Browsing by Author "Burgers, Wendy A"
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- ItemOpen AccessCase report: mechanisms of HIV elite control in two African women(BioMed Central, 2018-01-25) Moosa, Yumna; Tanko, Ramla F; Ramsuran, Veron; Singh, Ravesh; Madzivhandila, Mashudu; Yende-Zuma, Nonhlanhla; Abrahams, Melissa-Rose; Selhorst, Philippe; Gounder, Kamini; Moore, Penny L; Williamson, Carolyn; Abdool Karim, Salim S; Garrett, Nigel J; Burgers, Wendy ABackground: The majority of people living with HIV require antiretroviral therapy (ART) for controlling viral replication, however there are rare HIV controllers who spontaneously and durably control HIV in the absence of treatment. Understanding what mediates viral control in these individuals has provided us with insights into the immune mechanisms that may be important to induce for a vaccine or functional cure for HIV. To date, few African elite controllers from high incidence settings have been described. We identified virological controllers from the CAPRISA 002 cohort of HIV-1 subtype C infected women in KwaZulu Natal, South Africa, two (1%) of whom were elite controllers. We examined the genetic, clinical, immunological and virological characteristics of these two elite HIV controllers in detail, to determine whether they exhibit features of putative viral control similar to those described for elite controllers reported in the literature. Case presentation: In this case report, we present clinical features, CD4+ T cell and viral load trajectories for two African women over 7 years of HIV infection. Viral load became undetectable 10 months after HIV infection in Elite Controller 1 (EC1), and after 6 weeks in Elite Controller 2 (EC2), and remained undetectable for the duration of follow-up, in the absence of ART. Both elite controllers expressed multiple HLA Class I and II haplotypes previously associated with slower disease progression (HLA-A*74:01, HLA-B*44:03, HLA-B*81:01, HLA-B*57:03, HLA-DRB1*13). Fitness assays revealed that both women were infected with replication competent viruses, and both expressed higher mRNA levels of p21, a host restriction factor associated with viral control. HIV-specific T cell responses were examined using flow cytometry. EC1 mounted high frequency HIV-specific CD8+ T cell responses, including a B*81: 01-restricted Gag TL9 response. Unusually, EC2 had evidence of pre-infection HIV-specific CD4+ T cell responses. Conclusion: We identified some features typical of elite controllers, including high magnitude HIV-specific responses and beneficial HLA. In addition, we made the atypical finding of pre-infection HIV-specific immunity in one elite controller, that may have contributed to very early viral control. This report highlights the importance of studying HIV controllers in high incidence settings.
- ItemOpen AccessCharacterization of Mycobacterium tuberculosis-specific Th22 cells in HIV-TB co-infection(2020) Makatsa, Mohau Steven; Burgers, Wendy A; Riou, CatherineTuberculosis (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.
- ItemOpen AccessCharacterizing the cellular latent reservoir of HIV-1 and the effect of immune activation on characteristics of the reservoir(2022) Ismail, Sherazaan Dineo; Burgers, Wendy A; Williamson, Carolyn; Riou, Catherine; Abrahams, Melissa-RoseSince the advent of antiretroviral therapy (ART) and the resultant suppression of viraemia in the majority of people living with HIV-1 (PLWH) on ART, HIV-1 infection has become manageable and PLWH have similar life expectancies as uninfected persons. However, ART is not curative, is needed lifelong, and its cessation leads to the recrudescence of viraemia. This is due to the formation of a latent reservoir that is long-lived and stable over time, precluding HIV-1 cure. The factors affecting reservoir formation, establishment, and kinetics are not fully understood. Furthermore, differences exist at the population level in disease progression in PLWH depending on ethnicity, biological sex, and infecting viral subtype. Similarly, differences in the latent reservoir of HIV-1 have been described, although less extensively. Understanding what shapes the latent HIV-1 reservoir is critical for developing strategies for cure. Furthermore, it is imperative that cure research is undertaken in diverse populations to ensure coverage of knowledge across different demographics. The latter will ensure that a cure strategy can be developed that will be globally implementable. In the Introductory chapter of this thesis, I provide a detailed review of the current literature and address the need for cure research in low-and middle- income countries. If a global cure is to be achieved, the burden of HIV-1 will need to be addressed in many different populations, most notably African women, as women bear the burden of HIV-1 globally. In South Africa, the country in the sub-Saharan African region with the highest prevalence of HIV-1, women are roughly twice as likely to be living with HIV than men (aged 15 to 49), with a prevalence rate 6% higher than the national average of 19%. Since women are underrepresented in HIV-1 research in general and more specifically in cure studies due to the paucity of research in countries outside of the global North, reservoirs and cure strategies ii need to be characterized