Synsor: a tool for alignment-free detection of engineered DNA sequences.

DNA sequences of nearly any desired composition, length, and function can be synthesized to alter the biology of an organism for purposes ranging from the bioproduction of therapeutic compounds to invasive pest control. Yet despite offering many great benefits, engineered DNA poses a risk due to their possible misuse or abuse by malicious actors, or their unintentional introduction into the environment. Monitoring the presence of engineered DNA in biological or environmental systems is therefore crucial for routine and timely detection of emerging biological threats, and for improving public acceptance of genetic technologies. To address this, we developed Synsor, a tool for identifying engineered DNA sequences in high-throughput sequencing data. Synsor leverages the k-mer signature differences between naturally occurring and engineered DNA sequences and uses an artificial neural network to classify whether a DNA sequence is natural or engineered. By querying suspected sequences against the model, Synsor can identify sequences that are likely to have been engineered. Using natural plasmid and engineered vector sequences, we showed that Synsor identifies engineered DNA with >99% accuracy. We demonstrate how Synsor can be used to detect potential genetically engineered organisms and locate where engineered DNA is being introduced into the environment by analysing genomic and metagenomic data from yeast and wastewater samples, respectively. Synsor is therefore a powerful tool that will streamline the process of identifying engineered DNA in poorly characterized biological or environmental systems, thereby allowing for enhanced monitoring of emerging biological threats.

Continuous Sampling of Aerosolized Particles Using Stratified Two-Phase Microfluidics.

Health and security concerns have made it essential to develop integrated, continuous collection and sensing platforms that are compact and capable of real-time detection. In this study, we numerically investigate the flow physics associated with the single-step collection and enrichment of aerosolized polystyrene microparticles into a flowing liquid using a stratified air-water flow in a U-shaped microchannel. We validate our simulation results by comparing them to experimental data from the literature. Additionally, we fabricate an identical microfluidic device using PDMS-based soft lithography and test it to corroborate the previously published experimental data. Diversion and entrapment efficiencies are used as evaluation metrics, both of which increase with increasing particle diameter and superficial air inlet velocity. Overall, our ANSYS Fluent two-dimensional (2D) and three-dimensional (3D) multiphase flow simulations exhibit a good agreement with our experimental data and data in the literature (average deviation of ∼11%) in terms of diversion efficiency. Simulations also found the entrapment efficiency to be lower than the diversion efficiency, indicating discrepancies in the literature in terms of captured particles. The effect of the Dean force on the flow physics was also investigated using 3D simulations. We found that the effect of the Dean flow was more dominant relative to the centrifugal force on the smaller particles (e.g., 0.65 µm) compared to the larger particles (e.g., 2.1 µm). Increasing the superficial air inlet velocity also increases the effect of the centrifugal forces relative to the Dean forces. Overall, this experimentally validated multiphase model decouples and investigates the multiple and simultaneous forces on aerosolized particles flowing through a curved microchannel, which is crucial for designing more efficient capture devices. Once integrated with a microfluidic-based biosensor, this stratified flow-based microfluidic biothreat capture platform should deliver continuous sensor-ready enriched biosamples for real-time sensing.

Rinderpest: A Disease of the Past, and a Present Threat.

Rinderpest is a highly contagious viral disease that affects ungulates such as cattle, buffalo, yak, and various wildlife species, leading to significant morbidity and mortality. The global eradication of rinderpest was successfully accomplished in 2011 through extensive vaccination efforts. Today, safeguarding against the re-emergence of rinderpest in animal populations is paramount. The Food and Agriculture Organization of the United Nations and the World Organization for Animal Health are entrusted through a series of resolutions with the responsibility to prevent the re-emergence of rinderpest in animals.

Efficacy of therapeutically administered gepotidacin in a rabbit model of inhalational anthrax.

