Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine.

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.

Insight into structural diversity of influenza virus haemagglutinin.

Influenza virus infects host cells through membrane fusion, a process mediated by the low pH-induced conformational change of the viral surface glycoprotein haemagglutinin (HA). We determined the structures and biochemical properties of the HA proteins from A/Korea/01/2009 (KR01), a 2009 pandemic strain, and A/Thailand/CU44/2006 (CU44), a seasonal strain. The crystal structure of KR01 HA revealed a V-shaped head-to-head arrangement, which is not seen in other HA proteins including CU44 HA. We isolated a broadly neutralizing H1-specific monoclonal antibody GC0757. The KR01 HA-Fab0757 complex structure also exhibited a head-to-head arrangement of HA. Both native and Fab complex structures reveal a different spatial orientation of HA1 relative to HA2, indicating that HA is flexible and dynamic at neutral pH. Further, the KR01 HA exhibited significantly lower protein stability and increased susceptibility to proteolytic cleavage compared with other HAs. Our structures provide important insights into the conformational flexibility of HA.

Genetic populations of Bacillus anthracis isolates from Korea.

Bacillus (B.) anthracis is the pathogen that causes fatal anthrax. Strain-specific detection of this bacterium using molecular approaches has enhanced our knowledge of microbial population genetics. In the present study, we employed molecular approaches including multiple-locus variable-number tandem repeat analysis (MLVA) and canonical single-nucleotide polymorphism (canSNP) analysis to perform molecular typing of B. anthracis strains isolated in Korea. According to the MLVA, 17 B. anthracis isolates were classified into A3a, A3b, and B1 clusters. The canSNP analyses subdivided the B. anthracis isolates into two of the three previously recognized major lineages (A and B). B. anthracis isolates from Korea were found to belong to four canSNP sub-groups (B.Br.001/2, A.Br.005/006, A.Br.001/002, and A.Br.Ames). The A.Br.001/002 and A.Br.Ames sub-lineages are closely related genotypes frequently found in central Asia and most isolates were. On the other hand, B. anthracis CH isolates were analyzed that belonged to the B.Br.001/002 sub-group which found in southern Africa, Europe and California (USA). B.Br.001/002 genotype is new lineage of B. anthracis in Korea that was not found before. This discovery will be helpful for the creation of marker systems and might be the result of human activity through the development of agriculture and increased international trade in Korea.

Differences in the risk factors for surgical site infection between total hip arthroplasty and total knee arthroplasty in the Korean Nosocomial Infections Surveillance System (KONIS).

OBJECTIVE: To compare the characteristics and risk factors for surgical site infections (SSIs) after total hip arthroplasty (THA) and total knee arthroplasty (TKA) in a nationwide survey, using shared case detection and recording systems. DESIGN: Retrospective cohort study. SETTING: Twenty-six hospitals participating in the Korean Nosocomial Infections Surveillance System (KONIS). PATIENTS: From 2006 to 2009, all patients undergoing THA and TKA in KONIS were enrolled. RESULTS: SSI occurred in 161 (2.35%) of 6,848 cases (3,422 THAs and 3,426 TKAs). Pooled mean SSI rates were 1.69% and 2.82% for THA and TKA, respectively. Of the cases we examined, 42 (26%) were superficial-incisional SSIs and 119 (74%) were "severe" SSIs; of the latter, 24 (15%) were deep-incisional SSIs and 95 (59%) were organ/space SSIs. In multivariate analysis, a duration of preoperative hospital stay of greater than 3 days was a risk factor for total SSI after both THA and TKA. Diabetes mellitus, revision surgery, prolonged duration of surgery (above the 75th percentile), and the need for surgery due to trauma were independent risk factors for total and severe SSI after THA, while male sex and an operating room without artificial ventilation were independent risk factors for total and severe SSI after TKA. A large volume of surgeries (more than 10 procedures per month) protected against total and severe SSI, but only in patients who underwent TKA. CONCLUSIONS: Risk factors for SSI after arthroplasty differ according to the site of the arthroplasty. Therefore, clinicians should take into account the site of arthroplasty in the analysis of SSI and the development of strategies for reducing SSI.

