Browsing by Author "Burgers, Wendy"

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    Anti-vector immune responses to an MVA vaccine
    (2011) Muller, Tracey; Burgers, Wendy
    This study characterised the humoral and cellular immune responses to MVA from a candidate MVA-vectored HIV vaccine in non-human primates, and examined the effect of anti-vector immunity on the response to the HIV immunogens.
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    Characterising the magnitude and breadth of T cell responses to SARS-CoV-2 vaccination
    (2024) Chauke, Valencia Masego; Burgers, Wendy; Keeton, Roanneand; Mosala, Paballo
    Spike protein which serves as the immunogen in the current vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is targeted by the CD4 and CD8 T cells. While most studies frequently measure the magnitude of spike-specific CD4 and CD8 responses, only a few studies have characterised their breadth of targeting across the spike. This characterization is crucial for predicting cross-reactivity in T cell responses against emerging variants of concern (VOC) that may have mutations in the targeted regions. This study aimed to examine the magnitude and breadth of SARS-CoV-2 spike-specific memory CD4 and CD8 T cells induced by a heterologous prime-boost vaccination strategy consisting of Ad26.COV2.S and mRNA-1273 vaccines. Twenty healthcare workers (HCW) who participated in the SHERPA study where participants who had previously received either one (n=8) or two (n=12) doses of the Ad26.COV2.S vaccine received the mRNA-1273 vaccine booster at baseline (BL) were selected. The median time since last Ad26.COV2.S vaccine dose was 340 days [IQR: 335-355] and 187 days [IQR: 340-458] for the one and two prior dose groups, respectively. Ninety-five percent (19/20) of the participants had a history of SARS-CoV-2 infection. CD4 and CD8 T cell responses to the SARS-CoV-2 spike protein were characterized using intracellular cytokine staining interferon-γ (IFN-γ) or interleukin-2 (IL-2) and flow cytometry. The magnitude of T cell responses to full spike was examined, as well as crude T cell breadth, using 7 peptide pools spanning the entire length of the spike protein at BL and 4 weeks (W4) post-vaccination. Boosting with mRNA-1273 resulted in a significant increase in the magnitude of spike-specific CD4 responses (median % memory CD4 T cells: 0.085 vs. 0.157; p=0.04) and CD8 responses (median % memory CD8 T cells: 0 vs. 0.015; p=0.025). A strong positive correlation was observed between the magnitude of the full spike and the combined seven pools for both CD4 (p < 0.0001; r = 0.78) and CD8 (p < 0.0001; r = 0.74) responses. On average, participants had CD4 responses targeted against five pools (range 2-7) at BL. Following mRNA-1273 vaccination, the number of pools targeted slightly increased to a median of 5.5 (range 4-7). Only 8/20 (40%) participants had CD8 responses against the spike pools at BL (median: 0; range 0-6). At W4, the median number of targeted pools increased to one (range 0-6) among the participants. There was no statistical difference in CD4 or CD8 responses against the pools between those who had received one or two initial doses. All seven spike pools were targeted by the CD4 and CD8 cells at both timepoints. The highest median magnitude of CD4 responses was detected for pool 2 (0.025%) which covers the N terminal domain (131-315 aa), pool 5 (0.023%) which covers the fusion peptide domain (686- 890 aa), pool 6 (0.019%) which includes the heptapeptide repeat sequence and pool 3 (0.015%) covers a portion of the receptor binding domain (305-515 aa). Since most CD8 responders targeted only a single pool, no pool dominated in terms of the median magnitude. The data suggest that CD4 cells exhibit a broader breadth of responses compared to CD8 responses which were narrowly targeted against one region of the spike. This is consistent with what other published studies found. The broad targeting of the CD4 responses suggests that cross-reactivity against emerging VOCs may be better preserved than the CD8 responses, especially when mutations occur in the targeted epitopes. As new variants continue to emerge, it is important to introduce vaccines that induce broader CD8 responses to bolster cross reactivity.
