Immunogenicity and Protective Efficacy of Recombinant Protective Antigen Anthrax Vaccine (GC1109) in A/J Mice Model.

A recombinant protective antigen anthrax vaccine (GC1109) is being developed as a new-generation vaccine by the Korea Disease Control and Prevention Agency. In accordance with the ongoing step 2 of phase II clinical trials, the immunogenicity and protective efficacy of the booster dose of GC1109 were evaluated in A/J mice after 3 serial vaccinations at 4-week intervals. The results indicated that the booster dose significantly increased the production of anti-protective antigen (PA) IgG and toxin-neutralizing antibody (TNA) compared with those of the group without booster. An enhanced protective effect of the booster dose was not observed because the TNA titers of the group without booster were high enough to confer protection against spore challenge. Additionally, the correlation between TNA titers and probability of survival was determined for calculating the threshold TNA titer levels associated with protection. The threshold 50 % neutralization factor (NF50) of TNA showing 70 % probability of protection was 0.21 in A/J mice with 1,200 LD50 Sterne spores challenge. These results indicate that GC1109 is a promising candidate as a new-generation anthrax vaccine and that a booster dose might provide enhanced protection by producing toxin-neutralizing antibodies.

Deep Learning Techniques for Ear Diseases Based on Segmentation of the Normal Tympanic Membrane.

OBJECTIVES: Otitis media is a common infection worldwide. Owing to the limited number of ear specialists and rapid development of telemedicine, several trials have been conducted to develop novel diagnostic strategies to improve the diagnostic accuracy and screening of patients with otologic diseases based on abnormal otoscopic findings. Although these strategies have demonstrated high diagnostic accuracy for the tympanic membrane (TM), the insufficient explainability of these techniques limits their deployment in clinical practice. METHODS: We used a deep convolutional neural network (CNN) model based on the segmentation of a normal TM into five substructures (malleus, umbo, cone of light, pars flaccida, and annulus) to identify abnormalities in otoscopic ear images. The mask R-CNN algorithm learned the labeled images. Subsequently, we evaluated the diagnostic performance of combinations of the five substructures using a three-layer fully connected neural network to determine whether ear disease was present. RESULTS: We obtained the receiver operating characteristic (ROC) curve of the optimal conditions for the presence or absence of eardrum diseases according to each substructure separately or combinations of substructures. The highest area under the curve (0.911) was found for a combination of the malleus, cone of light, and umbo, compared with the corresponding areas under the curve of 0.737-0.873 for each substructure. Thus, an algorithm using these five important normal anatomical structures could prove to be explainable and effective in screening abnormal TMs. CONCLUSION: This automated algorithm can improve diagnostic accuracy by discriminating between normal and abnormal TMs and can facilitate appropriate and timely referral consultations to improve patients' quality of life in the context of primary care.

Draft Genome Sequence of Francisella tularensis subsp. holarctica Strain H0001, Isolated from a Tularemia Patient in the Republic of Korea.

Francisella tularensis is the etiological agent of the zoonosis tularemia. Here, we report the draft genome sequence of F. tularensis subsp. holarctica H0001, which was isolated from a tularemia patient in the Republic of Korea.

Early Laboratory Preparedness of the Korea Disease Control and Prevention Agency and Response to Unknown Pneumonia Outbreak from Wuhan, China, in January 2020.

We report the response process of the Laboratory Analysis Task Force (LATF) for Unknown Disease Outbreaks (UDOs) at the Korea Disease Control and Prevention Agency (KDCA) during January 2020 to coronavirus disease 2019 (COVID-19), which developed as a UDO in Korea. The advanced preparedness offered by the laboratory diagnostic algorithm for UDOs related to respiratory syndromes was critical for the rapid identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enabled us to establish and expand the diagnostic capacity for COVID-19 on a national scale in a timely manner.

Genetic Characterization of Clostridium botulinum Isolated from the First Case of Infant Botulism in Korea.

