Biodefense: Expanding Nursing Strategies After the SARS-CoV-2 Threat.

The COVID-19 pandemic has impacted the nursing profession and its existence in terms of preventing infection from spreading at the levels of patient care and management. Vigilance is essential in combating potential re-emerging diseases in the future. Hence, exploring a new framework, biodefense, is the best way to reframe nursing preparedness for new biological threats or new pandemics at any level of nursing care.

Categorizing Sequences of Concern by Function To Better Assess Mechanisms of Microbial Pathogenesis.

To identify sequences with a role in microbial pathogenesis, we assessed the adequacy of their annotation by existing controlled vocabularies and sequence databases. Our goal was to regularize descriptions of microbial pathogenesis for improved integration with bioinformatic applications. Here, we review the challenges of annotating sequences for pathogenic activity. We relate the categorization of more than 2,750 sequences of pathogenic microbes through a controlled vocabulary called Functions of Sequences of Concern (FunSoCs). These allow for an ease of description by both humans and machines. We provide a subset of 220 fully annotated sequences in the supplemental material as examples. The use of this compact (∼30 terms), controlled vocabulary has potential benefits for research in microbial genomics, public health, biosecurity, biosurveillance, and the characterization of new and emerging pathogens.

Biodefense and emergency use authorization: different originations, purposes, and evolutionary paths of institutions in the United States and South Korea.

BACKGROUND: Emergency-use-authorization (EUA) is the representative biodefense policy that allows the use of unlicensed medical countermeasures or off-label use of approved medical countermeasures in response to public health emergencies. This article aims to determine why the EUA policies of the United States and South Korea produced drastically different outcomes during the COVID-19 pandemic, and how these outcomes were determined by the originations and evolutionary paths of the two policies. METHOD: Historical institutionalism (HI) explains institutional changes-that is, how the institution is born and how it evolves-based on the concept of path dependency. However, the HI analytical narratives remain at the meso level of analysis in the context of structure and agency. This article discusses domestic and policy-level factors related to the origination of the biodefense institutions in the United States and South Korea using policy-learning concepts with the Event-related Policy Change Model. RESULTS: The 2001 anthrax letter attack (Amerithrax) and the 2015 Middle East Respiratory Syndrome (MERS) outbreak prompted the establishment of biodefense institutions in the United States and South Korea, respectively. Due to the different departure points and the mechanism of path dependency, the two countries' EUAs evolved in different ways-the United States EUA reinforced the Post-Exposure Prophylaxis (PEP) function, while the South Korea EUA strengthened the Non-Pharmaceutical Intervention (NPI) function. CONCLUSIONS: The evolution and outcomes of the two EUAs are different because both policies were born out of different needs. The United States EUA is primarily oriented toward protecting homeland security against CBRN (chemical, biological, radiological, and nuclear) threats, whereas the South Korea EUA is specifically designed for disease prevention against infectious disease outbreak.

Transcriptome analysis of molecular response to UVC irradiation in zebrafish embryos.

Ultraviolet (UV) rays can be both harmful and beneficial to humans. This study aimed to investigate the toxicity and safety of ultraviolet C (UVC) exposure in living organisms and the corresponding biodefense molecular mechanisms. Zebrafish embryos, at an early developmental stage (5-6 h post-fertilization), were irradiated with increasing UVC dosages using high-efficiency deep-ultraviolet light-emitting diodes (278 nm). Morphological phenotypes including survival rate, hatching rate, heart rate, and malformation rate were evaluated. Compared to un-irradiated controls, all zebrafish embryos exposed to 4.5 mJ/cm2 UVC survived and showed no significant difference in hatching and heart rate. However, 7.5 mJ/cm2 of UVC irradiation caused a significantly decreased survival rate (37.5%) and an increased malformation rate (81.8%). Therefore, 4.5 mJ/cm2 was chosen as the limit dosage that the internal biodefense system of zebrafish embryos can protect against UVC radiation. Transcriptome analysis (RNA sequencing) performed on 3 min and 3 days post-irradiation embryos (4.5 mJ/cm2) revealed the molecular mechanisms underlying the response of zebrafish embryos to irradiation. The embryos quickly responded to UVC-induced stress by activating the p53 signaling pathway. In addition, after 3 days of recuperation, the embryos showed activation of signal transducer and activator of transcription (STAT) signaling pathway. To our knowledge, this is the first study to evaluate the toxicological effects and the molecular mechanism of biodefense in zebrafish embryos upon 278 nm UVC irradiation.

Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens.

BACKGROUND: The state-of-the-art in nucleic acid based biodetection continues to be polymerase chain reaction (PCR), and many real-time PCR assays targeting biodefense pathogens for biosurveillance are in widespread use. These assays are predominantly singleplex; i.e. one assay tests for the presence of one target, found in a single organism, one sample at a time. Due to the intrinsic limitations of such tests, there exists a critical need for high-throughput multiplex assays to reduce the time and cost incurred when screening multiple targets, in multiple pathogens, and in multiple samples. Such assays allow users to make an actionable call while maximizing the utility of the small volumes of test samples. Unfortunately, current multiplex real-time PCR assays are limited in the number of targets that can be probed simultaneously due to the availability of fluorescence channels in real-time PCR instruments. RESULTS: To address this gap, we developed a pipeline in which the amplicons produced by a 14-plex end-point PCR assay using spiked samples were subsequently sequenced using Nanopore technology. We used bar codes to sequence multiple samples simultaneously, leading to the generation and subsequent analysis of sequence data resulting from a short sequencing run time (< 10 min). We compared the limits of detection (LoD) of real-time PCR assays to Oxford Nanopore Technologies (ONT)-based amplicon sequencing and estimated the sample-to-answer time needed for this approach. Overall, LoDs determined from the first 10 min of sequencing data were at least one to two orders of magnitude lower than real-time PCR. Given enough time, the amplicon sequencing approach is approximately 100 times more sensitive than real-time PCR, with detection of amplicon specific reads even at the lowest tested spiking concentration (around 2.5-50 Colony Forming Units (CFU)/ml). CONCLUSIONS: Based on these results, we propose amplicon sequencing assay as a viable alternative to replace the current real-time PCR based singleplex assays for higher throughput biodefense applications. We note, however, that targeted amplicon specific reads were not detectable even at the highest tested spike concentrations (2.5 X 104-5.0 X105 CFU/ml) without an initial amplification step, indicating that PCR is still necessary when utilizing this protocol.

Antivirals in medical biodefense.

The viruses historically implicated or currently considered as candidates for misuse in bioterrorist events are poxviruses, filoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses and a number of arboviruses causing encephalitis, including alpha- and flaviviruses. All these viruses are of concern for public health services when they occur in natural outbreaks or emerge in unvaccinated populations. Recent events and intelligence reports point to a growing risk of dangerous biological agents being used for nefarious purposes. Public health responses effective in natural outbreaks of infectious disease may not be sufficient to deal with the severe consequences of a deliberate release of such agents. One important aspect of countermeasures against viral biothreat agents are the antiviral treatment options available for use in post-exposure prophylaxis. These issues were adressed by the organizers of the 16th Medical Biodefense Conference, held in Munich in 2018, in a special session on the development of drugs to treat infections with viruses currently perceived as a threat to societies or associated with a potential for misuse as biothreat agents. This review will outline the state-of-the-art methods in antivirals research discussed and provide an overview of antiviral compounds in the pipeline that are already approved for use or still under development.

The threat of emerging and re-emerging pathogenic Sporothrix species.

