Francisella tularensis is the causative agent of tularemia and a Tier I bioterrorism agent. In the 1900s, several vaccines were developed against tularemia including the killed "Foshay" vaccine, subunit vaccines comprising F. tularensis protein(s) or lipoproteins(s) in an adjuvant formulation, and the F. tularensis Live Vaccine Strain (LVS); none were licensed in the U.S.A. or European Union. The LVS vaccine retains toxicity in humans and animals-especially mice-but has demonstrated efficacy in humans, and thus serves as the current gold standard for vaccine efficacy studies. The U.S.A. 2001 anthrax bioterrorism attack spawned renewed interest in vaccines against potential biowarfare agents including F. tularensis. Since live attenuated-but not killed or subunit-vaccines have shown promising efficacy and since vaccine efficacy against respiratory challenge with less virulent subspecies holarctica or F. novicida, or against non-respiratory challenge with virulent subsp. tularensis (Type A) does not reliably predict vaccine efficacy against respiratory challenge with virulent subsp. tularensis, the route of transmission and species of greatest concern in a bioterrorist attack, in this review, we focus on live attenuated tularemia vaccine candidates tested against respiratory challenge with virulent Type A strains, including homologous vaccines derived from mutants of subsp. holarctica, F. novicida, and subsp. tularensis, and heterologous vaccines developed using viral or bacterial vectors to express F. tularensis immunoprotective antigens. We compare the virulence and efficacy of these vaccine candidates with that of LVS and discuss factors that can significantly impact the development and evaluation of live attenuated tularemia vaccines. Several vaccines meet what we would consider the minimum criteria for vaccines to go forward into clinical development-safety greater than LVS and efficacy at least as great as LVS, and of these, several meet the higher standard of having efficacy ≥LVS in the demanding mouse model of tularemia. These latter include LVS with deletions in purMCD, sodBFt , capB or wzy; LVS ΔcapB that also overexpresses Type VI Secretion System (T6SS) proteins; FSC200 with a deletion in clpB; the single deletional purMCD mutant of F. tularensis SCHU S4, and a heterologous prime-boost vaccine comprising LVS ΔcapB and Listeria monocytogenes expressing T6SS proteins.

Smallpox (variola) virus is considered a Category A bioterrorism agent due to its ability to spread rapidly and the high morbidity and mortality rates associated with infection. Current recommendations recognize the importance of oral antivirals and call for having at least two smallpox antivirals with different mechanisms of action available in the event of a smallpox outbreak. Multiple antivirals are recommended due in large part to the propensity of viruses to become resistant to antiviral therapy, especially monotherapy. Advances in synthetic biology heighten concerns that a bioterror attack with variola would utilize engineered resistance to antivirals and potentially vaccines. Brincidofovir, an oral antiviral in late stage development, has proven effective against orthopoxviruses in vitro and in vivo, has a different mechanism of action from tecovirimat (the only oral smallpox antiviral currently in the US Strategic National Stockpile), and has a resistance profile that reduces concerns in the scenario of a bioterror attack using genetically engineered smallpox. Given the devastating potential of smallpox as a bioweapon, preparation of a multi-pronged defense that accounts for the most obvious bioengineering possibilities is strategically imperative.

Francisella tularensisis, the causative agent of tularemia has been classified as a category A bioterrorism agent. Here, we present the crystal structure of apo and adenine bound form of the adenine phosphoribosyltransferase (APRT) from Francisella tularensis. APRT is an enzyme involved in the salvage of adenine (a 6-aminopurine), converting it to AMP. The purine salvage pathway relies on two essential and distinct enzymes to convert 6-aminopurine and 6-oxopurines into corresponding nucleotides. The mechanism by which these enzymes differentiate different purines is not clearly understood. Analysis of the structures of apo and adenine-bound APRT from F. tularensis, together with all other available structures of APRTs, suggests that (a) the base-binding loop is stabilized by a cluster of aromatic and conformation-restricting proline residues, and (b) an N-H···N hydrogen bond between the base-binding loop and the N1 atom of adenine is the key interaction that differentiates adenine from 6-oxopurines. These observations were corroborated by bioinformatics analysis of ~ 4000 sequences of APRTs (with 80% identity cutoff), which confirmed that the residues conferring rigidity to the base-binding loop are highly conserved. Furthermore, an F23A mutation on the base-binding loop severely affects the efficiency of the enzyme. We extended our analysis to the structure and sequences of APRTs from the Trypanosomatidae family with a destabilizing insertion on the base-binding loop and propose the mechanism by which these evolutionarily divergent enzymes achieve base specificity. Our results suggest that the base-binding loop not only confers appropriate affinity but also provides defined specificity for adenine.