Avian influenza a (H5N1) virus antibodies in poultry cullers, South Korea, 2003-2004.

Transmission of influenza (H5N1) virus from birds to humans is a serious public health threat. In South Korea, serologic investigation among 2,512 poultry workers exposed during December 2003-March 2004 to poultry with confirmed or suspected influenza (H5N1) virus infection found antibodies in 9. Frequency of bird-to-human transmission was low.

Prospective nationwide surveillance of surgical site infections after gastric surgery and risk factor analysis in the Korean Nosocomial Infections Surveillance System (KONIS).

OBJECTIVE: To evaluate the risk factors for surgical site infection (SSI) after gastric surgery in patients in Korea. DESIGN: A nationwide prospective multicenter study. SETTING: Twenty university-affiliated hospitals in Korea. METHODS: The Korean Nosocomial Infections Surveillance System (KONIS), a Web-based system, was developed. Patients in 20 Korean hospitals from 2007 to 2009 were prospectively monitored for SSI for up to 30 days after gastric surgery. Demographic data, hospital characteristics, and potential perioperative risk factors were collected and analyzed, using multivariate logistic regression models. RESULTS: Of the 4,238 case patients monitored, 64.9% (2,752) were male, and mean age (± SD) was 58.8 (± 12.3) years. The SSI rates were 2.92, 6.45, and 10.87 per 100 operations for the National Nosocomial Infections Surveillance system risk index categories of 0, 1, and 2 or 3, respectively. The majority (69.4%) of the SSIs observed were organ or space SSIs. The most frequently isolated microorganisms were Staphylococcus aureus and Klebsiella pneumoniae. Male sex (odds ratio [OR], 1.67 [95% confidence interval (CI), 1.09-2.58]), increased operation time (1.20 [1.07-1.34] per 1-hour increase), reoperation (7.27 [3.68-14.38]), combined multiple procedures (1.79 [1.13-2.83]), prophylactic administration of the first antibiotic dose after skin incision (3.00 [1.09-8.23]), and prolonged duration (≥7 days) of surgical antibiotic prophylaxis (SAP; 2.70 [1.26-5.64]) were independently associated with increased risk of SSI. CONCLUSIONS: Male sex, inappropriate SAP, and operation-related variables are independent risk factors for SSI after gastric surgery.

Serological Correlate of Protection in Guinea Pigs for a Recombinant Protective Antigen Anthrax Vaccine Produced from Bacillus brevis.

OBJECTIVE: Recombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model. METHODS: Serological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminumhydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serumantibody titerswere determined by anti-PA IgGELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay. RESULTS: To examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA. CONCLUSION: Neutralizing-antibody titers can be used as a surrogate marker.

The poly-γ-D-glutamic acid capsule of Bacillus anthracis enhances lethal toxin activity.

The poly-γ-D-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguises B. anthracis from immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

Development and evaluation of a rapid influenza diagnostic test for the pandemic (H1N1) 2009 influenza virus.

We evaluated a new rapid influenza diagnostic test for the pandemic (H1N1) 2009 influenza virus by using real-time reverse transcription-PCR (rRT-PCR) and viral culture. The sensitivities were 68.5% and 64.5%, and the specificities were 98.4% and 97.6%, respectively. This kit should be used with caution, and negative results should be verified by a confirmative test.

Pathogenesis and Chronologic Localization of the Human Influenza A (H1N1) Virus in Cotton Rats.

OBJECTIVES: We aimed to evaluate the pathogenesis and chronologic localization of human influenza A (H1N1) virus in experimentally infected cotton rats. METHODS: The animals were intranasally inoculated with 10(7) plaque-forming units of A/Solomon Islands/3/2006 (H1N1) influenza virus and evaluated for pathogenicity for a period of 28 days. Virus replication kinetics and pathological properties were assessed chronologically. Acute antiviral responses were evaluated by mean of real-time polymerase chain reaction. RESULTS: Cotton rats infected with A/Solomon Islands/3/2006 virus lost weight until 6 days post-inoculation (DPI) and showed decreased activity until 3 DPI. At necropsy, focal areas of redness and consolidation of lungs were evident at 1, 2, and 3 DPI. Lung histopathology showed moderate to severe interstitial pneumonia, alveolitis and bronchiolitis. Influenza A specific viral protein was detected in bronchiolar epithelial cells, alveolar septa and pneumocytes. Influenza viruses were recovered from the lungs during the early period of infection and the titer peaked at 1 DPI. Viral proteins were detected from 4 hours to 6 hours DPI. These trends correlate with the up-regulation of mRNA expression of the IFN-α, Mx1, and Mx2 genes that play critical roles in the anti-influenza response at the early stage of infection. CONCLUSION: Our results provide evidence that supports the use of cotton rats for the study of influenza virus pathogenesis and the immune response.