Sporotrichosis is a neglected subcutaneous mycosis of humans and animals acquired by traumatic inoculation of soil and plant material (classical route) contaminated with infectious propagules of the pathogen or being bitten/scratched by infected cats (alternative route). Within a genus composed of 53 species displaying an essentially environmental core, there are only a few members which have considerable impacts on human or animal health. Infections are typically caused by S. brasiliensis, S. schenckii or S. globosa. Rare mammal pathogens include members of the S. pallida and S. stenocereus complexes. To illustrate the tremendous impact of emerging zoonotic sporotrichosis on public health, we discuss the main features of the expanding epidemics driven by S. brasiliensis in cats and humans. The cat entry in the transmission chain of sporotrichosis, causing epizooties (cat-cat) or zoonosis (cat-human), has contributed to the definition of new paradigms in Sporothrix transmission, reaching epidemic levels, making the disease a serious public health problem. Indeed, S. brasiliensis infection in humans and animals is likely to become even more important in the future, with projections of its expansion in biogeographic domains and host range, as well as greater virulence in mammals. Therefore, lessons from a long-standing outbreak in the state of Rio de Janeiro about the source and distribution of the etiological agents among outbreak areas can be used to create better control and prevention plans and increase awareness of sporotrichosis as a serious emerging zoonotic disease.

Antibodies against In Vivo-Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei.

Burkholderia mallei, a facultative intracellular bacterium and tier 1 biothreat, causes the fatal zoonotic disease glanders. The organism possesses multiple genes encoding autotransporter proteins, which represent important virulence factors and targets for developing countermeasures in pathogenic Gram-negative bacteria. In the present study, we investigated one of these autotransporters, BatA, and demonstrate that it displays lipolytic activity, aids in intracellular survival, is expressed in vivo, elicits production of antibodies during infection, and contributes to pathogenicity in a mouse aerosol challenge model. A mutation in the batA gene of wild-type strain ATCC 23344 was found to be particularly attenuating, as BALB/c mice infected with the equivalent of 80 median lethal doses cleared the organism. This finding prompted us to test the hypothesis that vaccination with the batA mutant strain elicits protective immunity against subsequent infection with wild-type bacteria. We discovered that not only does vaccination provide high levels of protection against lethal aerosol challenge with B. mallei ATCC 23344, it also protects against infection with multiple isolates of the closely related organism and causative agent of melioidosis, Burkholderia pseudomallei Passive-transfer experiments also revealed that the protective immunity afforded by vaccination with the batA mutant strain is predominantly mediated by IgG antibodies binding to antigens expressed exclusively in vivo Collectively, our data demonstrate that BatA is a target for developing medical countermeasures and that vaccination with a mutant lacking expression of the protein provides a platform to gain insights regarding mechanisms of protective immunity against B. mallei and B. pseudomallei, including antigen discovery.

Construction of Bacillus thuringiensis Simulant Strains Suitable for Environmental Release.

For a surrogate bacterium to be used in outdoor studies, it is important to consider environmental and human safety and ease of detection. Recently, Bacillus thuringiensis, a popular bioinsecticide bacterium, has been gaining attention as a surrogate bacterium for use in biodefense. In this study, we constructed simulant strains of B. thuringiensis with enhanced characteristics for environmental studies. Through transposon mutagenesis, pigment genes were inserted into the chromosome, producing yellow-colored colonies for easy detection. To prevent persistence of spores in the environment, a genetic circuit was designed to produce a spore without sporulation capability. Two loxP sites were inserted, one on each side of the spo0A gene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deleted spo0A during sporulation, producing spores that lacked the spo0A gene. In addition, two major α/ß-type small acid-soluble spore protein (SASP) genes, predicted by synteny analysis, were deleted. The spores of the mutant strain showed increased UV-C sensitivity and quickly lost viability when tested in a solar simulator. When the spores of the mutant strain were administered to the lungs of BALB/c mice, cells were quickly removed from the body, suggesting enhanced in vivo safety. All strains constructed in this study contain no antibiotic resistance markers and all heterologous genes were inserted into the chromosome, which are useful features for simulants to be released into the environment.IMPORTANCEB. thuringiensis has recently been receiving increasing attention as a good spore simulant in biodefense research. However, few studies were done to properly address many important features of B. thuringiensis as a simulant in environmental studies. Since spores can persist in the environment for years after release, environmental contamination is a big problem, especially when genetically engineered strains are used. To solve these problems, we report here the development of B. thuringiensis simulant strains that are capable of forming yellow colonies for easy detection, incapable of forming spores more than once due to a genetic circuit, and lacking in two major SASP genes. The genetic circuit to produce a spore without sporulation capability, together with the deletion of SASP genes, ensures the environmental and human safety of the simulant strains developed in this study. All of these features will allow wider use of B. thuringiensis as a simulant for Bacillus anthracis in environmental release studies.