Botulism is a neuroparalytic disease caused by a neurotoxin produced by Clostridium botulinum. This study aimed to genetically characterize C. botulinum strain isolated from the first case of infant botulism in Korea reported on June 17, 2019. We isolated C. botulinum strain CB-27 from a stool sample of the patient and analyzed the toxin types and toxin gene cluster compositions of the strain using a mouse bioassay, real-time PCR, and genome sequencing. Toxin gene cluster analysis showed that strain CB-27 possesses a C. botulinum neurotoxin type A harboring an unexpressed B gene. Although the nucleotide and amino acid sequences of toxin genes as well as the toxin gene cluster arrangements in strain CB-27 were identical to those of the known strain CDC_69094, the total nucleotide sequences of the toxin gene clusters of CB-27 differed from those of CDC_69094 by 0.47%, indicating genetic diversity of toxin gene clusters of CB-27 among other previously reported C. botulinum strains. To our knowledge, this is the first description of a C. botulinum strain with two separate toxin gene clusters in Korea.

A putative exosporium lipoprotein GBAA0190 of Bacillus anthracis as a potential anthrax vaccine candidate.

BACKGROUND: Bacillus ancthracis causes cutaneous, pulmonary, or gastrointestinal forms of anthrax. B. anthracis is a pathogenic bacterium that is potentially to be used in bioterrorism because it can be produced in the form of spores. Currently, protective antigen (PA)-based vaccines are being used for the prevention of anthrax, but it is necessary to develop more safe and effective vaccines due to their prolonged immunization schedules and adverse reactions. METHODS: We selected the lipoprotein GBAA0190, a potent inducer of host immune response, present in anthrax spores as a novel potential vaccine candidate. Then, we evaluated its immune-stimulating activity in the bone marrow-derived macrophages (BMDMs) using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Protective efficacy of GBAA0190 was evaluated in the guinea pig (GP) model. RESULTS: The recombinant GBAA0190 (r0190) protein induced the expression of various inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in the BMDMs. These immune responses were mediated through toll-like receptor 1/2 via activation of mitogen-activated protein (MAP) kinase and Nuclear factor-κB (NF-κB) pathways. We demonstrated that not only immunization of r0190 alone, but also combined immunization with r0190 and recombinant PA showed significant protective efficacy against B. anthracis spore challenges in the GP model. CONCLUSIONS: Our results suggest that r0190 may be a potential target for anthrax vaccine.

2019 Tabletop Exercise for Laboratory Diagnosis and Analyses of Unknown Disease Outbreaks by the Korea Centers for Disease Control and Prevention.

OBJECTIVES: The Korea Centers for Disease Control and Prevention has published "A Guideline for Unknown Disease Outbreaks (UDO)." The aim of this report was to introduce tabletop exercises (TTX) to prepare for UDO in the future. METHODS: The UDO Laboratory Analyses Task Force in Korea Centers for Disease Control and Prevention in April 2018, assigned unknown diseases into 5 syndromes, designed an algorithm for diagnosis, and made a panel list for diagnosis by exclusion. Using the guidelines and laboratory analyses for UDO, TTX were introduced. RESULTS: Since September 9th, 2018, the UDO Laboratory Analyses Task Force has been preparing TTX based on a scenario of an outbreak caused by a novel coronavirus. In December 2019, through TTX, individual missions, epidemiological investigations, sample treatments, diagnosis by exclusions, and next generation sequencing analysis were discussed, and a novel coronavirus was identified as the causal pathogen. CONCLUSION: Guideline and laboratory analyses for UDO successfully applied in TTX. Conclusions drawn from TTX could be applied effectively in the analyses for the initial response to COVID-19, an ongoing epidemic of 2019 - 2020. Therefore, TTX should continuously be conducted for the response and preparation against UDO.

Development of a double-antibody sandwich ELISA for sensitive detection of Yersinia pestis.

We developed a biotin-streptavidin-based sandwich ELISA for the sensitive and specific detection of Yersinia pestis. In this assay, the F1 capsular protein and Y. pestis were captured by anti-F1 mouse monoclonal antibody followed by detection with biotinylated-anti-F1 rabbit polyclonal antibody and HRP-conjugated streptavidin. The developed F1 ELISA could detect not only the F1 protein up to 29 and 17 pg/ml but also Y. pestis up to 177.8 and 129.2 CFU/ml in PBS buffer and human serum, respectively. In addition, the F1 ELISA did not show any cross-reactivity with various proteins and bacterial strains.

SERS-Based Immunoassays for the Detection of Botulinum Toxins A and B Using Magnetic Beads.

