to do so as welll, so this is not an indicator in any way of non-compliance. See: http://www.armscontrol.org/factsheets/pakistanprofile
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