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    Characterization of HIV-1 gag and nef in Cameroon: further evidence of extreme diversity at the origin of the HIV-1 group M epidemic
    (BioMed Central Ltd, 2013) Tongo, Marcel; Martin, Darren; Zembe, Lycias; Mpoudi-Ngole, Eitel; Williamson, Carolyn; Burgers, Wendy
    BACKGROUND: Cameroon, in west central Africa, has an extraordinary degree of HIV diversity, presenting a major challenge for the development of an effective HIV vaccine. Given the continuing need to closely monitor the emergence of new HIV variants in the country, we analyzed HIV-1 genetic diversity in 59 plasma samples from HIV-infected Cameroonian blood donors. Full length HIV gag and nef sequences were generated and phylogenetic analyses were performed. FINDINGS: All gag and nef sequences clustered within HIV-1M. Circulating recombinant form CRF02_AG predominated, accounting for 50% of the studied infections, followed by clade G (11%), clade D and CRF37_cpx (4% each), and clades A, F, CRF01_AE and CRF36_cpx (2% each). In addition, 22% of the studied viruses apparently had nef and gag genes from viruses belonging to different clades, with the majority (8/10) having either a nef or gag gene derived from CRF02_AG. Interestingly, five gag sequences (10%) and three (5%) nef sequences were neither obviously recombinant nor easily classifiable into any of the known HIV-1M clades. CONCLUSION: This suggests the widespread existence of highly divergent HIV lineages in Cameroon. While the genetic complexity of the Cameroonian HIV-1 epidemic has potentially serious implications for the design of biomedical interventions, detailed analyses of divergent Cameroonian HIV-1M lineages could be crucial for dissecting the earliest evolutionary steps in the emergence of HIV-1M.
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    Detection of natural infection with Mycobacterium intracellulare in healthy wild-caught Chacma baboons (Papio ursinus) by ESAT-6 and CFP-10 IFN-gamma ELISPOT tests following a tuberculosis outbreak
    (BioMed Central Ltd, 2008) Chege, Gerald; Warren, Robin; van Pittius, Nico; Burgers, Wendy; Wilkinson, Robert; Shephard, Enid; Williamson, Anna-Lise
    BACKGROUND:Both tuberculous and non-tuberculous mycobacteria can cause infection in nonhuman primates (NHP), indicating the existence of potential zoonotic transmission between these animals and visitors to zoos or animal handlers in primate facilities. Screening of mycobacterial infections in NHP is traditionally done by tuberculin skin test (TST), which is unable to distinguish between pathogenic and non-pathogenic mycobacterial infections. In this study, we investigated the use of ESAT-6 and CFP-10 for detection of mycobacterial infections in a wild-caught baboon colony after one baboon died of tuberculosis (TB). METHODS: Peripheral blood lymphocytes for interferon-gamma enzyme-linked immunospot assay (IFN-gamma ELISPOT) assay were obtained from TST positive baboons and those in contact with tuberculous baboons before being euthanased, autopsied and lung tissues taken for histology and mycobacterial culture. RESULTS: Both ESAT-6 and CFP-10 IFN-gamma ELISPOT assays were able to detect early M. tuberculosis but also M. intracellulare infection. Although this indicates potential cross-reactivity with M. intracellulare antigens, the method was able to distinguish M. bovis BCG vaccination from M. tuberculosis infection. This assay performed better than the TST, which failed to detect one M. tuberculosis and two early M. intracellulare infections. CONCLUSION: These results suggest that the IFN-gamma ELISPOT assay could improve the detection of M tuberculosis infections when screening NHP. There is some doubt, however, concerning specificity, as the assay scored positive three animals infected with M. intracellulare.