in this context. Furthermore, while early treatment is the WHO standard of care for people diagnosed with HIV, a large majority of PLWH only initiated treatment in chronic infection. Since early ART is known to restrict formation of the latent reservoir of HIV-1, research in both early and late ART initiators is necessary. This research focused on characterising the viral reservoir in South African women in a well-established cohort of women who were recruited during acute HIV infection and followed until treatment initiation (which occurred during chronic infection) and beyond. Overall, this thesis focuses on characterising immune activation and inflammation during the course of both untreated and treated HIV-1 infection in a cohort of South African women and subsequently determining whether clinical or immune measures influence characteristics of the latent reservoir of HIV-1. T cell activation and the levels of soluble inflammatory cytokines in plasma were determined in forty-six women in the CAPRISA 002 Acute infection cohort. Chapter 2 describes the cellular immune activation and inflammation profiles of these participants throughout the course of infection at the following timepoints: acute infection, oneyear post-infection, and within a year preceding ART initiation, and two- and four- years postART initiation. T cell activation peaked in chronic infection and reduced dramatically after ART initiation. CD4+ and CD8+ T cell activation reached a post-treatment nadir by two years after ART initiation. Cytokine measures were within the ranges reported in the literature for PLWH. Notably CXCL-10 levels in plasma decreased significantly between two- and four years post-ART, indicating that it may be a sensitive marker of ongoing systemic inflammation in people on ART. In short, the T cell activation and inflammation profiles of the women in this study reflected what has been observed in other cohorts. iii The size of the replication-competent HIV-1 reservoir, measured by quantitative viral outgrowth assay after 5 years of suppressive antiretroviral therapy (ART), was quantified in twenty women of the cohort. In Chapter 3, the clinical and immunological correlates of reservoir size were investigated. Predictive modelling showed that the size of the replicationcompetent reservoir is directly related to viral load and CD4+ T-cell counts over the course of infection, although these measures do not fully predict reservoir size. We found that, in addition to viral load and CD4+ T-cell count, CD8+ T-cell activation within the year preceding ART, nadir CD4+ T-cell count, and baseline as well as on-treatment CD4:CD8 ratio at the time of sizing was associated with replication-competent reservoir size. We provide evidence that the late CD8+ T-cell activation level before treatment, together with viral loads and CD4+ T-cell counts, are directly related to the size of the replication-competent reservoir of HIV-1. Our results are consistent with the hypothesis that the host immune milieu near the time of ART initiation plays an important role in shaping the durable reservoir of HIV infection that persists on ART. Another characteristic of the HIV-1 reservoir is persistence: the presence of all forms of HIV1 within cells and tissues that contribute to pathogenesis, including defective, non-induced, and non-integrated forms of HIV-1. In Chapter 4, total HIV-1 DNA levels were measured as a proxy for viral persistence in thirty-one participants, and the correlates thereof investigated. The HIV-1 DNA levels in this cohort were similar to those reported in the literature for other cohorts where participants initiated therapy in late chronic infection. HIV-1 DNA levels did not differ significantly between two- and four years post-ART, but there was a trend to lower HIV-1 DNA when measuring pol versus gag gene frequencies in peripheral blood mononuclear cells (PBMC). These findings indicate that HIV-1 DNA decay rates may differ depending on the gene being measured, even when using the same assay. A weak significant correlation was iv found between CD4+ T cell counts at ART initiation and the change in HIV-DNA levels between two-and four years on ART. There was a significant correlation between residual CD4+ T cell activation at four years post-ART initiation and gag copies per million PBMC. A trend towards a correlation was found between CD4+ T cell activation and pol copies per million PBMC at the same timepoint. Finally, we found significant correlations between several cytokines at one-year post-infection and within one year pre-ART. These findings further solidify the hypothesis that the immune milieu around the time of ART initiation and after may play a complex role in formation of the viral reservoir of HIV-1. Our studies show a significant link between chronic immune activation and replication competent reservoir size, and also ongoing immune activation and viral persistence on ART. Further studies into whether these immune measures affect the timing of establishment and clonality of the reservoir in this cohort are ongoing and will inform the field about whether differences in cure strategies will need to be explored for those PLWH who had high levels of chronic immune activation before treatment initiation and subsequent shaping for the long-lived viral reservoir.
