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Development of a polychromatic flow cytometry panel for the evaluation of HIV-specific T cell responsesNaicker, Prinola January 2009 (has links)
Includes abstract. / Includes bibliographical references (leaves 99-115). / Investigating T cell responses in HIV infection has revealed several correlates of viral control, but their importance is not fully understood. Further studies to understand the relationship between HIV and the immune system are warranted. The advent of polychromatic flow cytometry has allowed for in depth analysis of T cell functions and phenotypes in HIV infection, including the measurement of T cells that can produce multiple immune molecules simultaneously. The aim of this study was to develop a polychromatic flow cytometry panel to measure multiple functional markers, and optimise a stimulation and staining protocol for use in the laboratory.
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Cytomegalovirus viraemia in immunocompromised children in Cape TownHsiao, Nei-Yuan January 2009 (has links)
Includes bibliographical references (leaves 54-62).
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Prokaryotic Production of Human Immunodeficiency VirusType 1 Subtype C Tat, Nef and Reverse Transcriptase andInvestigation of Antibody Responses to these proteins inHIV-1 Infected Individuals as well as Macaques Vaccinatedwith SAAVI DNA-C/C2 and SAAVKgatle, M M January 2010 (has links)
The purified Tat, Nef and RT recombinant proteins were used as antigens to investigate the prevalence of HIV-1 antibodies in sera of HIV-1 infected people. There was no reactivity in any of the 20 negative control sera in the Western blot assay. Analysis of 481 sera from HIV-1 infected individuals showed that the majority of serum samples had relatively high prevalence of anti-RT antibodies, ranging from 89.9% to 95% irrespective of the clinical stages. Anti-Nef antibodies were observed in 47.4% of individuals tested and there was a strong association between the prevalence of these antibodies with the clinical stage of CD4 count range of 201 to 499 cells/μl. The prevalence of anti-Tat antibodies was less frequent and only 7.5% of serum samples had anti-Tat antibodies. There was no association of anti-Tat and anti-RT antibodies with viral load or CD4 counts in sera of HIV-1 infected individuals. Altogether, 92% of serum samples tested had positive antibody responses to one or more antigens, indicating a good performance for the Western blot detection method.The prevalence of antibody response against purified HIV-1 subtype C antigens (Tat, Nef and RT) was also investigated in eleven sera of macaques vaccinated with SAAVI DNA-C or SAAVI-DNA-C2 and SAAVI MVA-C. Western blot assays showed that 36%, 27% and 40% of serum samples from vaccinated rhesus macaques had detectable antibodies against HIV-1 subtype C Tat, Nef and RT antigens, respectively. The prevalence of antibody responses to Tat, Nef and RT antigens in the sera of macaques is lower relative to the prevalence of antibody responses in the sera of HIV-1 infected individuals. A possible explanation is that the immune system of HIV-1 infected people is continuously exposed to viral antigens, thus resulting to high level of detectable anti- Tat, -Nef and -RT antibodies. In contrast, macaques were exposed to HIV-1 antigens expressed by the DNA and MVA vectors for only five times through vaccinations. However, these results indicate that Western blot assay based on purified HIV-1 Tat, Nef and RT proteins may be a useful research tool for detection of antibody responses to corresponding vaccine immunogen in macaque sera.In conclusion, HIV-1 subtype C Tat, Nef and RT were successfully purified from Salmonella enterica serovar Typhimurium. The purified Tat, Nef and RT recombinant antigens were antigenically recognised by anti-Tat, anti-Nef and anti-RT antibodies in the sera of HIV-1 infected individuals and rhesus macaques vaccinated with candidate HIV-1 vaccines. Application of these purified proteins as reagents in a Western blot assay to detect antibodies in serum samples of HIV-1 subtype C positive individuals demonstrated an association between the prevalence of anti-Nef antibodies and CD4 count range of 201 to 499 cells/μl. In addition, the detection of antibody responses to purified Tat, Nef and RT antigens in the sera of vaccinated macaques suggests a possible application of these purified proteins in HIV vaccine research and improvement of existing Western blot-based HIV diagnostic kits.
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Characterisation of HIV superinfection : genetic evolution and adaptive immune responsesHarvey, Hayley Janet January 2011 (has links)
In this thesis we aimed to determine the timing and frequency of intra-subtype C superinfection, and to determine if the reason for superinfection was a greater genetic distance within epitopes of the superinfecting virus compared to those of circulating strains from the same cohort.