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    Development of a polychromatic flow cytometry panel for the evaluation of HIV-specific T cell responses
    (2009) Naicker, Prinola; Burgers, Wendy
    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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    Immunology and virology of HIV-1 infection in Cameroon
    (2014) Tongo Passo, Aime Marcel Simon; Burgers, Wendy; Martin, Darren
    This study confirms the widespread existence of highly divergent HIV lineages in Cameroon. While the genetic complexity of the Cameroonian HIV-1 epidemic has potentially serious implications for the design of biomedical interventions, detailed analyses of divergent Cameroonian HIV-1 group M lineages could be crucial for dissecting the earliest evolutionary steps in the emergence of HIV-1 group M. In addition, the central nature of HIV-1 consensus M sequences resulted in their broad recognition, but failed to identify highly immunodominant peptides between homogeneous and diverse HIV epidemics. Further refinement of these immunogens may contribute to the development of a globally relevant vaccine. Finally, the use of PTE peptides did not increase the breadth of T cell recognition in Abstract Page xvi this divergent population when compared to consensus M peptides. This underlies the need to include more mosaic peptides representing the variety of viruses that circulate in the region.
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    Investigating cross-clade immune responses in HIV-1 subtype C-infected individuals from South Africa
    (2007) Zembe, Lycias; Burgers, Wendy
    The increasing diversity of HIV-1 in different geographic regions presents a challenge for the development of an HIV/AIDS vaccine. Even if proven effective, the ability of a vaccine to have cross-subtype and intra-subtype protection remains an important question. The aim of the study was to investigate cross- and intra-clade T cell immune responses in HIV-1 subtype C-infected individuals. The objectives were to genetically characterise full length gag gene sequences from individuals chronically infected with HIV-1 and then assess the degree of cross-reactive immune responses in an ELlS pot assay using peptide reagents based on vaccine candidates.
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    Investigating cross-clade immune responses in HIV-1 subtype C-infected individuals from South Africa: implications for HIV vaccine design
    (2012) Zembe, Lycias; Burgers, Wendy; Williamson, Carolyn
    Background An effective HIV vaccine remains the main hope for controlling the HIV epidemic and is a global health priority. The genetic diversity of the virus across the globe is a major impediment to developing an effective vaccine. Whether a universal vaccine is possible still remain elusive. Therefore, there is need to fully characterise clusters of commonly targeted regions across the different HIV-1 clades. Centralized sequences have been suggested as vaccine immunogens and peptide reagents for assessing vaccine-induced responses, but their cross-reactivity has not been fully assessed in larger cohorts of subtype C-infection and in regions of differing HIV epidemics. In addition, the functional profile of HIV-specific T-cells recognizing variant epitopes has not been fully characterized. Whether cross-reactivity observed by IFN-γ production in an ELISpot assay can be observed at physiological concentrations of the peptides and for other functions of HIV-specific T-cells is an important question that remains to be answered. Methods The cross-reactivity of HIV-specific T-cells was assessed using clade-specific peptide reagents forming part of current candidate vaccine inserts based on the HIV-1 Gag protein from clades CDu422, CCH, A, B and D in 40 clade C-infected study participants using the IFN-γ ELISpot assay. To test the reactivity of group M consensus peptide reagents, 66 individuals, 44 of whom were ARV naïve, were assessed for HIV-specific T cell responses to group M Gag and Nef peptides. A selection of these individuals was screened for HIVspecific T-cell responses to clade CDu422 Gag peptides. Cross-reactivity of peptide variants was assessed at physiologically relevant peptide concentrations by functional avidity studies using peptide dilution IFN-γ ELISpot assays. Additionally, the cytokine profile, cytotoxic potential and proliferative capacity of cross-reactive peptide variants was characterised using multiparameter flow cytometry. Results The magnitude and breadth of HIV-specific T-cell responses were similar between the two clade-C peptide reagents in a clade C-infected population. However, the magnitude and breadth of responses to peptides based on clades A, B and D were significantly lower compared to the clade C peptides. Clusters of commonly targeted regions cross-reactive across the four clades investigated resided predominantly in conserved regions. Interestingly, there were Gag regions that were exclusively recognized in the different clades that had significantly lower entropy scores for