Executive Summary: Part II (Concluding part)
NATO Advanced Research Workshop: Preparedness against bioterrorism and re-emerging infectious diseases
Janusz Kocik MD, PhD
Military Institute of Hygiene and Epidemiology
Warsaw, Poland

APPLIED RESEARCH
           Representatives of several scientific centers presented their achievements in development of new vaccines, detection and identification, physical protection and decontamination.

Antitoxins
           Antisera are generated by immunising animals, such a goats or sheep, with an inactivated form of the toxin (such as a toxoid) followed by purification of the antitoxin from repeated production bleeds. The plasma obtained is purified to IgG. Adverse effects such as analphylaxis are common when patients are given whole IgG products derived from a foreign host. Dstl Porton Down, UK is looking for ways of producing effective antisera from fragments of the IgG, namely F(ab')2, Fab' and Fab. The rational behind these studies is that the reactogenic Fc portion of the molecule has been removed and thus the smaller molecules should result in fewer side effects. The smaller size of the fragments has the added advantage that they may reach their site of action more quickly than IgG and be more effective for post exposure therapy. This approach is currently being investigated for antitoxins against botulinum toxin and ricin.

Smallpox
           The vaccines licensed for use during the WHO global eradication program are known as first-generation vaccines. These vaccines are unlikely to be re-licensed because they were manufactured under conditions that would not meet current good manufacturing practice (cGMP). The modern, second-generation vaccines that are currently being developed are manufactured under cGMP. However, to be licensed, these second generation vaccines must undergo thorough clinical testing to prove safety and efficacy. Only first-generation smallpox vaccines have been proven to be effective in endemic conditions. Since the eradication of smallpox, it is impossible to carry out field trials with a new generation of vaccine. In addition, for obvious reasons, it is not possible to carry out reliable animal challenge models with smallpox virus. Thus, the efficacy tests available for inclusion in the clinical programmes for new smallpox vaccines are as follows:
1. Animal challenge models using other members of the Orthopox virus family.
2. Human inoculation studies based on development of a 'take' - the recognized indicator that a vaccine has induced protective immunity.
3. Human and animal immunology studies comparing development of neutralizing antibodies.
           The original smallpox vaccine Dryvax® has been effective against smallpox under endemic conditions in America, Asia and Africa. ACAM1000 was derived from Dryvax® and ACAM2000 was derived from ACAM1000. As part of the contract with the US Government, Acambis has developed a plan for licensure of ACAM2000. Pre-clinical challenge studies in mice and monkeys have shown ACAM2000 to have efficacy comparable with Dryvax®. In addition, it appears that ACAM2000 has an improved safety profile compared with Dryvax®. Specifically, the neurovirulence of ACAM2000 for mice and monkeys is reduced, which is likely to be associated with a lower risk of post-viral encephalitis in humans. To date, over 300 human subjects have been inoculated with our vaccine strains, with no serious adverse events. Initial results will be discussed and the full clinical programme will be presented.

Anthrax
           BW defense vaccine research at US Army Medical Research Institute for Infectious Diseases (USAMRIID) has led to the development of two new vaccine candidates against Bacillus anthracis and Yersinia pestis. The next-generation anthrax vaccine consists of purified recombinant protective antigen (PA), the principal component of anthrax exotoxins. The vaccine has been tested in two animal models for immunogenicity and direct efficacy against lethal inhalational anthrax, and has shown outstanding performance with only a one- or two-dose immunization regimen. Furthermore, passive transfer studies conducted to date in rabbits and mice have demonstrated the potential for anti-PA circulating antibody to be used as a surrogate marker for protection.

Plague
           Two killed whole-cell plague vaccines are currently available for use. Since the protection induced is highly variable and high levels of adverse reactions occur, the killed vaccines are unsuitable for general use, so are restricted to individuals who work with the pathogen or are potentially exposed to virulent strains. The killed vaccine fails to induce secretory IgA and may not protect against pneumonic transmission. A live vaccine is also available, derived from strain EV76. Strain EV76 is poorly defined, and resulted in excessive adverse reactions and highly variable responses between individuals. A second-generation vaccine of recombinant proteins developed at CBS Porton Down is currently undergoing clinical trials. The antigens in the formulation are the capsular F1 antigen and the V antigen, a component of the Type III secretion system. The first challenge in developing this second generation vaccine was obtaining a source of antigens which could be produced on a large scale. The caf operon encoding F1 antigen is carried on one of the three Y. pestis virulence plasmids. Cloning the caf operon allowed high level expression of recombinant F1 antigen, a much safer source than isolating the antigen from plague cultures. V antigen is more difficult to produce than F1 antigen due to inherent instability of the protein. It is produced as a GST fusion, which is purified and subsequently cleaved to give pure V antigen. The F1 and V antigens have been shown to be able to induce a protective immune response individually, but a combination of the proteins had an additive effect. Work is now underway to develop a vaccine which is mucosally delivered. In addition to inducing immune responses in the respiratory tract, mucosal delivery has the advantage of being needle-free. Much work has been undertaken to deliver the antigens using biodegradable polymeric microencapsulation. A preparation suitable for nasal administration has been produced which is fully protective against aerosol challenge in the mouse model after just two doses.
           Similarly in USAMRIID, a novel recombinant plague vaccine candidate, consisting of a protein fusion between Y. pestis F1 capsular antigen and V antigen (F1-V) has also been evaluated in two animal models. Results of direct efficacy in F1-V immunized Cynomologus macaque monkeys have shown high-level protection against lethal aerosol exposure to Y. pestis. These data represent the first time direct efficacy against pneumonic plague in the non-human primate demonstrated by a subunit vaccine. As with anthrax vaccine candidate, passive transfer experiments suggest that circulating antibodies to F1 and V antigens contribute significantly to protection against plague in the immune host. Preliminary pre-clinical safety and stability studies were also conducted on both vaccine candidates and yielded positive results.
           The recombinant products for both anthrax and plague represent excellent candidates for the next-generation vaccines against these BW threats. Pre-clinical development of these products continues at USAMRIID and Porton Down, and will ultimately facilitate their licensure. Both candidates also represent ideal platforms for multi-component, third-generation vaccines. Basic research is ongoing at USAMRIID to identify and characterize these additional components.