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Construction, stability and immunogenicity of recombinant BCG expressing HIV-1 subtype C gag under the control of MtrA promoter, with or without the leader sequencesLebeko, Maribanyana R January 2011 (has links)
This study aimed to compare recombinant mycobacteria expressing HIV-1 gag under the control of different promoters and leader sequences. This was done to determine whether the genetic stability of the recombinant mycobacteria could be improved by modification of these vector features and to gain insight into what types of immune responses may be elicited in mice.
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Characterization of Mycobacterium tuberculosis-specific Th22 cells in HIV-TB co-infectionMakatsa, Mohau Steven 19 November 2020 (has links)
Tuberculosis (TB) remains the infectious disease causing the greatest global mortality, with an estimated 10 million incident cases of TB and 1.45 million deaths in 2018. Although there is a cure for TB, the success of the treatment is hampered by multidrug resistant TB and HIV infection. There is an urgent need for an effective TB vaccine to prevent ongoing transmission. The development of a new and efficacious TB vaccine will likely be dependent on our understanding of protective immunity to TB. Although it is well established that Th1 cells are crucial in the response against Mycobacterium tuberculosis (Mtb), Th1 cytokines may not be sufficient to control Mtb infection. A major focus of this thesis is the contribution of an understudied Th subset in Mtb immunity, namely Th22 cells, producing the cytokine IL-22. IL-22 functions to preserve mucosal barriers and induce antimicrobial peptides, contributing to protective immunity to a range of extracellular and intracellular bacteria. A recent study in IL-22-deficient mice described a protective role for IL-22 during the development of TB. In humans, soluble IL-22 has been detected at sites of extra-pulmonary tuberculosis (TB), and a polymorphism in the IL-22 promoter has been linked to TB susceptibility. However, much remains to be understood about Th22 cells and their role in protective immunity to Mtb. In this study, we investigated the contribution of Th22 cells to TB immune responses by providing a detailed characterisation of Mycobacterium tuberculosis-specific Th22 cells in latent TB infection (LTBI), TB disease and HIV co-infection, using flow cytometric techniques. In Chapter 2, we optimised detection of IL-22 and determined the factors that contribute to Mtb-specific IL-22 production by CD4+ T cells, as well as characterising some aspects of Th22 cell biology. In Chapter 3, we examined the impact of TB disease and HIV infection on Th22 cells, compared to Th1 and Th17 cells. Finally, in Chapter 4, we explored Mtb-specific cytokine production by CD8+ T cells and CD4+ T cells following Mtb peptide stimulation, and the effect of TB disease and HIV infection. We detected significant IL-22 production from CD4+ T cells in healthy individuals following whole blood stimulation with Mtb whole cell lysate (MtbL). However, IL-22 responses were poorly detectable when peripheral blood mononuclear cells (PBMC) were stimulated with MtbL. Therefore, we sought to investigate conditions that influence IL-22 detection in whole blood and PBMC, and characterise Th22 cells further. We found that PBMC are able to produce IL-22 in response to Mtb but appear to lack the physiological environment for optimal induction of IL-22. We also discovered that TCR blocking inhibited Mtb-specific IL-22 production, suggesting that responses are stimulated through recognition of Mtb antigen by the TCR, rather than through bystander activation. IL-22 is produced by CD4+ T cells that appear to be conventional, rather than MAIT, γδ or iNKT cells. Indeed, analysis of the TCR clonality using vβ repertoire typing revealed similar repertoire usage between IL-22, IFN-γ-producing CD4+ T cells, and total CD4+ T cells. Overall, these data shed more light on the biology of IL-22-producing CD4+ T cells. Next, we examined the effects of HIV infection and TB disease on the magnitude, memory profile and activation phenotype of Mtb-specific Th22 cells, compared them to Th1 and Th17 cells. Blood samples were collected from 72 individuals classified into four groups based on their HIV-1 and TB status, namely HIV-/LTBI, HIV+/LTBI HIV-/active TB and HIV+/active TB. Blood was stimulated with MtbL and analysed for cytokine production using multiparameter flow cytometry. We observed similar frequencies of IL-22 to IFN-γ-producing CD4+ T cells in LTBI. Mtb-specific Th22 cells were reduced to a greater extent than Th1 cells by a combination of HIV infection and TB disease. Th22 cells demonstrated differences in their memory and activation phenotype compared to Th1 and Th17 cells. In the context of active TB, Th1 cells were characterised by a high expression of the activation marker HLA-DR. In contrast, Th22 cells did not demonstrate activation using this marker during TB disease. Similarly, Th1 cells were more differentiated in TB disease irrespective of HIV status, while there was no difference in the memory phenotype of Th22 cells during different disease states. Finally, we characterised Mtb peptide-specific CD4+ and CD8+ T cell responses in LTBI, active TB and HIV infection. CD4+ T cells did not produce detectable IL-22 when blood was stimulated with Mtb peptides, and there was also no IL-22 response from CD8+ T cells. Th1 cytokines IFN-γ and TNF-α were detectable from CD4+ and CD8+ T cells in response to Mtb peptides. Consistent with previous studies, there was a higher proportion of individuals with detectable CD8+ responses during active TB and HIV co-infection compared to HIV-infected LTBI individuals, but no difference is the magnitude of response was observed. Interestingly, HIV infection and TB disease induced similar levels of activation in Mtb-specific CD8+ compared to CD4+ T cells. Moreover, active TB and HIV co-infection impaired memory differentiation of Mtb-specific CD8+ T cells towards a less differentiated profile, compared to LTBI. These results confirm that both CD4+ and CD8+ T cells contribute to TB immune responses. In summary, we confirm that Th22 cells constitutes a substantially portion of CD4+ T cell response to Mtb . IL-22 appears to be produced by conventional CD4+ T cells but may require specific antigen presentation requirements to optimally induce its production. Interestingly, HIV infection during TB disease led to a near absence of Th22 cells in blood. Our results warrant further study of the role of Th22 cells in TB immunity, which may lead to insights that could assist the development of an effective vaccine against TB.
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Evolution of HIV-1 subtype C immune responses during acute and chronic HIV infectionGamieldien, Hoyam January 2011 (has links)
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).
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An investigation of HIV-1 diversity in Southern Africans, and characterisation of viral populations in recently infected womenRademeyer, Cecilia January 2003 (has links)
Bibliography: leaves 135-171.
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In vitro characterization of the antiviral activity of Secomet V against vaccinia virus infectionsRangel Lopes de Campos, Walter January 2004 (has links)
Includes bibliographical references (leaves 65-77). / The anti-poxvirus agent SECOMET V was the reference name for a plant extract produced in a bioreactor from primary plant stem cells, whose antiviral activity has been widely reported in folklore medicine. It exerted its anti-vaccinia virus activity by neutralizing cell-free virus rather than interfering with the downstream events following adsorption.
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Characterizing the genotypic and phenotypic diversity of Gardnerella vaginalis from vaginal clinical samplesMasete, Kopano Valerie 31 January 2019 (has links)
Bacterial vaginosis (BV) is a common vaginal condition affecting reproductive-age women, especially in sub-Saharan Africa. With poor treatment outcomes, BV has been associated with pregnancy complications, pelvic inflammatory disease as well as acquisition and transmission of sexually transmitted diseases. While the etiology of BV is not well characterized, it is understood that Gardnerella vaginalis plays a critical role in BV by initiating the formation of the polymicrobial biofilm that characterizes BV and by degrading protective vaginal mucus through the release of sialidase. Recent evidence suggests that the G. vaginalis species is more heterogeneous that initially thought and that not all G. vaginalis may be involved BV. The aim of this study was thus to characterize the genotypic and phenotypic diversity of G. vaginalis isolates. This was achieved in vitro, using 109 G. vaginalis isolates that were previously purified from vaginal samples of 109 French women who were BV-positive (n = 75), BV-intermediate (n = 20) or BV-negative (n = 14), as diagnosed by Nugent scoring. To determine the genotypic diversity of G. vaginalis isolates, 90 isolates were successfully genotyped using their chaperonin-60 (cpn60) sequences, revealing the presence of four phylogenetic clades (subgroups A-D) made up of 13 subgroup A, 17 subgroup B, 58 subgroup C and 2 subgroup D isolates. To determine the phenotypic diversity of G. vaginalis isolates, sialidase activity, biofilm formation and susceptibility to antibiotics used to treat BV were measured. Sialidase activity was not detected in subgroup A and D isolates but was detected, at similar levels, in subgroup B and C isolates. Isolates from all subgroups of G. vaginalis could form similar amounts of biofilm. G. vaginalis isolates (n = 45) were largely resistant to metronidazole (71%), but sensitive to clindamycin (100%), moxifloxacin (91%) and augmentin (100%). The presence of prophages in G. vaginalis isolates was also investigated, revealing the presence of bacteriophage (phage)-like particles that could not be classified into any known phage families, whose phage status remains to be confirmed. In conclusion, G. vaginalis subgroup B and C isolates were the only ones that formed biofilm as well as had detectable sialidase activity suggesting that G. vaginalis subgroups B and C are most likely to be involved in BV. These results contribute to our knowledge of BV and could be useful in future studies that aim to design better treatment strategies for BV.
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