In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of γ-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic, evidence-based method for preparing, concentrating, irradiating, and verifying inactivation of spore materials was developed. We demonstrate consistency of spore preparations across multiple biological replicates and show that two different DoD institutions arrive independently at comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 1010 colony forming units (CFU). Spore preparations from three different institutions in three strain backgrounds yielded similar decimal-reduction (D10) values and irradiation doses required to assure sterility (DSAL) to the point at which the probability of detecting a viable spore is 10-6 Furthermore, spores of a genetically-tagged strain of B. anthracis Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques.Importance: The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from Biological Select Agents and Toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide the data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated and tested such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for detection of agents of biological warfare and bioterrorism.

Terrorism [Byers et al.] is the intentional use of indiscriminate violence to create fear and terror as a means to achieve an ideological, financial, religious, or political aim. It is often used against non-combatant targets. Terror tactics may include biologic, chemical, nuclear, or explosive events. Biologic Bioterrorism is the intentional release of biological agents to cause illness or death in humans, animals, or plants. These agents may be bacteria, fungi, toxins, or viruses. They may be naturally occurring or human-modified. The agents are typically found in nature, but they may be altered in a laboratory to increase their resistance to antibiotics, and ability to spread in the environment. Biological and chemical agents may be spread through the air, food, or water. Terrorists use biological agents because they are often difficult to detect and illness onset may be delayed for hours to days increasing dispersal. Bioterrorism agents are a common choice for terrorists because they easy and inexpensive to produce, easy to disseminate, and can cause widespread effects. The challenge with bioweapons is that they may affect both enemy and friendly forces. Terrorists use biologic weapons as a method of creating mass panic. In the history of humanity, the intentional infliction of casualties on civilians was considered inappropriate. Civilians are usually not attacked for their own sake unless they happen to be living or working in an area that has tactical or strategic value. Unfortunately, bioterrorism agents are difficult to control and affect military personnel as well as civilian men, woman, and children. In the last 100 years, the United States and the international community have experienced multiple acts of terrorism and bioterrorism which have targeted civilians. World War I: Germany launched a biological sabotage campaign in France, Romania, Russia, and the United States by infecting horses and mules with glanders, a virulent disease. 1972: Two college students, Allen Schwander and Stephen Pera, were arrested for planning to poison the Chicago water supply with typhoid bacteria. 1984: The Bhagwan Shree Rajneesh followers in Oregon attempted to affect a local election by infecting doorknobs and salad bars in restaurants with Salmonella typhimurium bacteria. 1993: Aum Shinrikyo religious group released anthrax in Tokyo. 1995: Bombing of the Murrah Federal Building in Oklahoma City. 2001: Airline attack on the World Trade Center. 2001: Anthrax-laced of infectious anthrax were delivered to news media offices and the US Congress. 2009: Medical center attack at a Fort Hood, Texas. 2014: Bombing of the Boston Marathon. Present: Multiple bombings worldwide. Chemical During World War I there was widespread use of chemical weapons that often affected the civilian population resulting in many deaths. Since this war, most countries have agreed not to use chemical and biologic weapons. Unfortunately, with our present age of terrorism from individuals, splinter groups, and dictators, there is an ever-present risk that these agents will be used. Nuclear To date, other than the dropping of nuclear bombs by the United States to end World War II, there have been no recent acts of nuclear attack or nuclear terrorism. Nuclear terrorism is an act of terrorism in which a terrorist organization detonates a nuclear device. The possibility of terrorist organizations using nuclear is considered plausible as terrorists could acquire a nuclear weapon. However, despite thefts of small amounts of fissile material, all low-concern, there is no credible evidence any terrorist group has succeeded in obtaining the necessary multi-kilogram critical mass amounts of weapons-grade plutonium required to make a nuclear weapon. Explosive It is, unfortunately, becoming a common event on an international scale when terrorists use explosive devices to inflict casualties on the civilian population. Terrorism Definitions What is terrorism? What is a mass casualty incident? The United Nations definition of terrorism describes it as “an anxiety-inspiring method of repeated violent action, employed by (semi-) clandestine individual, group, or state actors, for idiosyncratic, criminal, or political reasons, whereby—in contrast to assassination—the direct targets of violence are not the main targets.” The Department of Defense defines terrorism as “the unlawful use of violence or threat of violence, often motivated by religious, political, or other ideological beliefs, to instill fear and coerce governments or societies in pursuit of goals that are usually political.”  The Federal Emergency Management Agency defines terrorism as “the use of force or violence against persons or property in violation of the criminal laws of the United States for purposes of intimidation, coercion, or ransom. Terrorists often use threats to create fear among the public, to try to convince citizens that their government is powerless to prevent terrorism, and to get immediate publicity for their cause”.  The US Code of Federal Regulations defines terrorism as “the unlawful use of force and violence against persons or property to intimidate or coerce a government, the civilian population, or any segment thereof, in furtherance of political or social objectives.” A mass casualty incident is defined terrorist act that generates more patients than available resources can manage using routine procedures. Terrorism is generally considered to be the use of force or violence outside the law to create fear among citizens with the intent to coerce some sort of action. Health professionals should be aware bioterrorism is a perfect vehicle for terrorists to strike fear into the hearts and minds of citizens in the hopes they will bend the will of the people to support their agendas. All health professionals need to be prepared for this potentially catastrophic event. [Aven et al. 2015]