Rapid and sensitive detection of botulinum neurotoxins (BoNTs) is important for immediate treatment with proper antitoxins. However, it is difficult to detect BoNTs at the acute phase of infection, owing to its rarity and ambiguous symptoms. To resolve this problem, we developed a surface-enhanced Raman scattering (SERS)-based immunoassay technique for the rapid and sensitive detection of BoNTs. Magnetic beads and SERS nanotags as capture substrates and detection probes, respectively, and Nile Blue A (NBA) and malachite green isothiocyanate (MGITC) as Raman reporter molecules were used for the detection of two different types of BoNTs (types A and B), respectively. The corresponding limits of detection (LODs) were determined as 5.7 ng/mL (type A) and 1.3 ng/mL (type B). Total assay time, including that for immunoreaction, washing, and detection, was less than 2 h.

A therapeutic human antibody against the domain 4 of the Bacillus anthracis protective antigen shows protective efficacy in a mouse model.

Since Bacillus anthracis is a high-risk pathogen and a potential tool for bioterrorism, numerous therapeutic methods including passive immunization have been actively developed. Using a human monoclonal antibody phage display library, we screened new therapeutic antibodies for anthrax infection against protective antigen (PA) of B. anthracis. Among 5 selected clones of antibodies based on enzyme-linked immunosorbent assay (ELISA) results, 7B1 showed neutralizing activity to anthrax lethal toxin (LT) by inhibiting binding of the domain 4 of PA (PD4) to its cellular receptors. Through light chain shuffling process, we improved the productivity of 7B1 up to 25 folds. The light chain shuffled 7B1 antibody showed protective activity against LT both in vitro and in vivo. Furthermore, the antibody also conferred protection of mice from 3 × LD50 challenges of fully virulent anthrax spores. Our result expands the possibility of developing a new therapeutic antibody for anthrax cure.

Muramyl dipeptide potentiates a Bacillus anthracis poly-γ-d-glutamic acid capsule surrogate that induces maturation and activation of mouse dendritic cells.

Poly-γ-d-glutamic acid (PGA) of anthrax is an important pathogenic factor due to its anti-phagocytic activity. Additionally, PGA has the ability to activate mouse macrophages for the secretion of cytokines through Toll-like receptor (TLR) 2. Peptidoglycan (PGN), a major bacterial cell-wall component, induces inflammatory responses in the host. We assessed whether PGA can induce maturation and cytokine expression in immature mouse dendritic cells (DCs) in the existence of muramyl dipeptide (MDP), the minimum motif of PGN with immunostimulatory activity. Stimulation of immature DCs with PGA or MDP alone augmented expression of costimulatory molecules and MHC class II proteins, which are all cell surface markers indicative of maturation. The observed effects were further enhanced by costimulation of PGA and MDP. PGA alone was sufficient to induce expression of TNF-α, IL-6, MCP-1, and MIP1-α, whereas MDP alone did not under the same conditions. Treatment with MDP enhanced PGA-induced expression of the tested inflammatory mediators; however, the synergistic effect found for PGA and MDP was not observed in TLR2- or nucleotide-binding oligomerization domain (NOD) 2-knockout DCs. Additionally, MDP augmented PGA-induced MAP kinases and NF-κB activation, which is crucial for expression of cytokines. Furthermore, MAP kinase and NF-κB inhibitors attenuated MDP enhancement of PGA-induced cytokine production. In addition, co-culture of splenocytes and PGA/MDP-matured DCs induced higher expression of IL-2 and IFN-γ compared to that of splenocytes and PGA-matured DCs. Collectively, our results suggest that PGA and MDP cooperatively induce inflammatory responses in mouse DCs through TLR2 and NOD2 via MAP kinase and NF-κB pathways, subsequently leading to lymphocyte activation.

Vibrio cholerae OmpU induces IL-8 expression in human intestinal epithelial cells.

Although Vibrio cholerae colonizes the small intestine and induces acute inflammatory responses, less is known about the molecular mechanisms of V. cholerae-induced inflammatory responses in the intestine. We recently reported that OmpU, one of the most abundant outer membrane proteins of V. cholerae, plays an important role in the innate immunity of the whole bacteria. In this study, we evaluated the role of OmpU in induction of IL-8, a representative chemokine that recruits various inflammatory immune cells, in the human intestinal epithelial cell (IEC) line, HT-29. Recombinant OmpU (rOmpU) of V. cholerae induced IL-8 expression at the mRNA and protein levels in a dose- and time-dependent manner. Interestingly, IL-8 was secreted through both apical and basolateral sides of the polarized HT-29 cells upon apical exposure to rOmpU but not upon basolateral exposure. rOmpU-induced IL-8 expression was inhibited by interference of lipid raft formation with nystatin, but not by blocking the formation of clathrin-coated pits with chlorpromazine. In addition, rOmpU-induced IL-8 expression was mediated via ERK1/2 and p38 kinase pathways, but not via JNK signaling pathway. Finally, V. cholerae lacking ompU elicited decreased IL-8 expression and adherence to HT-29 cells compared to the parental strain. Collectively, these results suggest that V. cholerae OmpU might play an important role in intestinal inflammation by inducing IL-8 expression in human IECs.