Bacillus anthracis is a Gram-positive Centers for Disease Control and Prevention category "A" biothreat pathogen. Without early treatment, inhalation of anthrax spores with progression to inhalational anthrax disease is associated with high fatality rates. Gepotidacin is a novel first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and is being evaluated for use against biothreat and conventional pathogens. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode and has in vitro activity against a collection of B. anthracis isolates including antibacterial-resistant strains, with the MIC90 ranging from 0.5 to 1 µg/mL. In vivo activity of gepotidacin was also evaluated in the New Zealand White rabbit model of inhalational anthrax. The primary endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. The trigger for treatment was the presence of anthrax protective antigen in serum. New Zealand White rabbits were dosed intravenously for 5 days with saline or gepotidacin at 114 mg/kg/d to simulate a dosing regimen of 1,000 mg intravenous (i.v.) three times a day (TID) in humans. Gepotidacin provided a survival benefit compared to saline control, with 91% survival (P-value: 0.0001). All control animals succumbed to anthrax and were found to be blood- and organ culture-positive for B. anthracis. The novel mode of action, in vitro microbiology, preclinical safety, and animal model efficacy data, which were generated in line with Food and Drug Administration Animal Rule, support gepotidacin as a potential treatment for anthrax in an emergency biothreat situation.

Study of the reusability and stability of nylon nanofibres as an antibody immobilisation surface.

In the case of a biological threat, early, rapid, and specific detection is critical. In addition, ease of handling, use in the field, and low-cost production are important considerations. Immunological devices are able to respond to these needs. In the design of these immunological devices, surface antibody immobilisation is crucial. Nylon nanofibres have been described as a very good option because they allow for an increase in the surface-to-volume ratio, leading to an increase in immunocapture efficiency. In this paper, we want to deepen the study of other key points, such as the reuse and stability of these nanofibres, in order to assess their profitability. On the one hand, the reusability of nanofibres has been studied using different stripping treatments at different pH values on the nylon nanofibres with well-oriented antibodies anchored by protein A/G. Our study shows that stripping with glycine buffer pH 2.5 allows the nanofibres to be reused as long as protein A/G has been previously anchored, leaving both nanofibre and protein A/G unchanged. On the other hand, we investigated the stability of the nylon nanofibres. To achieve this, we analysed any loss of immunocapture ability of well-oriented antibodies anchored both to the nylon nanofibres and to a specialised surface with high protein binding capacity. The nanofibre immunocapture system maintained an unchanged immunocapture ability for a longer time than the specialised planar surface. In conclusion, nylon nanofibres seem to be a very good choice as an antibody immobilisation surface, offering not only higher immunocapture efficiency, but also more cost efficiency as they are reusable and stable.

A simple undergraduate laboratory exercise for teaching the role of sentinel-level clinical microbiology testing in biological threat identification.

Undergraduate students in the biomedical sciences are mostly unaware of how clinical microbiology laboratories handle suspected agents of bioterrorism or emerging infectious diseases. The Public Health Security Bioterrorism Preparedness and Response Act of 2002 requires the US Department of Health and Human Services (HHS) to maintain a list of microbes that pose serious biological threats to human health and safety, including Tier 1 agents with the potential for use in bioterrorism. The Laboratory Response Network (LRN), founded by the Centers for Disease Control and Prevention, the Federal Bureau of Investigation, and the Association of Public Health Laboratories, coordinates the response of sentinel, reference, and national laboratories to these biothreats. The sentinel laboratories, which comprise most hospital-based and commercial laboratories, are the first to encounter a suspicious agent. For this reason, the LRN has published a series of testing guidelines to assist the sentinel laboratories in deciding whether a microbial isolate should be considered potentially hazardous and thus sent to a reference or national laboratory for further characterization. Here, we describe a simple laboratory exercise that teaches sentinel-level testing requirements in the context of an applied setting of a potential outbreak of anthrax that would require a sentinel laboratory to recognize a potential threat, attempt to rule it out, and refer to a national laboratory for identification.

Vacuum Oven Drying: A Cost-Effective Way of Producing Field-Deployable Reagents for In-house Real-Time PCR Methods.

The polymerase chain reaction (PCR), is a widely used, sensitive and reliable method for detecting pathogens. However, technical limitations may restrict its use outside sophisticated laboratories, e.g. for detecting pathogens at the site of a disease outbreak. In this study, real-time PCR reagents specific to four bacteria (Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella spp.) and to the Influenza A virus were dried using a vacuum oven drying method. The performance of the dried reagents stored at different temperatures, was monitored using both a standard-size and a portable real-time PCR instrument. The vacuum oven dried real-time PCR reagents were stable and retained the sensitivity for at least 14 months when stored in a refrigerator (+ 4 °C). When stored at room temperature, DNA assays remained stable for at least 10 weeks and Influenza A RNA assay for 3 weeks. These results demonstrate the feasibility of vacuum oven dried real-time PCR reagents and a portable thermocycler for the rapid and reliable detection of pathogens. The drying protocol presented here is cost-effective and easy to use, and could be applied to real-time PCR methods specific to other pathogens as well. In addition, this in-house drying protocol reduces reliance on commercial PCR tests during a time of shortage, such as that experienced during the Corovirus disease (COVID-19) crisis.