Therapeutic Antibodies for Biodefense.

Diseases can be caused naturally by biological agents such as bacteria, viruses and toxins (natural risk). However, such biological agents can be intentionally disseminated in the environment by a State (military context) or terrorists to cause diseases in a population or livestock, to destabilize a nation by creating a climate of terror, destabilizing the economy and undermining institutions. Biological agents can be classified according to the severity of illness they cause, its mortality and how easily the agent can be spread. The Centers for Diseases Control and Prevention (CDC) classify biological agents in three categories (A, B and C); Category A consists of the six pathogens most suitable for use as bioweapons (Bacillus anthracis, Yersinia pestis, Francisella tularensis, botulinum neurotoxins, smallpox and viral hemorrhagic fevers). Antibodies represent a perfect biomedical countermeasure as they present both prophylactic and therapeutic properties, act fast and are highly specific to the target. This review focuses on the main biological agents that could be used as bioweapons, the history of biowarfare and antibodies that have been developed to neutralize these agents.

Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution.

The newly emerging Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes a Severe Acute Respiratory Syndrome-like disease with ∼43% mortality. Given the recent detection of virus in dromedary camels, zoonotic transfer of MERS-CoV to humans is suspected. In addition, little is known about the role of human neutralizing Ab (nAb) pressure as a driving force in MERS-CoV adaptive evolution. Here, we used a well-characterized nonimmune human Ab-phage library and a panning strategy with proteoliposomes and cells to identify seven human nAbs against the receptor-binding domain (RBD) of the MERS-CoV Spike protein. These nAbs bind to three different epitopes in the RBD and human dipeptidyl peptidase 4 (hDPP4) interface with subnanomolar/nanomolar binding affinities and block the binding of MERS-CoV Spike protein with its hDPP4 receptor. Escape mutant assays identified five amino acid residues that are critical for neutralization escape. Despite the close proximity of the three epitopes on the RBD interface, escape from one epitope did not have a major impact on neutralization with Abs directed to a different epitope. Importantly, the majority of escape mutations had negative impacts on hDPP4 receptor binding and viral fitness. To our knowledge, these results provide the first report on human nAbs against MERS-CoV that may contribute to MERS-CoV clearance and evolution. Moreover, in the absence of a licensed vaccine or antiviral for MERS, this panel of nAbs offers the possibility of developing human mAb-based immunotherapy, especially for health-care workers.

Expert Views on Biological Threat Characterization for the U.S. Government: A Delphi Study.

Biological threat characterization (BTC) involves laboratory research conducted for the purpose of biological defense. BTC research is important for improving biological risk assessment and informing resource prioritization. However, there are also risks involved in BTC work, including potential for escape from the laboratory or the misuse of research results. Using a modified Delphi study to gather opinions from U.S. experts in biosecurity and biodefense, this analysis explores what principles and safeguards can maximize the benefits of BTC research and ensure that it is conducted safely and securely. Delphi participants were asked to give their opinions about the need for BTC research by the U.S. government (USG); risks of conducting this research; rules or guidelines that should be in place to ensure that the work is safe and accurate; components of an effective review and prioritization process; rules for when characterization of a pathogen can be discontinued; and recommendations about who in the USG should be responsible for BTC prioritization decisions. The findings from this research reinforce the need for BTC research at the federal level as well as a need for continued review and oversight of this research to maximize its effectiveness and reduce the risks involved. It also demonstrates the need for further discussion of what would constitute a "red line" for biothreat characterization research-research that should not be performed for safety, ethical, or practical reasons-and guidelines for when there is sufficient research in a given topic area so that the research can be considered completed.

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies.

Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors.

Safely balancing a double-edged blade: identifying and mitigating emerging biosecurity risks in precision medicine.

Tools and methods of precision medicine are developing rapidly, through both iterative discoveries enabled by innovations in biomedical research (e.g., genome editing, synthetic biology, bioengineered devices). These are strengthened by advancements in information technology and the increasing body of data-as assimilated, analyzed, and made accessible-and affectable-through current and emerging cyber-and systems- technologies. Taken together, these approaches afford ever greater volume and availability of individual and collective human data. Machine learning and/or artificial intelligence approaches are broadening this dual use risk; and in the aftermath of COVID-19, there is growing incentive and impetus to gather more biological data from individuals and their environments on a routine basis. By engaging these data-and the interventions that are based upon them, precision medicine offer promise of highly individualized treatments for disease and injury, optimization of structure and function, and concomitantly, the potential for (mis) using data to incur harm. This double-edged blade of benefit and risk obligates the need to safeguard human data from purloinment, through systems, guidelines and policies of a novel discipline, cyberbiosecurity, which, as coupled to ethical precepts, aims to protect human privacy, agency, and safety in ways that remain apace with scientific and technological advances in biomedicine. Herein, current capabilities and trajectories precision medicine are described as relevant to their dual use potential, and approaches to biodata security (viz.- cyberbiosecurity) are proposed and discussed.

A Biparatopic Intrabody Renders Vero Cells Impervious to Ricin Intoxication.

Expression of camelid-derived, single-domain antibodies (VHHs) within the cytoplasm of mammalian cells as "intrabodies" has opened-up novel avenues for medical countermeasures against fast-acting biothreat agents. In this report, we describe a heterodimeric intrabody that renders Vero cells virtually impervious to ricin toxin (RT), a potent Category B ribosome-inactivating protein (RIP). The intrabody consists of two structurally defined VHHs that target distinct epitopes on RT's enzymatic subunit (RTA): V9E1 targets RTA's P-stalk recruitment site, and V2A11 targets RTA's active site. Resistance to RT conferred by the biparatopic VHH construct far exceeded that of either of the VHHs alone and effectively inhibited all measurable RT-induced cytotoxicty in vitro. We propose that targeted delivery of bispecific intrabodies to lung tissues may represent a novel means to shield the airways from the effects of inhalational RT exposure.

A bacteriophage cocktail targeting Yersinia pestis provides strong post-exposure protection in a rat pneumonic plague model.

Yersinia pestis, one of the deadliest bacterial pathogens ever known, is responsible for three plague pandemics and several epidemics, with over 200 million deaths during recorded history. Due to high genomic plasticity, Y. pestis is amenable to genetic mutations as well as genetic engineering that can lead to the emergence or intentional development of pan-drug-resistant strains. Indeed, antibiotic-resistant strains (e.g., strains carrying multidrug-resistant or MDR plasmids) have been isolated in various countries and endemic areas. Thus, there is an urgent need to develop novel, safe, and effective treatment approaches for managing Y. pestis infections. This includes infections by antigenically distinct strains for which vaccines (none FDA approved yet) may not be effective and those that cannot be managed by currently available antibiotics. Lytic bacteriophages provide one such alternative approach. In this study, we examined post-exposure efficacy of a bacteriophage cocktail, YPP-401, to combat pneumonic plague caused by Y. pestis CO92. YPP-401 is a four-phage preparation effective against a panel of at least 68 genetically diverse Y. pestis strains. Using a pneumonic plague aerosol challenge model in gender-balanced Brown Norway rats, YPP-401 demonstrated ~88% protection when delivered 18 h post-exposure for each of two administration routes (i.e., intraperitoneal and intranasal) in a dose-dependent manner. Our studies provide proof-of-concept that YPP-401 could be an innovative, safe, and effective approach for managing Y. pestis infections, including those caused by naturally occurring or intentionally developed multidrug-resistant strains.IMPORTANCECurrently, there are no FDA-approved plague vaccines. Since antibiotic-resistant strains of Y. pestis have emerged or are being intentionally developed to be used as a biothreat agent, new treatment modalities are direly needed. Phage therapy provides a viable option against potentially antibiotic-resistant strains. Additionally, phages are nontoxic and have been approved by the FDA for use in the food industry. Our study provides the first evidence of the protective effect of a cocktail of four phages against pneumonic plague, the most severe form of disease. When treatment was initiated 18 h post infection by either the intranasal or intraperitoneal route in Brown Norway rats, up to 87.5% protection was observed. The phage cocktail had a minimal impact on a representative human microbiome panel, unlike antibiotics. This study provides strong proof-of-concept data for the further development of phage-based therapy to treat plague.