Four isothermal recombinase polymerase amplification (RPA) assays were developed for fast in-field identification of Bacillus anthracis The RPA assays targeted three specific sequences (i.e., the BA_5345 chromosomal marker, the lethal factor lef [from pXO1], and the capsule-biosynthesis-related capA [from pXO2]) and a conserved sequence in the adenylate cyclase gene (adk) for the Bacillus cereus group. B. anthracis-specific RPA assays were tested first with purified genomic DNAs (n = 60), including 11 representatives of B. anthracis, and then with soil (n = 8) and white powder (n = 8) samples spiked with inactivated B. anthracis spores and/or other biological agents. The RPA assays were also tested in another laboratory facility, which blindly provided DNA and lysate samples (n = 30, including 20 B. anthracis strains). RPA assays displayed 100% specificity and sensitivity. The hands-off turnaround times at 42°C ranged from 5 to 6 min for 102 genomic copies. The analytical sensitivity of each RPA assay was ∼10 molecules per reaction. In addition, the BA_5345 and adk RPA assays were assessed under field conditions with a series of surface swabs (n = 13, including 11 swabs contaminated with B. thuringiensis spores) that were blindly brought to the field laboratory by a chemical, biological, radiological, and nuclear (CBRN) sampling team. None of the 13 samples, except the control, tested positive for B. anthracis, and all samples that had been harvested from spore-contaminated surfaces tested positive with the adk RPA assay. All three B. anthracis-specific RPA assays proved suitable for rapid and reliable identification of B. anthracis and therefore could easily be used by first responders under field conditions to quickly discriminate between a deliberate release of B. anthracis spores and a hoax attack involving white powder.IMPORTANCE In recent decades, particularly following the 11 September 2001 and Amerithrax attacks, the world has experienced attempts to sow panic and chaos in society through thousands of white-powder copycats using household powders to mimic real bioterrorism attacks. In such circumstances, field-deployable detection methods are particularly needed to screen samples collected from the scene. The aim is to test the samples directly using a fast and reliable assay for detection of the presence of B. anthracis While this would not preclude further confirmatory tests from being performed in reference laboratories, it would bring useful, timely, and relevant information to local crisis managers and help them make appropriate decisions without having to wait for quantitative PCR results (with turnaround times of a few hours) or phenotypic identification and sequencing (with turnaround times of a few days). In the current investigation, we developed a set of isothermal RPA assays for the rapid screening and identification of B. anthracis in powders and soil samples, with the purpose of discriminating a deliberate release of B. anthracis spores from a hoax attack involving white powder; this would also apply to dispersion by spraying of aerosolized forms of B. anthracis Further work is now ongoing to confirm the first observations and validate the on-site use of these assays by first responders.