Ceftobiprole Medocaril Is an Effective Post-Exposure Treatment in the Fischer 344 Rat Model of Pneumonic Tularemia.

Francisella tularensis subspecies tularensis is a category-A biothreat agent that can cause lethal tularemia. Ceftobiprole medocaril is being explored as a medical countermeasure for the treatment of pneumonic tularemia. The efficacy of ceftobiprole medocaril against inhalational tularemia was evaluated in the Fischer 344 rat model of infection. The dose was expected to be effective against F. tularensis isolates with ceftobiprole minimum inhibitory concentrations ≤0.5 µg/mL. Animals treated with ceftobiprole medocaril exhibited a 92% survival rate 31 days post-challenge, identical to the survival of levofloxacin-treated rats. By comparison, rats receiving placebo experienced 100% mortality. Terminally collected blood, liver, lung, and spleen samples confirmed disseminated F. tularensis infections in most animals that died prior to completing treatments (placebo animals and a rat treated with ceftobiprole medocaril), although levels of bacteria detected in the placebo samples were significantly elevated compared to the ceftobiprole-medocaril-treated group geometric mean. Furthermore, no evidence of infection was detected in any rat that completed ceftobiprole medocaril or levofloxacin treatment and survived to the end of the post-treatment observation period. Overall, survival rates, body weights, and bacterial burdens consistently demonstrated that treatment with ceftobiprole medocaril is efficacious against otherwise fatal cases of pneumonic tularemia in the rat model.

In Vitro Activity of Novel Topoisomerase Inhibitors against Francisella tularensis and Burkholderia pseudomallei.

Antimicrobial resistance is a global issue, and the investigation of alternative therapies that are not traditional antibiotics are warranted. Novel bacterial type II topoisomerase inhibitors (NBTIs) have recently emerged as a novel class of antibiotics with reduced potential for cross-resistance to fluoroquinolones due to their novel mechanism of action. This study investigated the in vitro activity of a series of cyclohexyl-oxazolidinone bacterial topoisomerase inhibitors against type strains of Francisella tularensis and Burkholderia pseudomallei. Broth microdilution, time-kill, and cell infection assays were performed to determine activity against these biothreat pathogens. Two candidates were identified that demonstrated in vitro activity in multiple assays that in some instances was equivalent to ciprofloxacin and doxycycline. These data warrant the further evaluation of these novel NBTIs and future iterations in vitro and in vivo.

Improved understanding of biorisk for research involving microbial modification using annotated sequences of concern.

Regulation of research on microbes that cause disease in humans has historically been focused on taxonomic lists of 'bad bugs'. However, given our increased knowledge of these pathogens through inexpensive genome sequencing, 5 decades of research in microbial pathogenesis, and the burgeoning capacity of synthetic biologists, the limitations of this approach are apparent. With heightened scientific and public attention focused on biosafety and biosecurity, and an ongoing review by US authorities of dual-use research oversight, this article proposes the incorporation of sequences of concern (SoCs) into the biorisk management regime governing genetic engineering of pathogens. SoCs enable pathogenesis in all microbes infecting hosts that are 'of concern' to human civilization. Here we review the functions of SoCs (FunSoCs) and discuss how they might bring clarity to potentially problematic research outcomes involving infectious agents. We believe that annotation of SoCs with FunSoCs has the potential to improve the likelihood that dual use research of concern is recognized by both scientists and regulators before it occurs.

Efficacy of Intravenously Administered Gepotidacin in Cynomolgus Macaques following a Francisella tularensis Inhalational Challenge.

