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.

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.

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.

Influenza sequence and epitope database.

Influenza epidemics arise through the acquisition of viral genetic changes to overcome immunity from previous infections. An increasing number of complete genomes of influenza viruses have been sequenced in Asia in recent years. Knowledge about the genomes of the seasonal influenza viruses from different countries in Asia is valuable for monitoring and understanding of the emergence, migration and evolution of strains. In order to make full use of the wealth of information from such data, we have developed an integrated user friendly relational database, Influenza Sequence and Epitope Database (ISED), that catalogs the influenza sequence and epitope information obtained in Asia. ISED currently hosts a total of 13,020 influenza A and 2984 influenza B virus sequence data collected in 17 countries including 9 Asian countries, and a total of approximately 545 amantadine-resistant influenza virus sequences collected in Korea. ISED provides users with prebuilt application tools to analyze sequence alignment and different patterns and allows users to visualize epitope-matching structures, which is freely accessible at http://influenza.korea.ac.kr and http://influenza.cdc.go.kr.

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.

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.

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.

IDBD: infectious disease biomarker database.

Biomarkers enable early diagnosis, guide molecularly targeted therapy and monitor the activity and therapeutic responses across a variety of diseases. Despite intensified interest and research, however, the overall rate of development of novel biomarkers has been falling. Moreover, no solution is yet available that efficiently retrieves and processes biomarker information pertaining to infectious diseases. Infectious Disease Biomarker Database (IDBD) is one of the first efforts to build an easily accessible and comprehensive literature-derived database covering known infectious disease biomarkers. IDBD is a community annotation database, utilizing collaborative Web 2.0 features, providing a convenient user interface to input and revise data online. It allows users to link infectious diseases or pathogens to protein, gene or carbohydrate biomarkers through the use of search tools. It supports various types of data searches and application tools to analyze sequence and structure features of potential and validated biomarkers. Currently, IDBD integrates 611 biomarkers for 66 infectious diseases and 70 pathogens. It is publicly accessible at http://biomarker.cdc.go.kr and http://biomarker.korea.ac.kr.

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.

Complete genome sequence of Neisseria gonorrhoeae NCCP11945.

Neisseria gonorrhoeae is an obligate human pathogen that is the etiological agent of gonorrhea. We explored variations in the genes of a multidrug-resistant N. gonorrhoeae isolate from a Korean patient in an effort to understand the prevalence, antibiotic resistance, and importance of horizontal gene transfer within this important, naturally competent organism. Here, we report the complete annotated genome sequence of N. gonorrhoeae strain NCCP11945.

Development of real-time PCR assays for detection and quantification of human bocavirus.

BACKGROUND: Human bocavirus (HBoV) is a parvovirus that has been recently detected in patients with respiratory illness. OBJECTIVES: We developed a sensitive, specific, and quantitative real-time PCR assay based on the TaqMan method for HBoV detection and quantification in respiratory specimens. STUDY DESIGN: Three individual real-time PCR assays were designed to amplify HBoV NS1, NP-1, and VP1 genes. For clinical evaluation, 506 nasal aspirates obtained from patients with acute respiratory tract infections during December 2006 to May 2007 were tested. RESULTS: Each assay had a broad dynamic range (50 x 10(7) to 5 x 10(7)copies of plasmid DNA) and high inter- and intra-assay reproducibility. The detection limit of each assay was 10 genome copies per reaction, and no crossreactivity with other major respiratory viruses or bacteria was detected. Clinical evaluation revealed that 11 (2.1%) of 506 patients diagnosed with upper respiratory tract infections, pneumonia, bronchitis, pharyngitis, or sinusitis had HBoV detected by all three assays, with viral loads ranging from 8.2 x 10(4) to 8.1 x 10(9)copies/ml of specimen. CONCLUSIONS: The three assays for HBoV diagnosis and quantification are highly sensitive, specific real-time tools for the reliable epidemiological and pathogenetic study of HBoV infection.

Development of enrichment semi-nested PCR for Clostridium botulinum types A, B, E, and F and its application to Korean environmental samples.

An enrichment semi-nested PCR procedure was developed for detection of Clostridium botulinum types A, B, E, and F. It was applied to sediment samples to examine the prevalence of C. botulinum in the Korean environment. The first pair of primers for the semi-nested PCR was designed using a region shared by the types A, B, E, and F neurotoxin gene sequences, and the second round employed four nested primers complementary to the BoNT/A, /B, /E, and /F encoding genes for simultaneous detection of the four serotypes. Positive results were obtained from the PCR analysis of five of 44 sediments (11%) collected from Yeong-am Lake in Korea; all were identified as deriving from type B neurotoxin (bontb) genes. Two of the C. botulinum type B organisms were isolated, and their bontb genes sequenced. The deduced amino acid sequences of BoNT/B showed 99.5 and 99.8% identity with the amino acid sequence of accession no. AB084152. Our data suggest that semi-nested PCR is a useful tool for detecting C. botulinum in sediments, and renders it practicable to conduct environmental surveys.

Determination of neurotoxin gene expression in Clostridium botulinum type A by quantitative RT-PCR.

