Browsing by Author "Baloglu, Simge"

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    Applicability of vaccinia virus as cloning and expression vector for bacterial genes: mice immune responses to vaccinia virus expressing Brucella abortus and Listeria monocytogenes antigens
    Baloglu, Simge (Virginia Tech, 2001-07-27)
    Previous studies by our group showed that vaccinia virus recombinants expressing Brucella abortus (BA) antigens heat shock protein GroEL, 18 kDa protein and Cu/Zn SOD, were unable to induce protective immune responses against Brucella challenge. This dissertation analyzes the possible reasons for this phenomenon, by using other genes/proteins from BA and Listeria monocytogenes (LM), various shuttle plasmids (pSC65, pSC11) and immune response modulators (CpG, IL-12, B7-1). As the first objective, a vaccinia virus recombinant (WRL7/L12), expressing the BA L7/L12 gene was generated. L7/L12 ribosomal protein was used as a T-cell reactive antigen, with protective potential to Brucella challenge. The WRL7/L12 was able to express the gene of interest and induce IgG2A type antibody response, but not a protective immune response against Brucella challenge. As a control, an antigen from LM proven to induce CTL and protective immune responses, was used to test the efficacy of vaccinia virus to induce protection. A portion of hly gene, encoding partial listeriolysin (pLLO), was inserted into the same vaccinia virus stain. This recombinant (WRpLLO) was able to induce protection against a Listeria challenge. Next another vaccinia virus recombinant expressing Brucella abortus Cu/Zn SOD was analyzed. Although a variety of approaches, including the enhancement of the protein expression by the pMCO2 synthetic promoter, booster immunization, addition of the oligomer CpG adjuvant (WRSODCpG) to enhance Th1 type response, were used, the SOD recombinant failed to protect mice against Brucella challenge. Lastly, vaccinia virus produces a family of proteins that bind cytokines, chemokines and interferons to evade the host defensive systems. Therefore, a vaccinia virus strain co-expressing murine IL-12, and cofactor B7-1, were used to generate the recombinant WRIL12L7/L12. In order to further boost the induction of Th 1 type response, the adjuvant CpG was used. A similar recombinant, WRIL12pLLO, was generated with partial hly gene to serve as a positive control for protection. Mice immune responses to these recombinants, with and without adjuvant CpG, were analyzed, and compared with the recombinants generated with vaccinia strain WR. Co-expression of IL12 and B7 abrogated the protective efficacy of the vaccinia/ pLLO recombinant.
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    Assessment of the Expression of Brucella Abortus Heat Shock Protein, Groel, in Vaccinia Virus to Induce Protection Against a Brucella Challenge in Balb/C Mice
    Baloglu, Simge (Virginia Tech, 1997-07-08)
    B. abortus is an intracellular facultative bacterial pathogen which causes abortion in cattle and undulant fever in humans. Cattle vaccines such as B. abortus strains 19 and RB51 are live vaccine strains which protect approximately 75% of the vaccinated animals. No effective vaccines are available for the prevention of brucellosis in humans. We are developing vaccinia virus recombinants expressing various B. abortus proteins to prevent brucellosis in susceptible mammalian species. In this work the B. abortus groEL gene encoding the antigenic heat shock protein GroEL was subcloned into vaccinia virus via homologous recombination. Expression of the GroEL protein in vaccinia infected cells in-vivo was confirmed by immunoblotting. Groups of 5 female BALB/C mice were injected with the vaccinia recombinant or appropriate positive and negative control vaccines. Mice were bled and their humoral immune responses assessed. In addition, mice were challenged with virulent B. abortus strain 2308 and protection measured by the rate of splenic clearance of live Brucella. In spite of demonstrating specific GroEL antibodies in recombinant vaccinia injected mice, no significant level of protection was demonstrable. Preliminary lymphocyte transformation assays were carried out to establish if a cell mediated immune response to GroEL was induced in the vaccinated animals.