Browsing by Author "Gamieldien, Hoyam"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Open Access
    Comparing high-throughput methods to measure antibody dependent cellular cytotoxicity during HIV infection
    (2014) Mbodo, Iyaloo; Passmore, Jo-Ann; Gamieldien, Hoyam
    The prevalence of HIV-1 is highest in Sub-Saharan Africa. Protective immune responses directed against HIV are complex and involve both cellular and humoral immunity. Based on the recent finding that the best correlate of protection against the first protective prophylactic RV144 vaccine were HIV-specific antibody responses, including those mediating natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC), there has been considerable interest in measuring alternative roles for HIV-specific binding antibodies. The aim of this MSc dissertation was to optimise and compare two high-throughput flow cytometry based approaches - the GranToxilux and PanToxilux assays - to measure HIV-specific ADCC responses. To do this, NK cells from a panel of healthy HIV-negative individuals were screened for their ability to directly kill the tumour cell line K562, as a measure of direct NK cell cytotoxicity. The individual with the highest granzyme B and caspase activity against K562 cells was chosen as the universal NK cell donor for this study.
  • Thumbnail Image
    Item
    Open Access
    Evolution of HIV-1 subtype C immune responses during acute and chronic HIV infection
    (2011) Gamieldien, Hoyam; Passmore, Jo-Ann
    The aim of this study was to compare the magnitude and breadth of HIV-specific T cell responses to HIV Gag and Nef mounted during acute HIV infection with those that emerged during chronic infection and to investigate the association of these responses with subsequent HIV disease progression (CD4 counts and plasma viral loads).
  • Thumbnail Image
    Item
    Open Access
    In Silico Characterisation of Putative Prophages in Lactobacillaceae Used in Probiotics for Vaginal Health
    (2022-01-20) Happel, Anna-Ursula; Kullin, Brian R; Gamieldien, Hoyam; Jaspan, Heather B; Varsani, Arvind; Martin, Darren; Passmore, Jo-Ann S; Froissart, Rémy
    While live biotherapeutics offer a promising approach to optimizing vaginal microbiota, the presence of functional prophages within introduced Lactobacillaceae strains could impact their safety and efficacy. We evaluated the presence of prophages in 895 publicly available Lactobacillaceae genomes using Phaster, Phigaro, Phispy, Prophet and Virsorter. Prophages were identified according to stringent (detected by ≥4 methods) or lenient criteria (detected by ≥2 methods), both with >80% reciprocal sequence overlap. The stringent approach identified 448 prophages within 359 genomes, with 40.1% genomes harbouring at least one prophage, while the lenient approach identified 1671 prophages within 83.7% of the genomes. To confirm our in silico estimates in vitro, we tested for inducible prophages in 57 vaginally-derived and commercial Lactobacillaceae isolates and found inducible prophages in 61.4% of the isolates. We characterised the in silico predicted prophages based on weighted gene repertoire relatedness and found that most belonged to the Siphoviridae or Myoviridae families. ResFam and eggNOG identified four potential antimicrobial resistance genes within the predicted prophages. Our results suggest that while Lactobacillaceae prophages seldomly carry clinically concerning genes and thus unlikely a pose a direct risk to human vaginal microbiomes, their high prevalence warrants the characterisation of Lactobacillaceae prophages in live biotherapeutics.
  • Thumbnail Image
    Item
    Open Access
    Isolation and Characterization of Bacteriophages Targeting Uropathogenic E. coli
    (2019) Magwai, Thandi; Passmore, Jo-Ann; Froissart, Remy; Gamieldien, Hoyam
    Urinary tract infections (UTI)are one of the most common human bacterial infections, caused by uropathogenic E. coli (UPEC). Conventionally, UTI’s are treated with antibiotics, such as sulpha-mathoxazole/trimethoprim and ampicillin. However, a significant proportion of UPEC strains have become resistant to these antibiotics, resulting in a significant burden on the health care system worldwide. Bacteriophages (phages) that target E. coli strains could provide an alternative treatment for UTIs, particularly those resistant to antibiotics. This study aimed to screen a variety of environmental samples for phages that target a UPEC strains and characterize these phenotypically and genotypically, towards the development of a candidate phage preparation for treatment for antibiotic-resistant UPEC. Environmental samples (including faeces cattle, sheep, horse, goat faeces, river water, raw cow milk and mud) were collected from various sources around the City of Cape Town and screened for phages using an E. coli laboratory strain; K-12 MG1655. Lytic phages against E. coli MG1655 were purified and host-range testing was conducted against a panel of 11 UPEC strains form the E. coli reference collection (ECOR). These lytic phages were characterized phenotypically using transmission electron microscopy (TEM) using negative staining and genotypically using restriction enzyme HaeIII. In total, 41 phages that were lytic against MG1655 were isolated from six different environmental samples. Of these, 31/41 (76%) were lytic against 7/11 (64%) UPEC strains tested. Four phages (16-3, 16-4, 16-5 and 16-7; all isolated from raw milk) had the broadest host range of all the phages screened, being lyticagainst4/11(36%) UPEC strains. UPEC strain ECOR-40 was the most susceptible of the E. coli strains tested, being susceptible to 18/31 (58%) of the lytic phages isolated. In contrast, four UPEC strains (including ECOR-14, -60, -62 and -64) were not susceptible to any of the phages isolated. Ninety percent (28/31) of the isolated phages were structurally similar to four known phage families; including Myoviridae, Siphoviridae, Inoviridae and Rudiviridae. The remaining 10% (3/31) had structures that did not resemble any ofthe known phage families. DNA was x isolated from all 31 phage isolates to screen for similarities in restriction patterns of isolates. Of these, RFLP banding was clear for 5/31 samples; which showed that 5/5 phages were unique based on their banding patterns. In conclusion, this study demonstrated the existence of several unique E. coli phages in nature and their ability to target several of the UPEC strains known to cause UTI. Although time did not allow for sequencing of the full genomes of those isolates with unique characteristics in this study, this should be a priority for this research going forward.