Chancellery Research - Research Publications
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ItemVaccine potential of attenuated mutants of Corynebacterium pseudotuberculosis in sheep(AMER SOC MICROBIOLOGY, 1998-02)Corynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etiological agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel vaccines against CLA and as veterinary vaccine vectors. In this report, we have assessed the virulence of both aroQ and pld mutants of C. pseudotuberculosis in sheep and concurrently their capacity to act as vaccines against homologous challenge. The results suggest that aroQ mutants of C. pseudotuberculosis are attenuated with regard to both lymph node persistence and vaccination site reactogenicity. Immunologically, aroQ mutants failed to elicit detectable specific gamma interferon (IFN-gamma)-secreting lymphocytes and induced low levels of antibodies to C. pseudotuberculosis culture supernatant antigens. Following subcutaneous vaccination, the immune responses induced by aroQ mutants did not protect sheep from infection with the wild-type strain but did appear to reduce the clinical severity of disease resulting from challenge. Conversely, an attenuated C. pseudotuberculosis strain expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction of IFN-gamma-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The results suggest that aroQ mutants of C. pseudotuberculosis may be overly attenuated for use as a CLA vaccines or as vaccine vectors.
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ItemAttenuation and vaccine potential of aroQ mutants of Corynebacterium pseudotuberculosis(AMER SOC MICROBIOLOGY, 1997-08)Corynebacterium pseudotuberculosis, a gram-positive intracellular bacterial pathogen, is the etiological agent of the disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel live veterinary vaccine vectors. We have cloned and sequenced the aroB and aroQ genes from C. pseudotuberculosis C231. By allelic exchange, aroQ mutants of both C231, designated CS100, and a pld mutant strain TB521, designated CS200, were constructed. Infection of BALB/c mice indicated that introduction of the aroQ mutation into C231 and TB521 attenuated both strains. In sublethally infected BALB/c mice, both CS100 and CS200 were cleared from spleens and livers by day 8 postinfection. The in vivo persistence of these strains was increased when the intact aroQ gene was supplied on a plasmid in trans. Mice infected with TB521 harbored bacteria in organs at least till day 8 postinfection without ill effect. When used as a vaccine, only the maximum tolerated dose of CS100 had the capacity to protect mice from homologous challenge. Vaccination with TB521 also elicited protective immunity, and this was associated with gamma interferon (IFN-gamma) production from splenocytes stimulated 7 days postvaccination. The role of IFN-gamma in controlling primary infections with C. pseudotuberculosis was examined in mice deficient for the IFN-gamma receptor (IFN-gammaR(-/-) mice). IFN-gammaR(-/-) mice cleared an infection with CS100 but were significantly more susceptible than control littermates to infection with C231 or TB521. These studies support an important role for IFN-gamma in control of primary C. pseudotuberculosis infections and indicate that aroQ mutants remain attenuated even in immunocompromised animals. This is the first report of an aroQ mutant of a bacterial pathogen, and the results may have implications for the construction of aromatic mutants of Mycobacterium tuberculosis for use as vaccines.
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ItemDNA vaccines for bacterial infections(BLACKWELL SCIENCE, 1997-08)DNA vaccines are an exciting development in vaccine technology which may have a special role in preventing viral infections and as 'theracines' for cancer. Their use in preventing bacterial infections has, by comparison, been less well documented. While it is unlikely that traditional, highly successful and cheap vaccines for diseases such as diphtheria will be replaced by DNA vaccines, naked DNA may be particularly appropriate for preventing bacterial infections where cytotoxic T cells confer protection, or where a Th1 type T cell response mediates resistance. For example, DNA vaccines containing different mycobacterial antigens have been shown to inhibit overt infections by Mycobacterium tuberculosis in rodent models. The use of DNA vaccines in bacterial infections may be complicated by fundamental differences between prokaryotic and eukaryotic genes and gene products, including mRNA stability, codon bias, secondary structures surrounding native start sequences and glycosylation. These problems can be solved by re-synthesis of bacterial genes to produce 'new' sequences which are more highly expressed by eukaryotic cells.
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ItemCloning and manipulation of the Corynebacterium pseudotuberculosis recA gene for live vaccine vector development(OXFORD UNIV PRESS, 1996-09-01)Corynebacterium pseudotuberculosis is an intracellular bacterial pathogen causing a chronic abscessing disease in sheep and goats called caseous lymphadenitis. We are developing this bacterial species as a live vector system to deliver vaccine antigens to the animal immune system. Foreign genes expressed in bacterial hosts can be unstable so we undertook to delete the C. pseudotuberculosis chromosomal recA gene to determine whether a recA- background would reduce the frequency of recombination in cloned DNA. Homologous DNA recombination within an isogenic recA- C. pseudotuberculosis was 10-12-fold lower than that in the recA+ parental strain. Importantly, the recA mutation had no detectable affect upon the virulence of C. pseudotuberculosis in a mouse model. Taken together these results suggest that a recA- background may be useful in the further development of C. pseudotuberculosis as a vaccine vector.
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ItemUse of in vivo-regulated promoters to deliver antigens from attenuated Salmonella enterica var. typhimurium(AMER SOC MICROBIOLOGY, 1999-10)This study describes the construction and analysis of three in vivo-inducible promoter expression plasmids, containing pnirB, ppagC, and pkatG, for the delivery of foreign antigens in the DeltaaroAD mutant of Salmonella enterica var. Typhimurium (hereafter referred to as S. typhimurium). The reporter genes encoding beta-galactosidase and firefly luciferase were used to assess the comparative levels of promoter activity in S. typhimurium in vitro in response to different induction stimuli and in vivo in immunized mice. It was determined that the ppagC construct directed the expression of more beta-galactosidase and luciferase in S. typhimurium than the pnirB and pkatG constructs, both in vitro and in vivo. The gene encoding the C fragment of tetanus toxin was expressed in the aroAD mutant of S. typhimurium (BRD509) under the control of the three promoters. Mice orally immunized with attenuated S. typhimurium expressing C fragment under control of the pagC promoter [BRD509(pKK/ppagC/C frag)] mounted the highest tetanus toxoid-specific serum antibody response. Levels of luciferase expression in vivo and C-fragment expression in vitro from the pagC promoter appeared to be equivalent to if not lower than the levels of expression detected with the constitutive trc promoter. However, mice immunized with BRD509(pKK/ppagC/C frag) induced significantly higher levels of tetanus toxoid-specific antibody than BRD509(pKK/C frag)-immunized mice, suggesting that the specific location of foreign antigen expression may be important for immunogenicity. Mutagenesis of the ribosome binding sites (RBS) in the three promoter/C fragment expression plasmids was also performed. Despite optimization of the RBS in the three different promoter elements, the expression levels in vivo and overall immunogenicity of C fragment when delivered to mice by attenuated S. typhimurium were not affected. These studies suggest that in vivo-inducible promoters may give rise to enhanced immunogenicity and increase the efficacy of S. typhimurium as a vaccine vector.