Francisella tularensis (F. tularensis) is a Centers for Disease Control (CDC) category "A" Gram-negative biothreat pathogen. Inhalation of F. tularensis can cause pneumonia and respiratory failure and is associated with high mortality rates without early treatment. Gepotidacin is a novel, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode, has activity against multidrug-resistant target pathogens, and has demonstrated in vitro activity against diverse collections of F. tularensis isolates (MIC90 of 0.5 to 1 µg/mL). Gepotidacin was evaluated in the cynomolgus macaque model of inhalational tularemia, using the SCHU S4 strain, with treatment initiated after exposure and sustained fever. Macaques were dosed via intravenous (i.v.) infusion with saline or gepotidacin at 72 mg/kg/day to support a human i.v. infusion dosing regimen of 1,000 mg three times daily. The primary study endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. Gepotidacin treatment resulted in 100% survival compared to 12.5% in the saline-treated control group (P < 0.0001) at Day 43 postinhalational challenge. All gepotidacin-treated animals were blood and organ culture negative for F. tularensis at the end of the study. In contrast, none of the saline control animals were blood and organ culture negative. Gepotoidacin's novel mechanism of action and the efficacy data reported here (aligned with the Food and Drug Administration Animal Rule) support gepotidacin as a potential treatment for pneumonic tularemia in an emergency biothreat situation.

Point-of-care vertical flow immunoassay system for ultra-sensitive multiplex biothreat-agent detection in biological fluids.

This paper presents simple, fast, and sensitive detection of multiple biothreat agents by paper-based vertical flow colorimetric sandwich immunoassay for detection of Yersinia pestis (LcrV and F1) and Francisella tularensis (lipopolysaccharide; LPS) antigens using a vertical flow immunoassay (VFI) prototype with portable syringe pump and a new membrane holder. The capture antibody (cAb) printing onto nitrocellulose membrane and gold-labelled detection antibody (dAb) were optimized to enhance the assay sensitivity and specificity. Even though the paper pore size was relaxed from previous 0.1 µm to the current 0.45 µm for serum samples, detection limits as low as 0.050 ng/mL for LcrV and F1, and 0.100 ng/mL for FtLPS have been achieved in buffer and similarly in diluted serum (with LcrV and F1 LODs remained the same and LPS LOD reduced to 0.250 ng/mL). These were 40, 80, and 50X (20X for LPS in serum) better than those from lateral flow configuration. Furthermore, the comparison of multiplex format demonstrated low cross-reactivity and equal sensitivity to that of the singleplex assay. The optimized VFI platform thus provides a portable and rapid on-site monitoring system for multiplex biothreat detection with the potential for high sensitivity, specificity, reproducibility, and multiplexing capability, supporting its utility in remote and resource-limited settings.

Optimization of Oxford Nanopore Technology Sequencing Workflow for Detection of Amplicons in Real Time Using ONT-DART Tool.

An optimized, well-tested and validated targeted genomic sequencing-based high-throughput assay is currently not available ready for routine biodefense and biosurveillance applications. Earlier, we addressed this gap by developing and establishing baseline comparisons of a multiplex end-point Polymerase Chain Reaction (PCR) assay followed by Oxford Nanopore Technology (ONT) based amplicon sequencing to real time PCR and customized data processing. Here, we expand upon this effort by identifying the optimal ONT library preparation method for integration into a novel software platform ONT-DART (ONT-Detection of Amplicons in Real-Time). ONT-DART is a dockerized, real-time, amplicon-sequence analysis workflow that is used to reproducibly process and filter read data to support actionable amplicon detection calls based on alignment metrics, within sample statistics, and no-template control data. This analysis pipeline was used to compare four ONT library preparation protocols using R9 and Flongle (FL) flow cells. The two 4-Primer methods tested required the shortest preparation times (5.5 and 6.5 h) for 48 libraries but provided lower fidelity data. The Native Barcoding and Ligation methods required longer preparation times of 8 and 12 h, respectively, and resulted in higher overall data quality. On average, data derived from R9 flow cells produced true positive calls for target organisms more than twice as fast as the lower throughput FL flow cells. These results suggest that utilizing the R9 flowcell with an ONT Native Barcoding amplicon library method in combination with ONT-DART platform analytics provides the best sequencing-based alternative to current PCR-based biodetection methods.

Providing On-Site Laboratory and Biosafety Just-In-Time Training Inside a Box-Based Laboratory during the West Africa Ebola Outbreak: Supporting Better Preparedness for Future Health Emergencies.