Oseltamivir-resistant pandemic (H1N1) 2009 virus, South Korea.

To identify oseltamivir resistance, we analyzed neuraminidase H275Y mutations in samples from 10 patients infected with pandemic (H1N1) 2009 virus in South Korea who had influenza that was refractory to antiviral treatment with this drug. A neuraminidase I117M mutation that might influence oseltamivir susceptibility was detected in sequential specimens from 1 patient.

Replication and pathogenesis of the pandemic (H1N1) 2009 influenza virus in mammalian models.

This study aimed to characterize the replication and pathogenic properties of a Korean pandemic (H1N1) 2009 influenza virus isolate in ferrets and mice. Ferrets infected with A/Korea/01/2009 (H1N1) virus showed mild clinical signs. The virus replicated well in lungs and slightly in brains with no replication in any other organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected in the bronchiolar epithelial cells, in peribronchial glands with severe peribronchitis and in cells present in the alveoli. A/Korea/01/2009 (H1N1) virus-infected mice showed weight loss and pathological lung lesions including perivascular cuffing, interstitial pneumonia and alveolitis. The virus replicated highly in the lungs and slightly in the nasal tissues. Viral antigens were detected in bronchiolar epithelial cells, pneumocytes and interstitial macrophages. However, seasonal H1N1 influenza virus did not replicate in the lungs of ferrets, and viral antigens were not detected. Thus, this Korean pandemic (H1N1) 2009 isolate infected the lungs of ferrets and mice successfully and caused more pathological lesions than did the seasonal influenza virus.

Bloodstream infections and clinical significance of healthcare-associated bacteremia: a multicenter surveillance study in Korean hospitals.

Recent changes in healthcare systems have changed the epidemiologic paradigms in many infectious fields including bloodstream infection (BSI). We compared clinical characteristics of community-acquired (CA), hospital-acquired (HA), and healthcare-associated (HCA) BSI. We performed a prospective nationwide multicenter surveillance study from 9 university hospitals in Korea. Total 1,605 blood isolates were collected from 2006 to 2007, and 1,144 isolates were considered true pathogens. HA-BSI accounted for 48.8%, CA-BSI for 33.2%, and HCA-BSI for 18.0%. HA-BSI and HCA-BSI were more likely to have severe comorbidities. Escherichia coli was the most common isolate in CA-BSI (47.1%) and HCA-BSI (27.2%). In contrast, Staphylococcus aureus (15.2%), coagulase-negative Staphylococcus (15.1%) were the common isolates in HA-BSI. The rate of appropriate empiric antimicrobial therapy was the highest in CA-BSI (89.0%) followed by HCA-BSI (76.4%), and HA-BSI (75.0%). The 30-day mortality rate was the highest in HA-BSI (23.0%) followed by HCA-BSI (18.4%), and CA-BSI (10.2%). High Pitt score and inappropriate empirical antibiotic therapy were the independent risk factors for mortality by multivariate analysis. In conclusion, the present data suggest that clinical features, outcome, and microbiologic features of causative pathogens vary by origin of BSI. Especially, HCA-BSI shows unique clinical characteristics, which should be considered a distinct category for more appropriate antibiotic treatment.

Epidemiologic study of human influenza virus infection in South Korea from 1999 to 2007: origin and evolution of A/Fujian/411/2002-like strains.

Influenza epidemics arise through the accumulation of viral genetic changes, culminating in a novel antigenic type that is able to escape host immunity. Following an outbreak of the A/Fujian/411/2002-like strains in Asia, including China, Japan, and South Korea, in 2002, Australia and New Zealand experienced substantial outbreaks of the same strains in 2003, and subsequently worldwide outbreaks occurred in the 2003-2004 season. The emergence of A/Fujian/411/2002-like strains coincided with a higher level of influenza-like illness in South Korea than what is seen at the peak of a normal season, and there was at least a year's difference between South Korea and the United States. Genetic evolution of human influenza A/H3N2 viruses was monitored by sequence analysis of hemagglutinin (HA) genes collected in Asia, including 269 (164 new) HA genes isolated in South Korea from 1999 to 2007. The Fujian-like influenza strains were disseminated with rapid sequence variation across the antigenic sites of the HA1 domain, which sharply distinguished between the A/Moscow/10/1999-like and A/Fujian/411/2002-like strains. This fast variation, equivalent to approximately 10 amino acid changes within a year, occurred in Asia and would be the main cause of the disappearance of the reassortants, although the reassortant and nonreassortant Fujian-like strains circulated simultaneously in Asia.

Distribution of Virulence Genes and Their Association of Serotypes in Pathogenic Escherichia coli Isolates From Diarrheal Patients in Korea.

OBJECTIVES: To characterise the genetic and serological diversity of pathogenic Escherichia coli, we tested 111 E coli strains isolated from diarrhoeal patients in Korea between 2003 and 2006. METHODS: The isolates were tested through polymerase chain reaction (PCR) and slide agglutination method for the detection of virulence genes and serotypes, respectively. To compare the expression of Shiga toxin (stx)-1 and stx2 genes, real-time quantitative reverse-transcriptase PCR and rapid exprssion assay, reversed-passive latex agglutination, were performed. RESULTS: Forty-nine Shiga toxin-producing E coli (STEC) strains and 62 non-STEC strains, including 20 enteropathogenic E coli, 20 enterotoxigenic E coli, 20 enteroaggregative E coli, and 2 enteroinvasive E coli were randomly chosen from the strains isolated from diarrhoeal patients in Korea between 2003 and 2006. PCR analysis indicated that locus of enterocyte effacement pathogenicity island, that is, eaeA, espADB, and tir genes were present in STEC, enteropathogenic E coli, and enteroinvasive E coli. Quorum sensing-related gene luxS was detected in most of pathogenic E coli strains. Major serotypes of the STEC strains were O157 (26%) and O26 (20%), whereas the non-STEC strains possessed various serotypes. Especially, all the strains with serotype O157 carried stx2 and the tested virulence factors. Of the STEC strains, the data of real-time quantitative reverse-transcriptase PCR and reversed-passive latex agglutination tests showed that messenger RNA- and protein expression of stx2 gene were higher than those of stx1 gene. CONCLUSION: Our results provide the epidemiological information regarding the trend of STEC and non-STEC infections in the general population and show the fundamental data in association of serotypes with virulence genes in diarrhoeagenic E coli strains from Korea.

Bacillus anthracis capsule activates caspase-1 and induces interleukin-1beta release from differentiated THP-1 and human monocyte-derived dendritic cells.

The poly-gamma-d-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infection. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Recently, efforts have been made to include PGA as a component of anthrax vaccine; however, the innate immune response of PGA itself has been poorly investigated. In this study, we characterized the innate immune response elicited by PGA in the human monocytic cell line THP-1, which was differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and human monocyte-derived dendritic cells (hMoDCs). PGA capsules were isolated from the culture supernatant of either the pXO1-cured strain of B. anthracis H9401 or B. licheniformis ATCC 9945a. PGA treatment of differentiated THP-1 cells and hMoDCs led to the specific extracellular release of interleukin-1beta (IL-1beta) in a dose-dependent manner. Evaluation of IL-1beta processing by Western blotting revealed that cleaved IL-1beta increased in THP-1 cells and hMoDCs after PGA treatment. Enhanced processing of IL-1beta directly correlated with increased activation of its upstream regulator, caspase-1, also known as IL-1beta-converting enzyme (ICE). The extracellular release of IL-1beta in response to PGA was ICE dependent, since the administration of an ICE inhibitor prior to PGA treatment blocked induction of IL-1beta. These results demonstrate that B. anthracis PGA elicits IL-1beta production through activation of ICE in PMA-differentiated THP-1 cells and hMoDCs, suggesting the potential for PGA as a therapeutic target for anthrax.