Real time reverse transcription (RT)-PCR was used to quantify the expression of the botulinum neurotoxin type A (BoNT/A) gene (cntA) by normalization with the expression of 16S rRNA. The method were confirmed by monitoring the mRNA levels of cntA during growth in five type A strains. In all but one of the strains the expression of cntA mRNA was maximal in the late exponential phase, and approximately 35-fold greater than in the early exponential phase. The concentration of the extracellular BoNT/A complex detected by ELISA was highest in stationary phase. Sodium nitrite and sorbic acid completely inhibited growth at 20 ppm and 4 mg ml-1, respectively. CntA expression became lower in proportion to the concentration of sorbic acid, and this reduction was confirmed by mouse bioassay. Our results show that real time RT-PCR can be used to quantify levels of C. botulinum type A neurotoxin transcripts and to assess the effects of food additives on botulinal risk.

Outbreak of respiratory tract infections on an islet in Korea: possible Chlamydia pneumoniae infection.

In March 2004, we experienced an outbreak of Chlamydia pneumoniae infection on an islet of Korea. In order to assess the significance of the epidemic, we performed a mass examination of 137 students (7-16 years old; male, 69; female, 58) at a school. The examination consisted of a questionnaire inquiring about respiratory symptoms, a serum antibody test for C. pneumoniae using a microimmunofluorescence (MIF) method and enzyme-linked immunosorbent assay (ELISA), and nasopharyngeal swab tests to detect of the organism by specific PCR and cell culture. The results demonstrated that 72 (58.3%) of the students had respiratory symptoms such as rhinorrhea, a sore throat, and/or cough or fever. The PCR positivity of acute-phase patients was 63% (12/19) and PCR positivity using the culture sample was 94% (18/19). However, the existence of the organism was not confirmed fluorescein isothiocyanate (FITC). ELISA, one of the serological methods utilized, demonstrated, in the same patients, 48% (13/27) positive IgM antibodies at the acute phase of the outbreak, and 16% (3/19) positive IgM antibodies during the convalescent phase. The index value (ID) 3.0 for single-sera IgG was 19% (5/27) and that for IgA was 4% (1/27) at the acute phase; the corresponding percentages in the convalescent phase were 11% (2/19) and 5% (1/19), respectively. However, as regards paired sera, no patient demonstrated a 1.35 ELISA ID value at 2 weeks, or an increased value of 1.0 at 8 weeks after the onset of the outbreak. In the MIF experiment, the percent positivity of unpaired IgM from the acute phase was 58% (11/19). At convalescent phase, this percentage was 47% (9/19); however, the positivity of paired serum IgG was 26% (5/19). In the same sample, the percentage of positive cases demonstrated by both ELISA and MIF approaches for single IgM was 37% (7/19) at the acute phase and 11% (2/19) at the convalescent phase. We were unable to isolate C. pneumoniae by cell culture, but we did obtain sufficient serological and PCR data to consider C. pneumoniae as the causative agent of the outbreak. Meaningful results were acquired in terms of serology, and were compared to the healthy population in Korea. Although it remains necessary to investigate the possibility of co-infection and to determine whether or not this outbreak coincides with the prevalence of influenza, it was unequivocally concluded that this outbreak of C. pneumoniae infection has occurred on an islet of Korea.

Efficacy of non-toxic deletion mutants of protective antigen from Bacillus anthracis.

Current human anthrax vaccines available in the United States and Europe consist of alum-precipitated supernatant material from cultures of a toxigenic, nonencapsulated strain of Bacillus anthracis. The major component of human anthrax vaccine that confers protection is protective antigen (PA). A second-generation human vaccine using the recombinant PA (rPA) is being developed. In this study, to prevent the toxicity and the degradation of the native rPA by proteases, we constructed two PA variants, delPA (163-168) and delPA (313-314), that lack trypsin (S(163)-R(164)-K(165)-K(166)-R(167)-S(168)) or chymotrypsin cleavage sequence (F(313)-F(314)), respectively. These proteins were expressed in Bacillus brevis 47-5Q. The delPAs were fractionated from the culture supernatant of B. brevis by ammonium sulfate at 70% saturation, followed by anion exchange chromatography on a Hitrap Q, Hiload 16/60 superdex 200 gel filtration column and phenyl sepharose hydrophobic interaction column. In accordance with previous reports, both delPA proteins combined with lethal factor protein did not show any cytotoxicity on J774A.1 cells. The delPA (163-168) and delPA (313-314) formulated either in Rehydragel HPA or MPL-TDM-CWS (Ribi-Trimix), elicited a comparable amount of anti-PA and neutralizing antibodies to those of native rPA in guinea pigs, and confers full protection of guinea pigs from 50xLD50 of fully virulent B. anthracis spore challenges. Ribi-Trimix was significantly more effective in inducing anti-PA and neutralizing antibodies than Rehydragel HPA. These results indicate the possibility of delPA (163-168) and delPA (313-314) proteins being developed into nontoxic, effective and stable recombinant vaccine candidates.

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis.

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.

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.