Tularemia
           The Live Vaccine Strain (LVS) vaccine remains the only effective vaccine against tularemia developed to date. However, this vaccine is not currently available, though work to licence it is underway in the USA. In a range of other pathogens, the introduction of defined mutations into genes required for growth of the pathogen in vivo has yielded safe and effective vaccines. The construction of a defined attenuated mutant of F. tularensis could provide a safe, effective and licensable tularemia vaccine. The aromatic amino acid and purine biosynthetic pathways have already been identified from genome sequence information as targets for the construction of a defined attenuated mutant. However, the utility of this approach is limited because there are as yet no methods for the construction of allelic replacement mutants of F. tularensis. Work is currently underway at CBS Porton Down to develop methods to create isogenic allelic replacement mutants. In one of these mutants the transposon interrupts a gene required for purine biosynthesis, and as stated above, purine mutants are of interest because in other bacterial species they have been used as attenuated vaccine strains.
           A second approach followed in Porton Down is the evaluation of subunit vaccine antigens. One such potential protective subunit is lipopolysaccharide. This may form one part of a vaccine, but would require additional antigens for full protection. Analysis of the immune response to the LVS vaccine has shown heterogeneity of immunogenic epitopes recognized in humans and this indicates it is likely that a sub-unit vaccine will need to be composed of a number of protective antigens to provide protection against virulent strains.

New detection and identification tools
           The Defense Advanced Research Projects Agency (DARPA) is developing technologies for detecting biological materials in the natural environment. While several technologies show promise as broadband detectors, there is no "silver bullet" that detects all chemical and biological materials at the requisite levels of sensitivity and specificity. DARPA is developing enabling components for use in biological detectors. Recent advances in genomics and proteomics have generated new strategies for the development of nucleic acid- and immunochemical-based detection assays as well as rapid fingerprinting systems for biological warfare agents (BWA). The increasing list of sequenced and annotated microorganisms has enhanced the data obtained from comparative genomics and proteomics. In silico studies have facilitated the identification of putative signature sequences for various BWA and provided repetitive sequences that may be exploited in future rapid fingerprinting methods. Suppressive subtractive hybridization investigations of closely related strains and species of BWA have also pinpointed signature sequences for use in probe assays. Proteomics studies using 2 D-gels and MALDI-TOF of spores, secretomes under simulated host cell conditions, as well as vegetative cells have been used in determining differences that can be applied to probe assays. Protein chip SELDI-TOF can be used to observe differences of BWA outer membrane and exosporium proteins. All of these strategies are now being applied to technical platforms that will allow rapid detection and fingerprinting.

Phage Lytic Enzymes
           Phages have an ability to lyse bacteria using conserved catylitic enzymes (Muramidase, Glucosaminidase, Endopeptidase, L- alanine amidase) and variable cell-wall associated substrates which specifically recognize bacteria. The methods for obtaining pure phage associated lytic enzymes have been elaborated New Horizon Diagnostics, Columbia, USA. The effect is highly strain specific. B. anthracis-Specific Lytic Enzyme has been cloned from gamma-phage specific for B. anthracis and few closely related B. cereus such as RSVF1) The enzyme kills 5 different mutant strains of B. anthracis (delta Sterne, VNR1 delta 1, delta Ames, NNR1 delta 1 and delta NH1) and 10 virulent B. anthracis strains isolated worldwide A1a, A1b, A2, A3a, A3b, A4, B, Sterne, Vollum, Ames). It does not kill unrelated strains such as B. cereus or B. thuringiensis. Therefore phage lysins can potentially be used for specific decontamination. Moreover, as phage lysins lyse only the targeted pathogen enriching a sample in bacterial ATP, they may be used to augment luminescence signal in luminometer rapidly identifying specific bacteria.

