Microbiology & Immunology - Research Publications
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ItemDevelopment of recombinant S. Typhimurium, as a model for S. Typhi-based vaccine vectors(Faculty of Medicine, Universitas Indonesia, 1998-01-01)
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ItemStudies of the pathogenesis and immunology of attenuated mutants of Salmonella enterica var. Typhimurium: Lessons for human typhoid fever?(Faculty of Medicine, Universitas Indonesia, 1998-01-01)
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ItemIn vitro and in vivo stability of recombinant plasmids in a vaccine strain of Salmonella enterica var. Typhimurium(WILEY, 2003-07-15)This study examined the ability of different plasmid vectors encoding H(C) fragment, the non-toxic binding portion of tetanus toxin, to be stably retained by Salmonella enterica var. Typhimurium (Salmonella typhimurium) vaccine strain BRD509 and, upon immunisation, to induce an antibody response against the carried antigen. The H(C) fragment expression cassette containing the transcription/translation signals, H(C) fragment open reading frame and the downstream TrpA terminator, was excised from pTETtac4 and incorporated into the plasmids pIC20H, pBR322, pACYC184 and pRSF1010. The resulting constructs were transferred into attenuated S. typhimurium, BRD509, and the level of H(C) fragment expression was examined by Western blot analysis. The relative stability of each plasmid in S. typhimurium was determined in vitro in the absence of antibiotic selection, and in vivo following immunisation. The ability of each H(C) fragment-expressing strain to induce lipopolysaccharide- and tetanus toxoid-specific antibody responses was assayed by an enzyme-linked immunosorbent assay. These studies showed that all the vaccine vector constructs, except the S. typhimurium carrying the expression vector based on pIC20H, were able to elicit a high titre immune response. The level of tetanus toxoid-specific antibody induced by S. typhimurium directly correlated with the level of in vitro and in vivo stability of the H(C) fragment expression plasmid carried by the bacterium, and not with an increased copy number of the parent plasmid vector.
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ItemComparison of the abilities of different attenuated Salmonella typhimurium strains to elicit humoral immune responses against a heterologous antigen(AMER SOC MICROBIOLOGY, 1998-02)We compared the abilities of different Salmonella enterica var. Typhimurium (S. typhimurium) strains harboring mutations in the genes aroA, aroAD, purA, ompR, htrA, and cya crp to present the heterologous antigen, C fragment of tetanus toxin, to the mouse immune system. Plasmid pTETtac4, encoding C fragment, was transferred into the various S. typhimurium mutants, and the levels of antigen expression were found to be equivalent. After primary oral immunization of BALB/c mice, all attenuated strains were capable of penetrating the gut epithelium and colonizing the Peyer's patches and spleens of mice. Of all strains compared, the delta purA mutant colonized and persisted in the Peyer's patches at the lowest level, whereas the delta htrA mutant colonized and persisted in the spleen at the lowest level. The level of specific antibody elicited by the different strains against either S. typhimurium lipopolysaccharide or tetanus toxoid was strain dependent and did not directly correlate to the mutants' ability to colonize the spleen. The level of immunoglobulin G1 (IgG1) and IgG2a antibody specific for tetanus toxoid was determined in mice immunized with four S. typhimurium mutants. The level of antigen-specific IgG1 and IgG2a was significantly lower in animals immunized with S. typhimurium delta purA. Antigen-specific T-cell proliferation assays indicated a degree of variability in the capacity of some strains to elicit T cells to the heterologous antigen. Cytokine profiles (gamma interferon and interleukin-5) revealed that the four S. typhimurium mutants tested induced a Th1-type immune response. Mice were challenged with a lethal dose of tetanus toxin 96 days after oral immunization. With the exception of the S. typhimurium delta purA mutant, all strains elicited a protective immune response. These data indicate that the level of total Ig specific for the carried antigen, C fragment, does not correlate with the relative invasiveness of the vector, but it is determined by the carrier mutation and the background of the S. typhimurium strain.
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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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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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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.