Botulinum neurotoxin (BoNT) is the causative agent of botulism and a widely used pharmaceutical to treat a variety of neurological diseases. BoNTs are 150-kDa protein toxins organized into heavy chain (HC) and light chain (LC) domains linked by a disulfide bond. The HC selectively binds to neurons and aids cell entry of the enzymatically active LC. There are seven immunological BoNT serotypes (A to G); each serotype includes genetic variants, termed subtypes. Only two subtypes, BoNT/A1 and BoNT/B1, are currently used as therapeutics. BoNT serotype A (BoNT/A) subtypes A2 to A8 show distinct potency, duration of action, and pathology relative to BoNT/A1. Specifically, BoNT/A3 possesses shorter duration of action and elicits distinct symptoms in mice at high toxin doses. In this report, we analyzed the roles of LC and HC of BoNT/A3 for duration of action, neuronal cell entry, and mouse pathology by using clostridium-derived recombinant hybrid BoNTs consisting of reciprocal LC and HC (BoNTA1/A3 and BoNTA3/A1). Hybrid toxins were processed in their expression host to a dichain BoNT consisting of LC and HC linked via a disulfide bond. The LC and HC defined BoNT potency in mice and BoNT toxicity for cultured neuronal cells, while the LC defined the duration of BoNT action in cell and mouse models. Protein alignment identified a previously unrecognized region within the LC subtype A3 (LC/A3) relative to the other LC serotype A (LC/A) subtypes (low primary acid homology [LPH]) that correlated to intracellular LC localization. This study shows the utility of recombinant hybrid BoNTs with new therapeutic potential, while remaining sensitive to antitoxins and therapies to native BoNT.IMPORTANCE Botulinum neurotoxins are the most potent protein toxins for humans and potential bioterrorism threats, but they are also widely used as pharmaceuticals. Within the large family of BoNTs, only two subtypes are currently used as pharmaceuticals, with a large number of BoNT subtypes remaining as untapped potential sources for unique pharmaceuticals. Here, two recombinant hybrid toxins were engineered, consisting of domains from two BoNT subtypes that possess distinct duration of action and activity in human neurons and mice. We define the functional domains responsible for BoNT action and demonstrate creation of functional hybrid BoNTs with new therapeutic potential, while remaining sensitive to antitoxins and therapies to native BoNT.

Burkholderia pseudomallei is the causative agent of melioidosis and regarded as a bioterrorism threat. It can adapt to the nutrient-limited environment as the bacteria can survive in triple distilled water for 16 years. Moreover, B. pseudomallei exhibits intrinsic resistance to diverse groups of antibiotics in particular while growing in biofilms. Recently, nutrient-limited condition influenced both biofilm formation and ceftazidime (CAZ) tolerance of B. pseudomallei were found. However, there is no information about how nutrient-limitation together with antibiotics used in melioidosis treatment affects the structure of the biofilm produced by B. pseudomallei. Moreover, no comparative study to investigate the biofilm architectures of B. pseudomallei and the related B. thailandensis under different nutrient concentrations has been reported. Therefore, this study aims to provide new information on the effects of four antibiotics used in melioidosis treatment, viz. ceftazidime (CAZ), imipenem (IMI), meropenem (MEM) and doxycycline (DOX) on biofilm architecture of B. pseudomallei and B. thailandensis with different nutrient concentrations under static and flow conditions using confocal laser scanning microscopy. Impact of nutritional stress on drug susceptibility of B. pseudomallei and B. thailandensis grown planktonically or as biofilm was also evaluated. The findings of this study indicate that nutrient-limited environment enhanced survival of B. pseudomallei in biofilm after exposure to the tested antibiotics. The shedding planktonic B. pseudomallei and B. thailandensis were also found to have increased CAZ tolerance in nutrient-limited environment. However, killing activities of MEM and IMI were stronger than CAZ and DOX on B. pseudomallei and B. thailandensis both in planktonic cells and in 2-day old biofilm. In addition, MEM and IMI were able to inhibit B. pseudomallei and B. thailandensis biofilm formation to a larger extend compared to CAZ and DOX. Differences in biofilm architecture were observed for biofilms grown under static and flow conditions. Under static conditions, biofilms grown in full strength modified Vogel and Bonner's medium (MVBM) showed honeycomb-like architecture while a knitted-like structure was observed under limited nutrient condition (0.1×MVBM). Under flow conditions, biofilms grown in MVBM showed a multilayer structure while merely dispersed bacteria were found when grown in 0.1×MVBM. Altogether, this study provides more insight on the effect of four antibiotics against B. pseudomallei and B. thailandensis in biofilm under different nutrient and flow conditions. Since biofilm formation is believed to be involved in disease relapse, MEM and IMI may be better therapeutic options than CAZ for melioidosis treatment.