Poly-gamma-d-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea-pigs and rabbits.

Anthrax is a lethal infectious disease caused by the spore-forming Bacillus anthracis. The two major virulence factors of B. anthracis are exotoxin and the poly-gamma-d-glutamic acid (PGA) capsule. The three components of the exotoxin, protective antigen (PA), lethal factor and edema factor act in a binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of the anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of the anthrax vaccine. In this study, we report that PA-PGA conjugates induce expressions of anti-PA, anti-PGA and toxin-neutralizing antibodies in guinea-pigs and completely protect guinea-pigs against a 50 x LD(50) challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA-15mer offer a partial passive protection to guinea-pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. Our results demonstrate that PA-PGA conjugate vaccines are effective in the guinea-pig model, in addition to the previously reported mouse model.

Amantadine-resistant influenza A viruses isolated in South Korea from 2003 to 2009.

To investigate the frequency of amantadine resistance among influenza A viruses isolated in Korea during the 2003-2009 seasons, 369 (16.8%) 2199 A/H1N1 viruses and 780 (14.8%) of 5263 A/H3N2 viruses were randomly selected. The M2 and HA1 genes of each isolate were amplified by reverse transcription-polymerase chain reaction and followed by nucleotide sequencing. The results showed that the resistance rate to amantadine among A/H1N1 viruses increased significantly from 2004-2005 (33.3%) to 2007-2008 (97.8%) and then decreased dramatically in 2008-2009 (1.9%). The A/H1N1 isolates recently detected in 2008-2009 turned amantadine-sensitive containing two new substitutions at specific sites (S141N, G185A) in HA1. Compared with A/H1N1 viruses, the amantadine resistance among the A/H3N2 viruses increased from 2003-2004 (9.7%) to 2005-2006 (96.7%) and decreased in 2006-2007 (57.4%). During 2006-2007, both of amantadine-resistant and -sensitive A/H3N2 viruses co-circulated but clustered in different branches phylogenetically. All of A/H3N2 isolates tested during 2007-2009 appeared to cluster in the same group being resistant to amantadine.

Bacillus anthracis lethal toxin attenuates lipoteichoic acid-induced maturation and activation of dendritic cells through a unique mechanism.

Lethal toxin (LT), produced by the gram-positive bacterium Bacillus anthracis, was identified as a major etiologic agent causing anthrax due to its strong immunotoxicity. Gram-positive bacteria express lipoteichoic acid (LTA), which is considered as a counterpart to lipopolysaccharide (LPS) of gram-negative bacteria, but differs from LPS in the structure and function. Since dendritic cells (DCs) are essential for the appropriate initiation of immune response, we investigated the effect of LT on LTA-induced DC maturation using immature DCs prepared by differentiation of C57BL/6 mouse bone marrow cells. When immature DCs were matured with LTA in the presence of LT, the expression of representative markers for DC maturation such as CD80, CD83, and CD86 together with MHC class I and II molecules was inhibited. LT ameliorated the attenuation of endocytic capacity during DC maturation by LTA while such effect was not observed in LPS-matured DCs. Furthermore, exposure to LT resulted in a decrease in the expression of pro-inflammatory cytokines including IL-6, TNF-alpha, and IL-12p40 in LTA-stimulated DCs as in LPS-stimulated DCs. Interestingly, LT showed a minimal change in LTA-induced IL-1beta expression while LT highly enhanced the LPS-induced IL-1beta expression. Those inhibitory effects might be associated with LT interference of LTA-signaling pathways mediated through mitogen-activated protein kinases (MAPKs) since LT suppressed phosphorylation of MAPK, which was induced by LTA. Meanwhile, no change was observed in the expression of putative anthrax toxin receptors, TEM8 and CMG2, or Toll-like receptor 2. These results suggest that LT suppresses the maturation and activation of DCs stimulated with LTA, similar to the suppression in the LPS-stimulated DCs, but via a distinct mechanism.