Browsing by Author "Stutz, Helen"

Now showing 1 - 4 of 4
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    Isolation and characterization of n-alkane utilizing bacteria, which produce biomulsifiers
    (2003) Ghilamicael, Amanuel Menghs; Reid, Sharon J; Stutz, Helen
    Bacterial strains were isolated by enrichment cultures from oil-contaminated soil samples. In the present study, several strains, capable of growing on crude oil, were isolated. Isolates were screened for their inherent abilities to produce bioemulsifiers when they were grown on hydrocarbon substrates.
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    Priming with a recombinant pantothenate auxotroph of Mycobacterium bovis BCG and boosting with MVA elicits HIV-1 Gag specific CD8+ T cells
    (Public Library of Science, 2012) Chapman, Rosamund; Shephard, Enid; Stutz, Helen; Douglass, Nicola; Sambandamurthy, Vasan; Garcia, Irene; Ryffel, Bernhard; Jacobs, William; Williamson, Anna-Lise
    A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔ panCD ) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔ panCD [pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8 + T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4 + T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge.
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    Priming with recombinant auxotrophic BCG expressing HIV-1 Gag, RT and Gp120 and boosting with recombinant MVA induces a robust T cell response in mice
    (Public Library of Science, 2013) Chapman, Rosamund; Stutz, Helen; Jr, William Jacobs; Shephard, Enid; Williamson, Anna-Lise
    In previous studies we have shown that a pantothenate auxotroph of Myocbacterium bovis BCG (BCGΔ panCD ) expressing HIV-1 subtype C Gag induced Gag-specific immune responses in mice and Chacma baboons after prime-boost immunization in combination with matched rMVA and VLP vaccines respectively. In this study recombinant BCG (rBCG) expressing HIV-1 subtype C reverse transcriptase and a truncated envelope were constructed using both the wild type BCG Pasteur strain as a vector and the pantothenate auxotroph. Mice were primed with rBCG expressing Gag and RT and boosted with a recombinant MVA, expressing a polyprotein of Gag, RT, Tat and Nef (SAAVI MVA-C). Priming with rBCGΔ panCD expressing Gag or RT rather than the wild type rBCG expressing Gag or RT resulted in higher frequencies of total HIV-specific CD8 + T cells and increased numbers of T cells specific to the subdominant Gag and RT epitopes. Increasing the dose of rBCG from 10 5 cfu to 10 7 cfu also led to an increase in the frequency of responses to subdominant HIV epitopes. A mix of the individual rBCGΔ panCD vaccines expressing either Gag, RT or the truncated Env primed the immune system for a boost with SAAVI MVA-C and generated five-fold higher numbers of HIV-specific IFN-γ-spot forming cells than mice primed with rBCGΔ panCD containing an empty vector control. Priming with the individual rBCGΔ panCD vaccines or the mix and boosting with SAAVI MVA-C also resulted in the generation of HIV-specific CD4 + and CD8 + T cells producing IFN-γ and TNF-α and CD4 + cells producing IL-2. The rBCG vaccines tested in this study were able to prime the immune system for a boost with rMVA expressing matching antigens, inducing robust, HIV-specific T cell responses to both dominant and subdominant epitopes in the individual proteins when used as individual vaccines or in a mix.
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    The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen
    (Public Library of Science, 2014) Chapman, Rosamund; Bourn, William R; Shephard, Enid; Stutz, Helen; Douglass, Nicola; Mgwebi, Thandi; Meyers, Ann; Chin'ombe, Nyasha; Williamson, Anna-Lise
    Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10 7 CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10 6 splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge.