Gyeung Haeng Hur

Effects of calmodulin antagonists and anesthetics on the skin lesions induced by 2-chloroethylethyl sulfide.

The effects of calmodulin antagonists and anesthetics on the skin lesions induced by an alkylating vesicant, 2-chloroethylethyl sulfide, were investigated using female hairless mice. 2-Chloroethylethyl sulfide, topically applied (0.6 microliter/5 mm in diameter) on the back skin of hairless mice, induced mild to moderate petechiae on the 1st day, and ulcers with a thick scab after 3 days. The healing process started after 6 days, resulting in shedding of scabs on 9.52 days. Water-soluble ointment bases showed some beneficial effects, whereas oily bases made the skin lesions worse. Trifluoperazine (0.5-1%) and thioridazine (2%), potent calmodulin antagonists, in Pluronic F-127 base substantially prevented the development of 2-chloroethylethyl sulfide-induced skin lesions. A similar effect was achieved with pentamidine (10%), another type of calmodulin antagonist, but not with ketoconazole, a weak calmodulin antagonist. In addition, anesthetics, such as lidocaine and pentobarbital, showed some protection, although at high concentrations (> 5%). As judged by the microscopic appearance, trifluoperazine successfully reduced the hemorrhage and the infiltration of inflammatory cells in early skin lesions, and the formation of thick scabs, which leads to granulomatous scar tissue in late lesions. These results suggest that some calmodulin antagonists and anesthetics in water-soluble bases might be a choice for the treatment of 2-chloroethylethyl sulfide-induced skin burns.

Enhanced Immune Response to DNA Vaccine Encoding Bacillus anthracis PA-D4 Protects Mice against Anthrax Spore Challenge

Anthrax has long been considered the most probable bioweapon-induced disease. The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of anthrax. In the current study, we evaluated the efficiency of a genetic vaccination with the fourth domain (D4) of PA, which is responsible for initial binding of the anthrax toxin to the cellular receptor. The eukaryotic expression vector was designed with the immunoglobulin M (IgM) signal sequence encoding for PA-D4, which contains codon-optimized genes. The expression and secretion of recombinant protein was confirmed in vitro in 293T cells transfected with plasmid and detected by western blotting, confocal microscopy, and enzyme-linked immunosorbent assay (ELISA). The results revealed that PA-D4 protein can be efficiently expressed and secreted at high levels into the culture medium. When plasmid DNA was given intramuscularly to mice, a significant PA-D4-specific antibody response was induced. Importantly, high titers of antibodies were maintained for nearly 1 year. Furthermore, incorporation of the SV40 enhancer in the plasmid DNA resulted in approximately a 15-fold increase in serum antibody levels in comparison with the plasmid without enhancer. The antibodies produced were predominantly the immunoglobulin G2 (IgG2) type, indicating the predominance of the Th1 response. In addition, splenocytes collected from immunized mice produced PA-D4-specific interferon gamma (IFN-γ). The biodistribution study showed that plasmid DNA was detected in most organs and it rapidly cleared from the injection site. Finally, DNA vaccination with electroporation induced a significant increase in immunogenicity and successfully protected the mice against anthrax spore challenge. Our approach to enhancing the immune response contributes to the development of DNA vaccines against anthrax and other biothreats.

Bacillus anthracis Lethal Toxin Induces Cell-type-specific Cytotoxicity in Human Lung Cell Lines

Inhalational anthrax is caused by the entry of Bacillus anthracis spores into the lung. Inhaled spores are phagocytosed by alveolar macrophages. Bacilli then escape from the macrophage and spread to other cells, initiating a systemic anthrax infection. Based on the pathological studies of primate and human inhalational anthrax cases, it appears that lung tissue injury is a lethal consequence of the disease. Although the cytotoxicity of anthrax lethal toxin to macrophages is well known, it is not clear how anthrax toxin affects the various lung cell types.

A novel staphylococcal enterotoxin B subunit vaccine candidate elicits protective immune response in a mouse model

ABSTRACT Staphylococcal enterotoxin B (SEB), produced by the gram‐positive bacterium Staphylococcus aureus, is responsible for food poisoning and toxic shock syndrome, and is considered a potential bioterrorism agent. Unfortunately, still now no approved vaccines are available against SEB. In this study, we constructed a series of nontoxic SEB mutants (mSEBs) and examined whether these mSEBs provide protective immunity against SEB challenge. These mSEB vaccine candidates did not demonstrate superantigen activity in mouse splenocyte cultures. Immunization with the vaccine candidates triggered the production of IgG‐antibodies with neutralizing activity. In addition, increased production of IgG1 and IgG3 was observed after immunization, which signifies both Th1‐ and Th2‐induced immune responses. Among the vaccine candidates tested, S9, a double mutant (N23A and Y90A) and S19, a quadruple mutant (N23A, Y90A, R110A, and F177A), demonstrated complete protection against a lethal SEB challenge. Altogether, our results strongly suggest that these mSEBs could be an effective recombinant SEB vaccine candidates for further/future preclinical and clinical studies. HIGHLIGHTSSEB mutants were proposed as vaccine candidates.Vaccine candidates contain amino acid mutations on the TCR binding site.No superantigenic activity was observed in vitro.Full protection against 10 LD50 of SEB toxin was confirmed.

Protection against lethal vaccinia virus infection in mice using an siRNA targeting the A5R gene.