- ItemOpen AccessIntra-and inter-clade cross-reactivity by HIV-1 Gag specific T-cells reveals exclusive and commonly targeted regions: implications for current vaccine trials(Public Library of Science, 2011) Zembe, Lycias; Burgers, Wendy A; Jaspan, Heather B; Bekker, Linda-Gail; Bredell, Helba; Stevens, Gwynneth; Gilmour, Jill; Cox, Josephine H; Fast, Patricia; Hayes, PeterThe genetic diversity of HIV-1 across the globe is a major challenge for developing an HIV vaccine. To facilitate immunogen design, it is important to characterize clusters of commonly targeted T-cell epitopes across different HIV clades. To address this, we examined 39 HIV-1 clade C infected individuals for IFN-γ Gag-specific T-cell responses using five sets of overlapping peptides, two sets matching clade C vaccine candidates derived from strains from South Africa and China, and three peptide sets corresponding to consensus clades A, B, and D sequences. The magnitude and breadth of T-cell responses against the two clade C peptide sets did not differ, however clade C peptides were preferentially recognized compared to the other peptide sets. A total of 84 peptides were recognized, of which 19 were exclusively from clade C, 8 exclusively from clade B, one peptide each from A and D and 17 were commonly recognized by clade A, B, C and D. The entropy of the exclusively recognized peptides was significantly higher than that of commonly recognized peptides (p = 0.0128) and the median peptide processing scores were significantly higher for the peptide variants recognized versus those not recognized (p = 0.0001). Consistent with these results, the predicted Major Histocompatibility Complex Class I IC 50 values were significantly lower for the recognized peptide variants compared to those not recognized in the ELISPOT assay (p<0.0001), suggesting that peptide variation between clades, resulting in lack of cross-clade recognition, has been shaped by host immune selection pressure. Overall, our study shows that clade C infected individuals recognize clade C peptides with greater frequency and higher magnitude than other clades, and that a selection of highly conserved epitope regions within Gag are commonly recognized and give rise to cross-clade reactivities.
- ItemOpen AccessInvestigating cytokine responses to different strains of Mycobacterium tuberculosis(2021) Booi, Zandile; Burgers, Wendy A; Keeton, RoanneMycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), is one of the world's most prevalent fatal pathogens, infecting one third of the global population. The highly specialised pathogen that infects humans today evolved from a group of bacteria, known as the Mycobacterium tuberculosis complex (MTBC). It is divided into different lineages, based on phylogeography and genetic structure. It is well established that infection with different strains can lead to different types of innate immune responses. Recent studies indicate differences in the adaptive immune response (IL-22 and IL-17) induced by various Mtb strains, however these differences have not been clearly defined. The aim of this study was to investigate CD4+ T cell adaptive cytokine responses (IFN-γ, IL-22 and IL-17) in blood from human volunteers with prior exposure to Mtb, when stimulated with different strains within the MTBC, using a whole blood assay, flow cytometry and enzyme-linked immunosorbent assay (ELISA). My results indicate that the frequency of IFN-γ and IL-22-producing CD4+ T cells was similar for the two strains, as no significant differences were observed. In contrast, IL-17 responses for HN878 was almost 2-fold higher than H37Rv (p=0.04), consistent with published observations. Moreover, three additional Mtb strains (Indo-oceanic, East African-Indian and the Euro-American outbreak strain CDC1551) were tested for their ability to detect cytokine responses. While all five strains enabled the detection of Mtb-specific CD4+ T cell IFN-γ, IL22 and IL-17, no significant differences were observed when comparing the responses between strains. The highest responses were observed for IFN-γ, followed byIL-22 and IL17. In addition to flow cytometry, soluble cytokine concentrations of IFN-γ and IL-22 were examined in plasma in whole blood stimulated with Mtb lysate derived from H37Rv and HN878, using an ELISA. Responses were tested at both 24 and 48 hours of stimulation, and while median responses were higher at the later time point, they were not significantly different. IFN-γ showed no difference between H37Rv and HN878 at 24 hours in the ELISA. In contrast, HN878 IFN-γ responses were lower than H37Rv at 48 hours (p=0.04). The amount of secreted IL-22 did not differ significantly between the twos trains at either time point. Further work is required to confirm these findings in a larger cohort to understand differences in adaptive responses to different Mtb strains. In conclusion, understanding Mtb strain modulation of host immune function is of major importance, as it may provide better insights into human TB immunity and could assist in the development of an effective TB vaccine.