The Biological Light Fieldable Laboratory for Emergencies (B-LiFE) is a box-based modular laboratory with the capacity to quickly deploy on-site in cases of uncontrolled spread of infectious disease. During the 2014-2015 West Africa Ebola outbreak, this tent laboratory provided diagnostic support to the N'Zerekore Ebola Treatment Center (ETC), Guinea, for three months. One of the objectives of B-LiFE deployment was to contribute, as much as possible, to national capacity building by training local scientists. Two Guinean biologists were selected according to their basic biological knowledge and laboratory skills among 50 candidate trainees, and were integrated into the team through "just-in-time training" (JiTT), which helped the biologists acquire knowledge and laboratory skills beyond their expertise. The JiTT program was conducted according to standard laboratory procedures, in line with international biosafety guidelines adapted to field conditions. Supervised acquisition of field-laboratory practices mainly focused on biochemical testing and Ebola viral load quantification using routine PCR-based detection, including the Biofire FilmArray® system (BFA), a novel, as yet non-validated, automated assay for diagnostic testing of Ebola virus disease at the time of B-LiFE deployment. During the JiTT, the two biologists were closely involved in all laboratory activities, including BFA validation and biosafety procedures. Meanwhile, this successful JiTT enhanced the B-LiFE in-field operational capacity and contributed to national capacity building. A post-training evaluation and contacts were organised to assess the evolution and technical skills gained by the two researchers during the B-LiFE mission. At the end of the B-LiFE mission, both biologists were enrolled in follow-on programmes to curb the epidemic spreading in Africa. These results demonstrate that during infectious disease outbreaks or major crises, the JiTT approach can rapidly expand access to critical diagnostic testing and train local staff to do so.

Development and evaluation of a multiplex droplet digital polymerase chain reaction method for simultaneous detection of five biothreat pathogens.

Biothreat agents pose a huge threat to human and public health, necessitating the development of rapid and highly sensitive detection approaches. This study establishes a multiplex droplet digital polymerase chain reaction (ddPCR) method for simultaneously detecting five high-risk bacterial biothreats: Yersinia pestis, Bacillus anthracis, Brucella spp., Burkholderia pseudomallei, and Francisella tularensis. Unlike conventional multiplex real-time PCR (qPCR) methods, the multiplex ddPCR assay was developed using two types of probe fluorophores, allowing the assay to perform with a common two-color ddPCR system. After optimization, the assay performance was evaluated, showing a lower limit of detection (LOD) (0.1-1.0 pg/µL) and good selectivity for the five bacteria targets. The multiplex assay's ability to simultaneously detect two or more kinds of targets in a sample was also demonstrated. The assay showed strong sample tolerance when testing simulated soil samples; the LOD for bacteria in soil was 2 × 102-2 × 103 colony-forming unit (CFU)/100 mg soil (around 5-50 CFU/reaction), which was 10-fold lower than that of the single-target qPCR method. When testing simulated soil samples at bacterial concentrations of 2 × 103-2 × 104 CFU/100 mg soil, the assay presented a higher sensitivity (100%, 35/35) than that of the qPCR method (65.71%, 23/35) and a good specificity (100%, 15/15). These results suggest that the developed 5-plex ddPCR method is more sensitive than conventional qPCR methods and is potentially suitable for rapidly detecting or screening the five selected bacterial biothreats in suspicious samples.

Evaluation of Delafloxacin against a Burkholderia pseudomallei Efflux Mutant Panel.

In vitro activities of delafloxacin and ciprofloxacin were evaluated against Burkholderia pseudomallei mutants expressing or lacking defined resistance-nodulation-cell division (RND) efflux pumps using CLSI methodology at pHs of 5.8 and 7.2. Delafloxacin MIC values were as much as 8-fold lower at pH 5.8 than those at pH 7.2, while ciprofloxacin MICs increased as much as 8-fold. The data from this study suggest that compared to ciprofloxacin, delafloxacin may have improved efflux avoidance, notably at acidic pH. In contrast to ciprofloxacin, delafloxacin may thus retain its therapeutic potential, even in BpeEF-OprC efflux-pump-expressing B. pseudomallei strains that compromise the use of fluoroquinolones, such as ciprofloxacin. IMPORTANCE Resistance-nodulation-cell division (RND) efflux pumps play a major role in intrinsic and acquired antibiotic resistance in Burkholderia pseudomallei, and these pumps are its only known multidrug resistance determinants. Fluoroquinolones have performed poorly in clinical settings and are currently not recommended for treatment of B. pseudomallei infections. While the reasons for the poor clinical performance of this pathogen remain unclear, efflux may be partially responsible since fluoroquinolones like ciprofloxacin are prone to efflux by RND pumps, notably BpeEF-OprC. In vitro efficacy testing using a panel of efflux-proficient and efflux-deficient strains allows identification of fluoroquinolones that compared to ciprofloxacin are less prone to efflux.