BACKGROUND Although the World Health Organization has declared the eradication of smallpox in 1980, the fear of its potential use in bioterrorism has become a reality. Since the effectiveness of current vaccines and antiviral drugs is limited, development of new therapeutic strategies is needed. In this study, we investigated small interfering RNA (siRNA) as a therapeutic approach for preventing and treating smallpox infection. METHODS Eight siRNA sequences were designed and evaluated for antiviral activity against vaccinia virus (VACV) in vitro and in vivo. RESULTS Of eight siRNAs, A5R1 siRNA targeted the A5R gene and reduced VACV replication in cell culture by up to 85% at 100 nM concentration without inducing cytotoxicity. A prolonged prophylactic as well as therapeutic effect of siRNA was observed. In addition, real-time PCR analysis showed that A5R1 siRNA can especially reduce the target mRNA. Finally, intraperitoneal delivery of A5R1 siRNA in Balb/c mice significantly protected these animals from lethal challenge with VACV. CONCLUSIONS This study suggests the potential of A5R1 siRNA as a therapeutic antiviral agent against smallpox.

A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication

ABSTRACT Botulinum neurotoxins (BoNTs) are the most potent toxins to mammals. A toxoid vaccine was previously used for prevention of botulinum intoxication; however, this vaccine is no longer available. Currently, no approved botulinum vaccines are available from the Food and Drug Administration (FDA). Recently, a recombinant host cell receptor-binding subunit created for use as a potential vaccine completed phase 2 clinical trials. The current study designed a vaccine candidate against BoNT type A (BoNT/A) using a structural design. Our vaccine candidate was the BoNT/A heavy chain C-terminal region (HCR) that contained the point mutation BA15 (R1269A) within the ganglioside-binding site. A Biacore affinity test showed that the affinity of BA15 for ganglioside GT1b was 100 times lower than that of the HCR. A SNAP25 cleavage assay revealed that immunized sera blocked SNAP25 cleavage of the BoNT/A toxin via BA15. In an in vivo experiment, mice and guinea pigs immunized with BA15 produced neutralizing antibodies that protected against 3,000 LD50 of BoNT/A. In conclusion, the results of both in vitro and in vivo assays showed that our BA15 vaccine candidate was similar to the recombinant host cell receptor-binding subunit vaccine. The inability of BA15to bind ganglioside shows that BA15 is a potential safe vaccine candidate.

Immunogenicity and Protective Efficiency in Mice of a Smallpox DNA Vaccine Candidate

The adverse reactions of the current live smallpox vaccine, and potential use of smallpox as a bioterrorism weapon, have highlighted the need to develop a new effective vaccine for this infectious disease. In the present study, a DNA vaccine vector was produced, which was optimized for expression of the vaccinia virus L1 antigen in a mouse model. Plasmid-encoded IgM-tL1R, which contains a truncated L1R gene fused to an IgM signal sequence, was constructed and expressed under the regulation of an SV40 enhancer. The expressed recombinant tL1 proteins were successfully secreted into the culture media. The DNA vaccine was administered to mice by electroporation, and animals were subsequently challenged with the lethal dose of vaccinia virus. We observed that immunization with IgM-tL1R induced robust neutralizing antibody responses and provided complete protection against a vaccinia virus infection. Isotyping studies revealed a lower IgG1/IgG2a ratio following vaccination with IgM-tL1R, suggesting the stimulation of Th1 immune responses. Our results propose that an optimized DNA vaccine, IgM-tL1R, can be effective in eliciting an anti-vaccinia virus immune response and provide protection against lethal orthopoxvirus challenge.

Structure of the staphylococcal enterotoxin B vaccine candidate S19 showing eliminated superantigen activity

Mutations introduced into S19, a recombinant staphylococcal enterotoxin B vaccine candidate, contribute to its reduced cytokine induction in vivo by removing hydrogen bonds and allowing interaction with the T-cell receptor beta chain and major histocompatibility complex class II.

Role of chondroitin sulfate C in the action of anthrax toxin

Anthrax toxin is produced by Bacillus anthracis, the causative agent of anthrax, and is responsible for the majority of disease symptoms. The toxin consists of 3 proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), which combine to form lethal and edema toxin. Glycosaminoglycans, which are present on the surface of cells, were investigated with regard to their role in toxicity resulting from anthrax toxin exposure. Lethal toxin-induced cytotoxicity of the RAW 264.7 cells was significantly inhibited by the addition of chondroitin sulfate C as determined by the MTT assay. By contrast, several other glycosaminoglycans, including heparin, heparan sulfate, and dermatan sulfate did not show significant levels of inhibition. Studies utilizing fluorescence-labeled PA demonstrated decreased PA binding to RAW 264.7 cells with the addition of chondroitin sulfate C. Formation of PA oligomers at the surface of cells after binding was also inhibited by chondroitin sulfate C. Interestingly, enzymatic degradation of endogenous chondroitin sulfate C from the cell surface with chondroitinase ABC was accompanied by increased sensitivity to the toxin. These findings were further confirmed by pretreating cells with sodium chlorate to reduce the degree of cell surface glycosaminoglycans sulfation. In addition, chondroitin sulfate C effectively inhibits edema toxin-induced cAMP accumulation in cells. Our results indicate that chondroitin sulfate C may play an important role in the toxicity of anthrax toxin.