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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.

Clostridium botulinum produces one of the most potent biological toxins and causes botulism, a rare but potentially fatal neuroparalytic disease. In 2014, a foodborne botulism case was reported in Korea, and a strain (CB-2014001) was isolated. Initial characterization identified it as a BoNT/B-producing strain based on mouse bioassay and conventional PCR. However, subsequent genomic analysis revealed the presence of dual BoNT gene clusters, bont/B and bont/F, corresponding to subtypes B5 and F2, respectively. Therefore, we aimed to analyze the genetic characteristics and toxin expression profiles of the isolated strain. The strain showed high sequence identity with Bf-type strains such as CDC 3281 and An436. Functional assays confirmed simultaneous expression of both BoNT/B and /F toxins at 35 °C, and temperature-dependent assays revealed predominant expression of BoNT/F at 30 °C and BoNT/B at 37 °C, indicating that toxin expression is influenced by environmental temperature. These findings highlight the potential for differential pathogenicity based on culture conditions and underscore the importance of developing diagnostic tools capable of detecting multiple bont genes. To our knowledge, this is the first report of a dual-toxin-producing C. botulinum strain associated with foodborne botulism in Korea, providing important insights into botulism diagnosis, treatment strategies, and public health preparedness.

Background Monitoring immunity levels nationwide and identifying disparities are important to prepare for future pandemics. However, data regarding changes in the seroprevalence of SARS‐CoV‐2 and disparities in consecutive epidemic waves using existing surveillance systems are limited. Methods We conducted a cross‐sectional serosurvey using 11,506 residual serum samples collected from the Korean National Health and Nutrition Examination Survey between January 2021 and December 2022. Antibodies to the SARS‐CoV‐2 spike (anti‐S) protein, indicative of vaccination or past infection of SARS‐CoV‐2, and nucleocapsid (anti‐N) protein, indicating infection, were quantified. Then, we applied post‐stratification weighting through the bootstrap resampling based on the age and sex distribution of the South Korean population. We used regression models to identify any disparities in the seropositivity prevalence ratio (PR) across different epidemic waves of SARS‐CoV‐2 and demographics in the population. Results We identified that the anti‐S seropositivity gradually increased after the COVID‐19 vaccination rollout, whereas anti‐N seropositivity increased after the SARS‐CoV‐2 Omicron variant was introduced in Korea. Anti‐S seropositivity PR was 0.12–0.76 times lower in individuals < 18 years than in elderly individuals ≥ 65 years during Waves 4–5 (July 2021 to June 2022). Anti‐N seropositivity PR was 1.25–1.83 times higher in individuals less than 64 years than in elderly individuals during Waves 5–7 (January 2022 to December 2022). Conclusions Our findings provide insights into the dynamic changes in immunity levels among the Korean population after the COVID‐19 vaccine rollout and the introduction of the Omicron variant. Identifying the disparity in seroprevalence in the study population during the pandemic by using the existing surveillance system provides helpful information to develop future pandemic preparedness plans for the population.

Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.

Background Anthrax and smallpox are high-risk infectious diseases, and considered as potential agents for bioterrorism. To develop an effective countermeasure for these diseases, we constructed a bivalent vaccine against both anthrax and smallpox by integrating a gene encoding protective antigen (PA) of Bacillus anthracis to the genome of the attenuated vaccinia virus strain, KVAC103. Results Immunization with this bivalent vaccine induced antibodies against both PA and vaccinia virus in a mouse model. We also observed that the efficacy of this vaccine can be enhanced by combined immunization with immunoadjuvant-expressing KVAC103. Mouse groups co-immunized with PA-expressing KVAC103 and either interleukin-15 (IL-15) or cholera toxin subunit A (CTA1)-expressing KVAC103 showed increased anti-PA IgG titer and survival rate against B. anthracis spore challenge compared to the group immunized with PA-expressing KVAC103 alone. Conclusions We demonstrated that the attenuated smallpox vaccine KVAC103 is an available platform for a multivalent vaccine and co-immunization of immunoadjuvants can improve vaccine performance.

Background: Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are potent protein toxins that can cause botulism, which leads to death or neuroparalysis in humans by targeting the nervous system. BoNTs comprise three functional domains: a light-chain enzymatic domain (LC), a heavy-chain translocation domain (HCN), and a heavy-chain receptor-binding domain (HCC). The HCC domain is critical for binding to neuronal cell membrane receptors and facilitating BoNT internalization via endocytosis. Accordingly, it may serve as a vaccine candidate, inducing anti-BoNT-neutralizing antibodies in animals. Here, we aimed to develop a vaccine capable of simultaneously defending against both BoNT/A and B. Methods: We combined the HCC domains of botulinum neurotoxin type A (BoNT/A) and botulinum neurotoxin type B (BoNT/B) in Escherichia coli to produce a recombinant protein (rHCCB-L-HCCArHCcB) that offers dual protection against both toxins by inhibiting their receptor binding. To evaluate the efficacy of the vaccine, mice were immunized intramuscularly with rHCCB-L-HCCA plus alum thrice at 2-week intervals, followed by the assessment of immunogenicity and protective efficacy. Results: The antibody titer in mice immunized with rHCCB-L-HCCA was significantly higher than that in mice immunized with alum alone, protecting them from the lethal challenges of BoNT/A (105 50% lethal dose, LD50) and B (103 LD50). Conclusion: These findings suggest that rHCCB-L-HCCA may simultaneously be an effective vaccine candidate against BoNT/A and B.