Integrated SERS-Based Microdroplet Platform for the Automated Immunoassay of F1 Antigens in Yersinia pestis.

The development of surface-enhanced Raman scattering (SERS)-based microfluidic platforms has attracted significant recent attention in the biological sciences. SERS is a highly sensitive detection modality, with microfluidic platforms providing many advantages over microscale methods, including high analytical throughput, facile automation, and reduced sample requirements. Accordingly, the integration of SERS with microfluidic platforms offers significant utility in chemical and biological experimentation. Herein, we report a fully integrated SERS-based microdroplet platform for the automatic immunoassay of specific antigen fraction 1 (F1) in Yersinia pestis. Specifically, highly efficient and rapid immunoreactions are achieved through sequential droplet generation, transport, and merging, while wash-free immunodetection is realized through droplet-splitting. Such integration affords a novel multifunctional platform capable of performing complex multistep immunoassays in nL-volume droplets. The limit of detection of the F1 antigen for Yersinia pestis using the integrated SERS-based microdroplet platform is 59.6 pg/mL, a value approximately 2 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays. This assay system has additional advantages including reduced sample consumption (less than 100 µL), rapid assay times (less than 10 min), and fully automated fluid control. We anticipate that this integrated SERS-based microdroplet device will provide new insights in the development of facile assay platforms for various hazardous materials.

The Poly-γ-D-Glutamic Acid Capsule of Bacillus licheniformis, a Surrogate of Bacillus anthracis Capsule Induces Interferon-Gamma Production in NK Cells through Interactions with Macrophages.

The poly-γ-D-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, provides protection of the bacterium from phagocytosis and allows its unimpeded growth in the host. We investigated crosstalk between murine natural killer (NK) cells and macrophages stimulated with the PGA capsule of Bacillus licheniformis, a surrogate of the B. anthracis capsule. PGA induced interferon-gamma production from NK cells cultured with macrophages. This effect was dependent on macrophage-derived IL-12 and cell-cell contact interaction with macrophages through NK cell receptor NKG2D and its ligand RAE-1. The results showed that PGA could enhance NK cell activation by inducing IL-12 production in macrophages and a contact-dependent crosstalk with macrophages.

Variations in Spike Glycoprotein Gene of MERS-CoV, South Korea, 2015.

An outbreak of nosocomial infections with Middle East respiratory syndrome coronavirus occurred in South Korea in May 2015. Spike glycoprotein genes of virus strains from South Korea were closely related to those of strains from Riyadh, Saudi Arabia. However, virus strains from South Korea showed strain-specific variations.

The poly-γ-d-glutamic acid capsule surrogate of the Bacillus anthracis capsule induces nitric oxide production via the platelet activating factor receptor signaling pathway.

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, confers protection of the bacillus from phagocytosis and allows its unimpeded growth in the host. PGA capsules released from B. anthracis are associated with lethal toxin in the blood of experimentally infected animals and enhance the cytotoxic effect of lethal toxin on macrophages. In addition, PGA capsule itself activates macrophages and dendritic cells to produce proinflammatory cytokine such as IL-1ß, indicating multiple roles of PGA capsule in anthrax pathogenesis. Here we report that PGA capsule of Bacillus licheniformis, a surrogate of B. anthracis capsule, induces production of nitric oxide (NO) in RAW264.7 cells and bone marrow-derived macrophages. NO production was induced by PGA in a dose-dependent manner and was markedly reduced by inhibitors of inducible NO synthase (iNOS), suggesting iNOS-dependent production of NO. Induction of NO production by PGA was not observed in macrophages from TLR2-deficient mice and was also substantially inhibited in RAW264.7 cells by pretreatment of TLR2 blocking antibody. Subsequently, the downstream signaling events such as ERK, JNK and p38 of MAPK pathways as well as NF-κB activation were required for PGA-induced NO production. In addition, the induced NO production was significantly suppressed by treatment with antagonists of platelet activating factor receptor (PAFR) or PAFR siRNA, and mediated through PAFR/Jak2/STAT-1 signaling pathway. These findings suggest that PGA capsule induces NO production in macrophages by triggering both TLR2 and PAFR signaling pathways which lead to activation of NF-kB and STAT-1, respectively.