the reactive variants than their nonreactive counterparts, suggesting that the variability in targeted regions could have been shaped by host immune pressure. For consensus group M peptides, the magnitude and breadth of Gag responses were significantly higher than that of Nef in clade C-infected individuals. In addition, consensus group M Gag peptides had significantly lower magnitude and breadth of HIV-specific T-cell responses compared to clade C peptide reagents, suggesting loss of responses by centralised reagents despite their central nature to group M viruses. On the contrary, the magnitude and breadth of responses to consensus group M Gag peptides were comparable to that of clade-mismatched peptides, namely clades A, B and D, suggesting that these reagents can be used interchangeably. Peptide dilution assays showed that amino acid mismatches have discordant effects on functional avidity and that some peptides are cross-reactive at physiological concentrations. Similarly, discordant effects (differences in functional avidity, cytokine and cytotoxic profiles and proliferative capacity) of amino acid mismatches on cytokine and cytotoxic potential profiles as well as proliferative capacity were observed. Conclusion People infected by a particular HIV clade can recognize HIV peptides based on other clades. However, the magnitude and breadth of responses are greater for the matched clades compared to mismatched clades, suggesting that there may be an advantage of using vaccines based on matched over unmatched clades. Group M based consensus sequences can be recognized in HIV-infected individuals, but with a lower frequency, magnitude and breadth of responses compared to clade-matched peptides, suggesting a limitation of these peptides both as reagents and vaccine immunogens. However, the frequency, magnitude and breadth T-cell reponses to consensus group M peptides were comparable to clademismatched responses, suggesting that these reagents may be used interchangeably. Furthermore, amino acid variations across corresponding viral regions have discordant effects on the functional avidity, cytokine profile, cytotoxic potential and proliferative capacity; implying that qualitative measures of cross-reactivity beyond IFN-γ frequencies need to be considered. These data may aid in the development of reagents for the assessment of vaccine-induced responses as well as in HIV vaccine immunogen design.
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    Investigating defects in monocyte responses to Mycobacterium tuberculosis in HIV-TB co-infection
    (2014) Thawer, Narjis Khatoon G; Burgers, Wendy
    Includes abstract. Includes bibliographical references.
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    Phenotypic and functional characterisation of cervical and peripheral HIV-1 specific T cell responses
    (2007) Liebenberg, Lenine Julie; Passmore, Jo-Ann; Burgers, Wendy
    Distinct HIV variants occur at the genital mucosa compared to in blood, which may similarly result in differences in HIV T cell responses. There have been no studies of the maturational status of HIV-specific T cells present at the female genital mucosa. This study aimed to characterise HIV-specific cervical immune responses and to determine if compartmentalized immune responses occur in chronic HIV infection by comparing the characteristics of T cells at the cervical mucosa to those in blood.
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    Studies on CD4+ T cell subsets in Mycobacterium tuberculosis immunity during HIV co-infection
    (2023) Nesamari, Rofhiwa; Burgers, Wendy; Riou, Catherine
    Tuberculosis (TB) is a global pandemic which resulted in 5.8 million disease cases and approximately 1.5 million deaths worldwide in 2020. TB disease is the leading cause of death worldwide from a single infectious agent, Mycobacterium tuberculosis (M.tb). It is estimated that a one fourth of the world's population is latently infected with TB and of these 5-10% will go on to develop active TB in their lifetime. Human immunodeficiency virus (HIV) infection is the greatest risk factor for developing active TB, with people living with HIV (PLWH) up to 19 times more likely to develop active TB compared to HIV uninfected individuals. Epidemiological studies show that there is increased risk of developing active TB (either reactivation of latent TB or new M.tb infection) throughout the course HIV infection, with the highest risk observed within the first year of infection, even when CD4+ T cell counts are still high. Previous studies have demonstrated that M.tb-specific CD4+ T cell responses are depleted early after HIV infection, within 3-12 months. This early defect in M.tb-specific CD4+ T cells may explain the elevated risk of TB even within the first year of HIV infection, prior to substantial immunosuppression. Antiretroviral therapy (ART) is an important strategy in preventing TB in PLWH. Earlier studies done in sub-Saharan Africa show that ART is associated with a 70-90% reduction in TB risk and a 52% decrease in TB related mortality. Several studies have