- ItemOpen AccessRestoration of cellular immunity in HIV-infected individuals on antiretroviral therapy(2017) Fatime Ramla, Tanko; Burgers, Wendy A; Riou, Catherine; Soares, AndreiaDuring the course of HIV pathogenesis, the virus induces multiple defects in immune cells, altering their functional ability to efficiently control HIV itself and other infections. Whilst the widespread implementation of antiretroviral therapy (ART) has led to reduced morbidity and mortality in most HIV-infected individuals having access to treatment, we still do not know whether full restoration of immune function occurs. The aim of this study was to assess the extent to which ART restores both phenotypic and functional T and B cell immunity. HIV-infected women were studied before and 1 year after ART initiation. In Chapter 2, the effect of ART on T cell activation and differentiation profiles was evaluated in HIV-infected individuals (n=28; pre- and post-ART), and compared to HIVuninfected age- and sex-matched controls (n=23). In Chapter 3, the restoration of copathogen specific CD4+ T cells was determined by comparing their cytokine secretion ability and memory differentiation profiles in response to Mycobacterium tuberculosis and cytomegalovirus in HIV-infected (n=15; pre- and post-ART), compared to uninfected (n=9) individuals. Finally, Chapter 4 examined changes in B cell activation and memory profiles in HIV-infected persons (n=19; pre- and post- ART), and compared profiles to HIV-uninfected individuals (n=19). Multiparameter flow cytometry was performed to address the study objectives. T cell activation, as measured by CD38 and HLA-DR expression, was significantly reduced one year after ART for both CD4+ and CD8+ T cells, but normalisation to levels in HIV-uninfected individuals did not occur, despite suppression of viral load. In addition, skewed CD4+ and CD8+ T cell memory profiles were not completely restored. Furthermore, no change in the cytokine production capacity and memory profile of pathogen-specific CD4+ T cells was found before and after ART, but pathogen-specific CD4+ T cells exhibiting a late differentiated profile (CD27- CD45RO+) had a lower ability to replenish (p=0.02; r = -0.5) compared to cells with an early differentiated profile (CD27+CD45RO+; p=0.04; r = 0.45) prior to ART. Similar to T cells, activated B cells (CD40+CD86+) were only partially normalised post-ART, and remained significantly higher than B cells of HIV-uninfected individuals. The frequency of all B cell memory subsets were comparable between HIV-treated and uninfected individuals, with the exception of plasmablasts, whose frequency was still significantly higher than in HIV-uninfected subjects. In summary, these results demonstrate that HIV-infected women on suppressive ART show a substantial but only partial normalisation of T cell and B cell memory subsets, and lower levels of T cell and B cell activation. In addition, restoration of co-pathogen specific memory CD4+ T cells upon treatment was dependent on their memory profile before ART. Understanding the impact of HIV on T and B cell dysfunction and restoration upon ART may provide important insights into the mechanisms of HIV pathogenesis in the era of ART.
- ItemOpen AccessSARS-CoV-2 Infection Is Associated with Uncontrolled HIV Viral Load in Non-Hospitalized HIV-Infected Patients from Gugulethu, South Africa(2022-06-03) Lambarey, Humaira; Blumenthal, Melissa J; Chetram, Abeen; Joyimbana, Wendy; Jennings, Lauren; Tincho, Marius B; Burgers, Wendy A; Orrell, Catherine; Schäfer, GeorgiaIn South Africa, high exposure to SARS-CoV-2 occurs primarily in densely populated, low-income communities, which are additionally burdened by highly prevalent Human Immunodeficiency Virus (HIV). With the aim to assess SARS-CoV-2 seroprevalence and its association with HIV-related clinical parameters in non-hospitalized patients likely to be highly exposed to SARS-CoV-2, this observational cross-sectional study was conducted at the Gugulethu Community Health Centre Antiretroviral clinic between October 2020 and June 2021, after the first COVID-19 wave in South Africa and during the second and beginning of the third wave. A total of 150 adult (median age 39 years [range 20–65 years]) HIV-infected patients (69% female; 31% male) were recruited. 95.3% of the cohort was on antiretroviral therapy (ART), had a median CD4 count of 220 cells/µL (range 17–604 cells/µL) and a median HIV viral load (VL) of 49 copies/mL (range 1–1,050,867 copies/mL). Furthermore, 106 patients (70.7%) were SARS-CoV-2 seropositive, and 0% were vaccinated. When stratified for HIV VL, patients with uncontrolled HIV viremia (HIV VL > 1000 copies/mL) had significantly higher odds of SARS-CoV-2 seropositivity than patients with HIV VL < 1000 copies/mL, after adjusting for age, sex and ART status (p = 0.035, adjusted OR 2.961 [95% CI: 1.078–8.133]). Although the cause–effect relationship could not be determined due to the cross-sectional study design, these results point towards a higher risk of SARS-CoV-2 susceptibility among viremic HIV patients, or impaired HIV viral control due to previous co-infection with SARS-CoV-2.