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.

To the best of our knowledge, no study has been conducted to date to directly compare the collagen metabolism of photoaged and naturally aged human skin. In this study, we compared collagen synthesis, matrix metalloproteinase-1 levels, and gelatinase activity of sun-exposed and sun-protected skin of both young and old subjects. Using northern blot analysis, immunohistochemical stain, and Western blot analysis, we demonstrated that the levels of procollagen type I mRNA and protein in photoaged and naturally aged human skin in vivo are significantly lower than those of young skin. Furthermore, we demonstrated, by northern blot analysis, that the procollagen α1(I) mRNA expression of photoaged skin is much greater than that of sun-protected skin in the same individual. In situ hybridization and immunohistochemical stain were used to show that the expression of type I procollagen mRNA and protein in the fibroblasts of photoaged skin is greater than for naturally aged skin. In addition, it was found, by Western blot analysis using protein extracted from the dermal tissues, that the level of procollagen type I protein in photoaged skin is lower than that of naturally aged skin. The level of matrix metalloproteinase-1 protein and the activity of matrix metalloproteinase-2 were higher in the dermis of photoaged skin than in naturally aged skin. Our results suggest that the natural aging process decreases collagen synthesis and increases the expression of matrix metalloproteinases, whereas photoaging results in an increase of collagen synthesis and greater matrix metalloproteinase expression in human skin in vivo. Thus, the balance between collagen synthesis and degradation leading to collagen deficiency is different in photoaged and naturally aged skin.

To understand the molecular alterations occurring during the aging process, we compared mitogen-activated protein (MAP) kinase activities in the intrinsically aged and photoaged skins in the same individuals. Furthermore, we investigated the molecular events related to MAP kinase changes in intrinsically aged and photoaged skins. We found that extracellular-signal-regulated kinase (ERK) activity in photoaged skin was reduced, and that the activities of c-Jun N-terminal kinase (JNK) and p38 kinase were increased compared with intrinsically aged skin in the same individuals. Phospho-c-Jun levels and activator protein 1 activities in photoaged skin were also higher than in intrinsically aged skin. Moreover, catalase activity was found to be much reduced in primary dermal fibroblasts from photoaged skin, and as a result, H2O2 accumulated more in primary dermal fibroblasts in photoaged skin. In addition, treating primary dermal fibroblasts from photoaged skin with catalase reduced H2O2 levels, reversed aging-dependent MAP kinase changes, and inhibited matrix metalloproteinase (MMP)-1 expression. Our results indicate that the accumulation of reactive oxygen species due to catalase attenuation may be a critical aspect of the MAP kinase signaling changes that may lead to skin aging and photoaging in human skin in vivo. Thus, the induction and regulation of endogenous antioxidant enzymes including catalase may offer a strategy for preventing and treating skin aging.

The novel anthrax vaccine candidate GC1109, composed of recombinant protective antigen, has shown robust antibody responses and safety in preclinical and clinical studies. However, the assessment of GC1109 vaccine efficacy was limited in a rodent model and could not be applied in clinical studies due to ethical issues. In this study, we aim to provide predictive insights for human applications of GC1109 by examining the correlation between anthrax toxin-neutralizing antibodies (TNAs) and protection against Bacillus anthracis infection in rabbit. The threshold level of TNAs necessary for protection was assessed following subcutaneous (s.c) challenge with lethal B. anthracis spores. In addition, the longevity of immunity in GC1109-vaccinated rabbits was investigated. A positive correlation between TNA levels and survival was observed, indicating their potential as a predictor of protection in humans. The 50 % neutralization factor (NF50) value of 0.5 was associated with a 70 % probability of survival, establishing this value as a surrogate marker for protective efficacy. Long-term protection was confirmed, with the GC1109-vaccinated group exhibiting significantly higher survival rates (91.7 %) than the control group (8.3 %) at 12 months post-vaccination. These findings highlight the protective efficacy and durability of GC1109-induced immunity against lethal B. anthracis challenge. The study supports the continued development of GC1109 as a viable anthrax vaccine candidate and underscores its potential for emergency preparedness in Korea. •GC1109 showed immunogenicity and protective efficacy in a rabbit model.•A surrogate marker for assessing anthrax vaccine-induced immunity was established.•A 50 % neutralization factor of 0.5 correlated with 70 % survival probability.•Long lasting immunogenicity against lethal anthrax spores were validated.