The Poly-γ-d-Glutamic Acid Capsule Surrogate of the Bacillus anthracis Capsule Is a Novel Toll-Like Receptor 2 Agonist.

Bacillus anthracis is a pathogenic Gram-positive bacterium that causes a highly lethal infectious disease, anthrax. The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of B. anthracis, along with exotoxins. PGA enables B. anthracis to escape phagocytosis and immune surveillance. Our previous study showed that PGA activates the human macrophage cell line THP-1 and human dendritic cells, resulting in the production of the proinflammatory cytokine interleukin-1ß (IL-1ß) (M. H. Cho et al., Infect Immun 78:387-392, 2010, http://dx.doi.org/10.1128/IAI.00956-09). Here, we investigated PGA-induced cytokine responses and related signaling pathways in mouse bone marrow-derived macrophages (BMDMs) using Bacillus licheniformis PGA as a surrogate for B. anthracis PGA. Upon exposure to PGA, BMDMs produced proinflammatory mediators, including tumor necrosis factor alpha (TNF-α), IL-6, IL-12p40, and monocyte chemoattractant protein 1 (MCP-1), in a concentration-dependent manner. PGA stimulated Toll-like receptor 2 (TLR2) but not TLR4 in Chinese hamster ovary cells expressing either TLR2 or TLR4. The ability of PGA to induce TNF-α and IL-6 was retained in TLR4(-/-) but not TLR2(-/-) BMDMs. Blocking experiments with specific neutralizing antibodies for TLR1, TLR6, and CD14 showed that TLR6 and CD14 also were necessary for PGA-induced inflammatory responses. Furthermore, PGA enhanced activation of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB), which are responsible for expression of proinflammatory cytokines. Additionally, PGA-induced TNF-α production was abrogated not only in MyD88(-/-) BMDMs but also in BMDMs pretreated with inhibitors of MAP kinases and NF-κB. These results suggest that immune responses induced by PGA occur via TLR2, TLR6, CD14, and MyD88 through activation of MAP kinase and NF-κB pathways.

A novel nanoprobe for the sensitive detection of Francisella tularensis.

Francisella tularensis is a human zoonotic pathogen and the causative agent of tularemia, a severe infectious disease. Given the extreme infectivity of F. tularensis and its potential to be used as a biological warfare agent, a fast and sensitive detection method is highly desirable. Herein, we construct a novel detection platform composed of two units: (1) Magnetic beads conjugated with multiple capturing antibodies against F. tularensis for its simple and rapid separation and (2) Genetically-engineered apoferritin protein constructs conjugated with multiple quantum dots and a detection antibody against F. tularensis for the amplification of signal. We demonstrate a 10-fold increase in the sensitivity relative to traditional lateral flow devices that utilize enzyme-based detection methods. We ultimately envision the use of our novel nanoprobe detection platform in future applications that require the highly-sensitive on-site detection of high-risk pathogens.

Fast and sensitive detection of an anthrax biomarker using SERS-based solenoid microfluidic sensor.

We report the application of a fully automated surface-enhanced Raman scattering (SERS)-based solenoid-embedded microfluidic device to the quantitative and sensitive detection of anthrax biomarker poly-γ-D-glutamic acid (PGA) in solution. Analysis is based on the competitive reaction between PGA and PGA-conjugated gold nanoparticles with anti-PGA-immobilized magnetic beads within a microfluidic environment. Magnetic immunocomplexes are trapped by yoke-type solenoids embedded within the device, and their SERS signals were directly measured and analyzed. To improve the accuracy of measurement process, external standard values for PGA-free serum were also measured through use of a control channel. This additional measurement greatly improves the reliability of the assay by minimizing the influence of extraneous experimental variables. The limit of detection (LOD) of PGA in serum, determined by our SERS-based microfluidic sensor, is estimated to be 100 pg/mL. We believe that the defined method represents a valuable analytical tool for the detection of anthrax-related aqueous samples.

Enterococcus faecalis lipoteichoic acid suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in human periodontal ligament cells.

Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.