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| The Department of Microbiology consists of two programs: 1) the Molecular Microbiology Program and 2) the Molecular Vaccinology Program. The Department's activities are supported by the Korea Research Foundation, the Ministries of Education and Human Resources Development and of Science and Technology of the Republic of Korea, the Korea Science and Engineering Foundation, and the Swedish International Development Cooperation Agency (SIDA). |
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| Molecular Microbiology Program |
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Ms. Seon Young Choi and Mr. Yoon Seong Jeon reading DNA microarray results.
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| Background |
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| Recent developments in the fields of molecular epidemiology and population genetics have altered how researchers classify the bacteria that cause disease. Molecular epidemiological studies demonstrate that bacterial pathogens, such as Salmonella, Shigella, Vibrio cholerae, and meningococci, are genotypically diverse. Specific complexes of related hyper virulent isolates have been responsible for epidemics across the world. Horizontal genetic exchange occurs continually within populations of pathogenic bacteria and provides a mechanism by which hyper virulent isolates continue to emerge. Genetic exchange enables the acquisition of genes that enhance pathogenicity, but it also facilitates the exchange of genes that encode variable antigens. This has important implications for the design of vaccines, since organisms may develop the ability to switch genes and thereby evade the immune response. Molecular epidemiology therefore provides powerful tools to monitor such changes at the genomic level. |
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| In addition to classical methods, such as ribotyping, multi-locus sequence typing (MLST), and pulsed-field gel electrophoresis, new methods that capitalize on the growing body of genomic data are being developed to improve the quality of databases for pathogens. An integrated system of databases is being developed with user-friendly interfaces for a variety of bioinformatic analyses, such as clustering and ordination methods. Overall, the databases developed by this program should prove useful to current and future epidemiological studies and other projects related to vaccine development. |
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| The Molecular Microbiology Program is also conducting extensive research to analyze the molecular mechanisms involved in the pathogenesis of certain infections, especially shigellosis. These studies could help identify new targets for vaccines and drugs to treat these infections. |
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| Goals |
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| The goals of the Molecular Microbiology Program are to: |
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Develop methods for identification and genetic characterization of microbial pathogens relevant to IVI's epidemiological studies and vaccine development programs; and
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Better understand the molecular mechanisms involved in the pathogenesis of diseases caused by microbial pathogens relevant to IVI's vaccine development programs.
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| Projects |
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| Genotyping of Vibrio cholerae and Salmonella: |
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| The Molecular Microbiology Program is setting up basic methods for genotyping Shigella, Salmonella, and Vibrio cholerae, including ribotyping and multi-locus sequence typing (MLST). MLST has also been successfully used to analyze isolates from a recent cholera outbreak in Mozambique(see paper in J Med Microbiol). The Program is also analyzing Salmonella typhi and S. paratyphi isolates collected during IVI's typhoid field studies, in collaboration with the Sanger Institute, to determine epidemiologic patterns and geographical distributions and to identify antibiotic-resistant isolates, which can then be analyzed to locate the resistance mechanism. The mechanism will be confirmed by reverse transformation. The Program has also collected about 100 isolates of Salmonella groups B and C from Indonesia. Using traditional serotyping methods, 12 S. paratyphi isolates were identified, serotyped and characterized with respect to antibiotic resistance, ribotype, and presence of a genetic element encoding multi-drug resistance. One of these strains is being considered for whole-genome sequencing at the Wellcome Trust Sanger Institute. |
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| Genotyping of Shigella: |
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| IVI has developed a multi-locus sequence typing (MLST) for genotyping of Shigella strains, in collaboration with the Korean National Institute of Health and Kyungpook National University School of Medicine. The development of this MLST for Shigella has resulted in the identification of several loci useful for specific Shigella typing. This new system has been tested and should help elucidate genomic relationships among nearly 3,000 isolates obtained from IVI's field studies in six Asian countries. The technique for identification of Single Nucleotide Polymorphism (SNP) will be adapted to Shigella in collaboration with the Wellcome Trust Sanger Institute in the UK. Such analyses should provide useful information regarding the geographic distribution, evolution and phylogenic relationships of Shigella species. |
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| Genotyping of respiratory pathogens: |
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| IVI will conduct genotyping of selected respiratory pathogens, such as influenza virus, M. tuberculosis, S. pneumoniae and SARS. Methods for genotyping influenza viruses are now being established. Standard operating procedures (SOPs) are being established and applied to the treatment of clinical specimens for preparing cDNAs for subsequent genotyping of virus isolates combined with bioinformatic management of data. |
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| IVI has acquired expertise in microbiological genotyping and bioinformatics and is building additional analytical resources to handle large numbers of specimens. A database for influenza virus genome sequences is under construction. Several in-house software tools and computer servers for general virus genotyping are currently under development. This system has been fine-tuned for influenza, especially avian influenza viruses. As part of an effort to become a resource for the Asia-Pacific region, IVI will establish SOPs and bioinformatics systems for avian flu genotyping. |
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| Establishment of enteric bacteria strain bank: |
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| A bacterial strain collection is being established, which will include standard reference strains as well as clinical isolates collected from IVI's study sites and partner laboratories. Genetic analyses of S. typhi, S. paratyphi and Shigella isolates will be used to generate corresponding databases. These databases should be especially useful to characterize emerging strains during epidemic outbreaks. The program will also develop a multi-user, intranet-based computer server, with a data management system to store information and a user-friendly interface for operating IVI databases. |
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| Research on molecular mechanisms involved in pathogenesis of Shigella: |