also shown the importance of early ART initiation for HIV pathogenesis and transmission. However, despite ART intervention studies in sub-Saharan Africa, as well as Europe and North America, show that in these regions rates of TB remain high in PLWH. These high TB rates are most likely linked to the incomplete restoration of TB immune responses during ART. Overall, this thesis focused on characterising M.tb-specific CD4+ T cell responses during the course of HIV infection, from the acute phase of infection to post ART initiation. In Chapter 3, we aimed to confirm and extend previous findings that show there is a rapid depletion of M.tb-specific CD4+ T cells soon after HIV infection. We examined the kinetics of M.tb-specific CD4+ T cell responses over the course of HIV infection in two cohorts, a cross sectional cohort (n=58) and a longitudinal cohort (n=17). Consistent with previous findings, our results showed that there is a significant decrease in the frequency of M.tb responders 3 months after HIV infection. However, not all participants experienced M.tb-specific CD4+ T cell loss after HIV infection, with half the cohort losing 50% or more of their responses and the other half maintaining their responses. In the group that maintained their M.tb responses after HIV infection, the majority had very little fluctuations in their responses during the acute and chronic phase of infection. When comparing the clinical characteristics and the function and phenotype of M.tb-specific CD4+ T cells between individuals who maintained their M.tb-specific response and those who don't, no significant differences were found. In Chapter 4, to investigate the impact of early ART on M.tb immunity, we characterised M.tb-specific CD4+ T cell responses in individuals who initiated ART at an early stage of HIV infection (median 7.5 months post infection, n=16) in comparison to those who started ART during the chronic phase of HIV infection (median 66 months post infection, n=22). In this study, we examined multiple parameters between the two groups including their clinical characteristics, as well as the magnitude, function and phenotype of their M.tb-specific CD4+ T cells. We show that 2 years after ART initiation (irrespective of its timing) induced a significant immune reconstitution, marked by an increase in CD4 T cell count and restoration of the C4/CD8 ratio, but had no significant impact on the magnitude, function or phenotype of M.tb-specific CD4+ T cells. In Chapter 5, we sought to characterise M.tb-specific T cell responses in a small (n=13) cohort of participants who developed active TB during the CAPRISA 002 study. We performed a descriptive study examining the magnitude and phenotype of M.tbspecific T cells longitudinally over the course of TB treatment: before TB treatment/diagnosis, during treatment and after successful treatment. Despite the limited number of participants in this sub-study, we showed that after TB treatment there was a decreasing trend of activated M.tb-specific CD4+ T cells coincident with an increase in IFN-+ IL-2+ dual functional cells. Overall, our data confirms previous findings that there is an early depletion of M.tbspecific CD4+ T cells within a year HIV infection. However, in our study we found that not all HIV infected individuals lose their M.tb-specific responses. Instead, there was a variation in M.tb-specific responses after HIV infection, with some individuals maintaining their responses and others completely losing them. Results of our study also showed there were no major differences between participants who initiated ART early and those who initiated later during chronic HIV infection. To our knowledge this is the first study which looks at the impact of ART timing on M.tb-specific immunity in PLWH. The study therefore provides further insight, for future research, into whether early ART preserves TB immunity and therefore reduce the early, elevated risk of TB in HIV infected individuals.
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    Studies on immunity in the male genital tract
    (2011) Olivier, Abraham Jacobus; Burgers, Wendy
    The male genital tract is a major site of HIV acquisition and transmission. It is an obvious site for inducing immune responses to candidate HIV vaccines, to prevent infection or halt the spread of the virus. There are relatively few published studies characterising T cells in the male genital tract. A challenge that hampers studies at this mucosal surface is obtaining samples with sufficient immune cells. Therefore, the first aim of this study was to establish an optimised method to isolate immune cells from the male genital tract. Cellular activation and inflammation in the genital tract have important implications for both transmission and acquisition of HIV, since they provide target cells for viral replication. Thus, the second and third aim of this study was to investigate mucosal T cell activation and inflammatory cytokine profiles in semen in HIV?infected and uninfected men, and compare the immune milieu of the genital tract with the systemic compartment.