A Novel Method for Training the Interdiction of Restricted and Hazardous Biological Materials by Detection Dogs.

The interdiction of restricted and hazardous biological agents presents challenges for any detection method due to the inherent complexity of sample type and accessibility. Detection capabilities for this category of agents are limited and restricted in their mobility, adaptability and efficiency. The potential for identifying biological agents through a volatile organic compound (VOC) signature presents an opportunity to use detection dogs in a real-time mobile capacity for surveillance and screening strategies. However, the safe handling and access to the materials needed for training detection dogs on restricted or hazardous biological agents prevents its broader application in this field. This study evaluated the use of a polymer-based training aid in a viral detection model using bovine viral diarrhea virus mimicking biosafety level 3+ agent conditions. After the biological agent-based odor was absorbed into the polymer, the aid was rendered safe for handling through a rigorous sterilization process. The viral culture-based training aid was then used to train a cohort of detection dogs (n = 6) to discriminate agent-based target odor in culture from relevant distractor odors including non-target biological agent-based odors. Following culture-based training, dogs were tested for generalization to aids with infected animal sample-based odors across five sample types (fecal, blood, nasal, saliva, and urine). Within the context of the polymer-based training aid system, dogs were successfully trained to detect and discriminate a representative biological viral agent-based odor from distractor odors with a 97.22% (±2.78) sensitivity and 97.11% (±1.94) specificity. Generalization from the agent-based odor to sample-based odors ranged from 65.40% (±8.98) to 91.90 % (±6.15) sensitivity and 88.61% (±1.46) to 96.00% (±0.89) specificity across the sample types. The restrictive nature for mimicking the access and handling of a BSL 3+ agent presented challenges that required a strict study design uncommon to standard detection dog training and odor presentation. This study demonstrates the need to further evaluate the utility and challenges of training detection dogs to alert to biological samples using safe and manageable training aids.

A multipathogen DNA vaccine elicits protective immune responses against two class A bioterrorism agents, anthrax and botulism.

The potential use of biological agents has become a major public health concern worldwide. According to the CDC classification, Bacillus anthracis and Clostridium botulinum, the bacterial pathogens that cause anthrax and botulism, respectively, are considered to be the most dangerous potential biological agents. Currently, there is no licensed vaccine that is well suited for mass immunization in the event of an anthrax or botulism epidemic. In the present study, we developed a dual-expression system-based multipathogen DNA vaccine that encodes the PA-D4 gene of B. anthracis and the HCt gene of C. botulinum. When the multipathogen DNA vaccine was administered to mice and guinea pigs, high level antibody responses were elicited against both PA-D4 and HCt. Analysis of the serum IgG subtype implied a combined Th1/Th2 response to both antigens, but one that was Th2 skewed. In addition, immunization with the multipathogen DNA vaccine induced effective neutralizing antibody activity against both PA-D4 and HCt. Finally, the protection efficiency of the multipathogen DNA vaccine was determined by sequential challenge with 10 LD50 of B. anthracis spores and 10 LD50 of botulinum toxin, or vice versa, and the multipathogen DNA vaccine provided higher than 50% protection against lethal challenge with both high-risk biothreat agents. Our studies suggest the strategy used for this anthrax-botulinum multipathogen DNA vaccine as a prospective approach for developing emergency vaccines that can be immediately distributed on a massive scale in response to a biothreat emergency or infectious disease outbreak. Key points • A novel multipathogen DNA vaccine was constructed against anthrax and botulism. • Robust immune responses were induced following vaccination. • Suggests a potential vaccine development strategy against biothreat agents.