Integrating Biomanufacturing with Research
           A flexible, creative, and rapidly responsive biomanufacturing infrastructure is an essential part of an effective overall strategy for bioterrorism preparedness and biological defense. A variety of approaches and technologies are evolving to provide the capacity to bring innovations in biological threat detection, prophylaxis, and therapeutics from the laboratory bench to advanced development and ultimately to the enduser and/or the marketplace. The products of biotechnology research, including affinity reagents, PCR probes and primers, molecular elements for microarray manufacture, therapeutic peptides, and vaccines, each have unique requirements for their production at useful scales. Another critical feature of a flexible bio-manufacturing facility is the ability to archive a variety of biological materials in a secure fashion. The multi-faceted approach to biological manufacturing being advanced by the U.S. Army Edgewood Chemical Biological Center and its partners in government, academia and industry. State-of-the-art biological manufacturing methods (efficient cell culture reactors, cost analysis studies) as well as traditional methods (fermentation) and an advanced cryorepository are being used to solve problems in biological agent detection, agent simulation, environmental decontamination and the production of biologicals for human clinical trials. Results from additional research on real-time optical monitoring of in vivo production of recombinant proteins are available.

Decontamination
           New plasma-chemical processes of decontamination of the biologically polluted water and liquid media are the ones with one of the most potential for theoretical and applied research. As a result of action of nonequilibrium low-temperature plasma the active particles, radicals and other compounds are generated in liquid promoting decontamination. The research at the establishments of the Ministry of Public Health of Ukraine and Russia has shown that as a result of processing of water and liquid media containing the pathogenic and conditionally - pathogenic microorganisms: According to the authors such technology can become reliable guarantee of safe consumption of drinking water during massive epidemics, including mass contamination of water in bioterrorism attack scenario.
           As shown by researchers from Ukrainian National Academy of Science metal-containing adsorbents, ultrathin synthetic fibres and various biologically active compounds (biophores) introduced in polyurethane matrixes during their synthesis can be another effective method of water and air purification.

NEW THREATS
Terrorist Threat to Food
           Supplies Historically, consumables are vulnerable to lower BW technology approach. Costs of the mass casualties causing attack and risk to perpetrators are also lower comparing to other means of dissemination. Food contamination allows for more select targeting. It results in loss of confidence in the food supply and has major economic impact. Distinguishing natural outbreak vs. terrorist activity is extremely difficult. Terrorists can contaminate food sources with biologic agents at all points from production sites, through supply channels, in processing and storage, to final preparation and serving. Preparedness and response system should identify points along food chains where threats to the safety are greatest and recommend security control measures to minimize threat potential in the food chain. Simple methodology should be developed to identify food supply chains and potential threats in that chain to be used for making location or situation specific vulnerability assessments. Security Public health, food service, transport and contracting personnel are the key points in chain of custody. Protection of food and water systems must be a balanced approach between an understanding of the threat, fully implementing routine food & water safety measures, application of technology, & improved safeguard and security measures. Detection is not perfect. No single/multiple technology or approach is available today to detect 100% of possible biological/chemical agents prior to consumption.

Genetically Modified Organisms (GMO) as means of bioterrorism.
It should be taken into account that food/feed have different value in different parts of the world. Novel technologies open the possibilities to treat food/feed not only as political weapon, but also as biowarfare. Preparation of food containing toxic products is easy. However, as described before its delivery (transportation and distribution) can be prevented on many levels. Nevertheless the sociological effect of the news "Poison in Food" should be taken into account. The public acceptance of genetic engineering is already minor and the society's awareness is relatively low. The sociological effect of "bio-socio-terror" would be tremendous. For today preparedness against this threat is null.

CONCLUSIONS
           This NATO Advanced Research Workshop was designed to examine all possible directions a country should aim for in building its preparedness and capacity to response to biological attack. It achieved its goal to gather specialists from relevant fields including: epidemiologists, infectious diseases specialists, NBC specialists, medical planners, researchers in the fields of vaccines, biological agents detection and identification, physical protection and decontamination. This forum allowed for the integration of thinking, elaboration of a common approach to the problem and the exchange of experiences. Building preparedness for response against bioterrorism is a very complex issue, which requires integration of work of the relevant institutions on the national level. However, the disease does not know boundaries so preparedness should be built from the beginning with "thinking-outside-of-the-box" approach, taking into account international interdependence. This requires unification of epidemiological and laboratory procedures as well as public health planning in the countries of the Region. However taking into account differences in the health systems of the countries, this leveling of the approaches seems to be a very complicated goal. NATO gives us a possibility to come near to this goal. The main recommendation that comes from this forum is the establishment of permanent forum for a further unification effort. Establishment of the NATO NBC training center in Central Europe could only help this effort of building public health preparedness for a unified response to biological attack. Aware of the current international situation, the Workshop participants recognized the need for international team(s) properly trained, equipped and vaccinated to be dispatched on very short notice in suspected BW contaminated areas in order to assess the situation and provide advise to the local authorities and relief organizations. WHO or other bodies, if in a position to do so, is invited to establish an inter-agency mechanism for information sharing and coordination of activities on preparedness and response to CBW threats to humans, animals and plants.

Editor's Note: Our congratulations to Dr. Janusz Kocik and the MIHE team for a super job in putting together this outstanding NATO ARW. The information transferred and knowledge gained, and the friendships acquired or renewed across this complex arena, says it all.