Neutrinos to Astrovirology: Signatures.

In the 20th century, the concept of terrestrial life's unity was solidified, and the 21st century saw the emergence and establishment of astrovirology. To date, life originating beyond Earth has not been identified. The singular instance where NASA investigated potential microfossils in Martian ejecta found on Earth has since been refuted. This report suggests that a more comprehensive discussion and analysis of life's biosignatures and communication methods are essential. Such approaches are crucial not only to avoid overlooking the possible existence of extra-terrestrial intelligence (ETI) but also to prevent potential human infections that could arise from extra-terrestrial contact. In addition terrestrial infections by microorganism that originally derived from Earth and were returned, require investigation due to potential mutations and subsequent increased pathogenicity.

Astrovirology and terrestrial life survival.

Microbial organisms have been implicated in several mass extinction events throughout Earth's planetary history. Concurrently, it can be reasoned from recent viral pandemics that viruses likely exacerbated the decline of life during these periods of mass extinction. The fields of exovirology and exobiology have evolved significantly since the 20th century, with early investigations into the varied atmospheric compositions of exoplanets revealing complex interactions between metallic and non-metallic elements. This diversity in exoplanetary and stellar environments suggests that life could manifest in forms previously unanticipated by earlier, more simplistic models of the 20th century. Non-linear theories of complexity, catastrophe, and chaos (CCC) will be important in understanding the dynamics and evolution of viruses.

Animal models for Francisella tularensis and Burkholderia species: scientific and regulatory gaps toward approval of antibiotics under the FDA Animal Rule.

The development and regulatory approval of medical countermeasures (MCMs) for the treatment and prevention of bacterial threat agent infections will require the evaluation of products in animal models. To obtain regulatory approval, these models must accurately recapitulate aspects of human disease, including, but not necessarily limited to, route of exposure, time to disease onset, pathology, immune response, and mortality. This article focuses on the state of animal model development for 3 agents for which models are largely immature: Francisella tularensis, Burkholderia mallei, and Burkholderia pseudomallei. An overview of available models and a description of scientific and regulatory gaps are provided.

Post-Vaccination Delivery of CpG ODNs Enhances the Th2-Associated Protective Immunity of the Smallpox DNA Vaccine.

Potential threat of smallpox bioterrorism and concerns related to the adverse effects of currently licensed live-virus vaccines suggest the need to develop novel vaccines with better efficacy against smallpox. Use of DNA vaccines containing specific antigen-encoding plasmids prevents the risks associated with live-virus vaccines, offering a promising alternative to conventional smallpox vaccines. In this study, we investigated the efficiency of toll-like receptor (TLR) ligands in enhancing the immunogenicity of smallpox DNA vaccines. BALB/c mice were immunized with a DNA vaccine encoding the vaccinia virus L1R protein, along with the cytosine-phosphate-guanine (CpG) motif as a vaccine adjuvant, and their immune response was analyzed. Administration of B-type CpG oligodeoxynucleotides (ODNs) as TLR9 ligands 24 h after DNA vaccination enhanced the Th2-biased L1R-specific antibody immunity in mice. Moreover, B-type CpG ODNs improved the protective effects of the DNA vaccine against the lethal Orthopoxvirus challenge. Therefore, use of L1R DNA vaccines with CpG ODNs as adjuvants is a promising approach to achieve effective immunogenicity against smallpox infection.