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| The Program has also been conducting research to better understand the molecular mechanisms leading to tissue-destructive inflammatory responses caused by Shigella spp. This work focuses on the characterization of cell signaling pathways involved in the activation of pro-inflammatory processes by Shigella virulence factors. In collaboration with Institut Pasteur in Paris, France, IVI scientist Dr. DW Kim, has shown that Shigella OspG (Other Secreted Protein G) interferes with host cell ubiquitination of IkB by binding to ubiquitin conjugating enzymes, thereby inhibiting the inflammatory process (by activation of NFkB)(see paper in Proc Natl Acad Sci USA). Work is ongoing to examine whether OspG can phosphorylate proteins involved in ubiquintination and, if so, can be considered a virulence factor for Shigella. |
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| Molecular Vaccinology Program |
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Mr. Byoung-Shick Shim analyzing images from bacterial DNA finger prints
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| Background |
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| Advances in microbial genetics and genetic engineering are part of the on-going vaccine revolution. Identifying the molecular basis of virulence and the microbial antigens essential for inducing successful defense mechanisms in the host enables the construction of "intelligent" vaccines, such as genetically-engineered, attenuated microorganisms or live vectors carrying foreign genes capable of inducing immune-mediated protection. For example, genetic modifications of S. typhi, V. cholerae, rotavirus, influenza virus, M. tuberculosis, measles virus and polio virus have led to the production of attenuated strains, and thus to the development of live vaccine candidates. Live attenuated strains can also be used to carry foreign genes in their bacterial genomes. Deciphering the entire genomes of the most important human pathogens has had a significant impact on vaccine development. The Molecular Vaccinology Program tackles the problems of vaccine development using the state-of-the-art techniques of molecular biology. |
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| Goals |
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| The objectives of the Molecular Vaccinology Program are to: |
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Develop a generic strategy for identifying protective antigens and/or defined moieties to be incorporated into candidate vaccines against selected intestinal and respiratory pathogens;
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Develop vectors for optimal delivery of vaccine antigens;
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Develop novel adjuvants for enhancing systemic and mucosal immune responses to vaccines.
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| Projects |
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| Research on DNA vaccines: |
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| The Molecular Vaccinology Program is conducting research on DNA vaccines. A Sindbis virus replicon system is being used as a naked DNA vaccine. Chiron has developed the vaccine pSINCP-85A, and this vaccine vector has demonstrated enhanced immunogenicity and long-term protection against tuberculosis, compared with the conventional DNA vaccine vector. Recently, Chiron has allowed IVI to use its vector to develop vaccines against tuberculosis and severe acute respiratory syndrome (SARS). Recent studies have compared the immune responses induced by the conventional vector and by pSINCP, which encodes several tuberculosis antigens, either codon optimized or not(see paper in J Virol). This research is being undertaken in collaboration with POSTECH and Yonsei University in Korea.
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| IVI has acquired expertise in the preparation of plasmid DNA vaccines, in vitro and in vivo detection of plasmid-encoded polypeptides, and engineering of live-attenuated recombinant delivery systems (adenoviruses, Mycobacteria) for mucosal (buccal, nasal) and systemic (intradermal, intramuscular) administration. The objective of this research is to identify, in pre-clinical models, protective antigens and/or defined moieties to be incorporated into candidate vaccines against such pathogens as Shigella and influenza virus, including H5N1. |
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| Research to identify novel protective antigens in Shigella: |
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| In addition, IVI scientists have identified a number of Shigella surface-expressed and secreted proteins common to all Shigella species, using comparative analyses of whole genome sequences from the four Shigella species. Several of these proteins have been subcloned into suitable expression vector systems and individual, dual and multiple knock-out strains are being engineered and further tested in animals. Several of these proteins have already been purified at laboratory scale and are being tested in animal models for shigellosis, including a new guinea pig model for intestinal shigellosis recently developed at IVI. Since there is considerable variation in the prevalence of different Shigella species and serotypes from country to country, and even from year to year within a given country, these new findings lend promise for the development of a vaccine that could protect across Shigella species and serovars. |
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| Development of novel adjuvants to enhance immune responses to vaccines: |
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| The Molecular Vaccinology Program is committed to developing novel adjuvants for amplifying systemic and mucosal immune responses to vaccines. Work is on-going to develop genetically-engineered mutants of existing bacterial toxins with known adjuvant properties, but which lack toxicity and which have been designed for improved cellular penetration. IVI is building a platform to identify small peptides capable of mimicking natural or synthetic (e.g. ODN) adjuvants. The aim of this activity is to characterize and produce in a scalable manner proprietary peptides that are not immunogenic and could be produced in a simple and cost-effective manner - either by synthesis or by recombinant over expression with appropriate vectors. To carry out this work, IVI is establishing strategic partnerships with Korean academic institutions and pharmaceutical firms. This platform will use state-of-the-art technologies, such as functional genomics, proteomics, cell biology (identification of markers associated with distinct signaling pathways), combined with high throughput screening of genes and/or peptides secreted by human dendritic cells and/or epithelial cells, to identify bioactive analogs and derivatives of known adjuvants. |
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| IVI is also joining forces with Inserm in France on a laboratory program - the IVI-Inserm Associated Laboratory - to work on the isolation and propagation of primary epithelial and dendritic cell lines of human origin. Generation of subtractive DNA libraries from cell lines exposed to adjuvants and inactive mutants thereof will be carried out to limit the number of potentially relevant genes through collaborative arrangements with Inserm and the University of Gothenburg. Parallel proteomic analyses of subcellular protein contents will be performed to identify corresponding peptides associated with adjuvanticity. Animal experiments will also be performed to establish the putative adjuvanticity of corresponding peptides identified and synthesized. |
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