Masters Degrees (Immunology)
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Item Impact of Duffy Antigen Receptor for Chemokines (DARC)-null linked Neutropenia on Neutrophil and Natural Killer cell Function in HIV-1 Infection.(2018) Naidoo, Kewreshini Kasturi.; Thobakgale-Tshabalala, Christina Fanesa.Sub-Saharan Africa carries a disproportionate burden of the HIV-1 epidemic. The Duffy Antigen Receptor for Chemokines (DARC)-null polymorphism is a predictor of ethnic neutropenia which commonly occurs in persons of African ethnicity and is thought to account for up to 11% of HIV-1 infections on the African continent. Neutrophils are recognised for their killing mechanisms and have been noted for their regulatory mechanisms in recent years. For example, a role for neutrophils in natural killer (NK) cell priming in the periphery has been suggested, and neutrophil deficiency has been implicated in contributing to NK cell immaturity and dysfunction. While the DARC-null genotype is well associated with lower absolute neutrophil counts (ANCs), studies that assess the effects of the polymorphism on neutrophil functionality are lacking and the impact of DARC-null linked neutropenia on HIV disease progression is debatable. Furthermore, the influence of DARC-null neutropenia on the NK cell compartment is unknown. In this cross sectional pilot study, we assessed the impact of the DARC-null trait on neutrophil effector functions and also characterised NK cell profiles in Zulu/Xhosa African individuals from a high incidence HIV setting in Durban, South Africa. We hypothesised that in the context of the DARC-null genotype and lower ANCs in our cohort participants, neutrophils would have impaired functionality and would be unable to efficiently prime NK cells; thus affecting NK cell maturation and function, and altering NK cell homeostatic activities such as survival and proliferation. We further hypothesised that the impaired cellular responses in DARC-null individuals would be more prominent in HIV-1 infected individuals compared to HIV negative individuals. Neutrophil killing mechanisms were measured in HIV-1 chronically infected (n=22) individuals and HIV negative (uninfected) controls (n=20). For assessment of key neutrophil effector functions, isolated neutrophils were evaluated for Fc receptor-mediated phagocytosis following uptake of IgG opsonised beads using flow cytometry; reactive oxygen species (ROS) emission was measured by chemi-luminesce after activation of neutrophils with phorbol 12-myristate 13-acetate (PMA). Activated neutrophils were also visualised by fluorescent microscopy for neutrophil extracellular trap (NET) quantification. Assessment of the NK cell compartment in chronically HIV-1 infected (n=18) and uninfected (n=20) individuals using multi-parametric flow cytometry determined NK cell subsets, maturation profiles, cytokine production and degranulation. Annexin V and propidium iodide assays were used to determine NK cell survival, whilst CFSE staining was used to examine cytokine-activated NK cell proliferation. Study subjects were genotyped for the DARC trait using TaqMan allelic discrimination assays and ANCs were measured by full blood count. Our findings confirmed a high prevalence of the DARC-null allele in the African population and the polymorphism was significantly associated with lower ANCs. Neutrophil functional analysis detected rapid and higher phagocytic activity in the absence of DARC at 10 minutes (p=0.05 and p=0.009) and 60 minutes (p=0.05 and p=0.07) in HIV negative and HIV-1 infected subjects respectively. ROS and NET production in neutrophils were mostly unaffected by DARC negativity irrespective of HIV status. The only exception to this was a reduction in NET production in neutrophils from DARC-null HIV infected subjects (p=0.04) following prolonged in vitro stimulation. In the NK cell compartment, individuals showed similar NK cell counts irrespective of HIV status. In HIV negative individuals, a marked reduction of total NK cell counts was noted in the absence of DARC (p=0.006) and this correlated with lower ANCs (p=0.002) and a weak trend towards higher CD56 bright subset proportions was noted in DARC-null individuals (p=0.08). HIV negative DARC-null subjects also displayed a less mature NK cell phenotype with higher proportions of hypo-responsive KIR-NKG2A- NK cells (p=0.06) and lower frequencies of terminally differentiated CD57 (p=0.02) expressing NK cells. However, this immature phenotype did not translate to differences in expression of NK cell activation markers CD69 or HLA-DR and exhaustion marker PD-1 by DARC state. Furthermore, no differences in relation to NK cell degranulation and cytokine production were detected in the absence of DARC in HIV negative subjects. In contrast to HIV negative individuals, HIV-1 infected subjects displayed NK cell subset redistribution marked by higher CD56 negative NK cells, marginally higher frequencies of less mature NK cells, accompanied by higher expression of activation and exhaustion markers and lower cytolytic potential. However, these observed phenotypic and functional differences were lost upon DARC stratification in HIV-1 infected persons. Lastly, examination of NK cell survival capacity demonstrated only marginal differences during HIV infection in the absence of DARC (p=0.09); no changes were detected in NK cell proliferation by DARC state in HIV negative or infected individuals. Together our data suggests that the DARC-null polymorphism and lower ANCs does not adversely affect neutrophil activity irrespective of HIV status. We also show that while HIV negative individuals with the DARC-null genotype displayed reduced NK cell counts with a less mature phenotype, the condition did not compromise NK cell functionality or homeostatic activities. Furthermore, no significant differences were exhibited in the context of DARC during HIV infection, suggesting that any advantage that the DARC-positive trait may offer pre-infection is lost in chronic infection. DARC-null associated neutropenia is considered a mild condition and thus our findings support reports that the effect of ethnic neutropenia is not as pronounced as exhibited in severe neutropenia. Overall the data presented here provides mechanistic evidence behind the asymptomatic clinical characteristics associated with benign ethnic neutropenia.markers.Item Sex differences in the Kinetics of immune reconstitution under antiretroviral therapy.(2017) Mazibuko, Noluthando Y.; Thobakgale, Christina Fanesa.; Ndung'u, Peter Thumbi.The human immunodeficiency virus type 1 (HIV-1) remains a global health threat and is increasingly becoming a female epidemic due to gender inequalities. The introduction of antiretroviral therapy (ART) has greatly increased the life span while reducing AIDS-related deaths in HIV-1-infected people. However, some patients experience adverse effects during ART due to an acute inflammatory response termed immune reconstitution inflammatory syndrome (IRIS), which is a paradoxical clinical worsening upon initiation of ART that is thought to be due to a hyperactive or uncontrolled immune restoration. Studies have shown that females infected with HIV-1 elicit a stronger immune response and faster disease progression compared to men with the same viral load. Given the above mentioned higher baseline levels of immune activation in HIV-1 infected females, we hypothesized that immune restoration during ART will have significant sex-based differential outcomes. The aim of this project was to understand the impact of immune reconstitution in HIV-1-infected men and women during ART by investigating antigen presenting cells (monocytes, myeloid dendritic cells (mDCs) and plasmacytoid dendritic (pDCs) cells) phenotype and function. In total, we investigated cryopreserved samples from eleven HIV-1-infected males and thirteen HIV-1-infected females from which peripheral blood mononuclear cells (PBMCs) and plasma samples were longitudinally collected at defined time points, including before the initiation of ART (TP01), after 1-4 months (TP02) and after 5-8 months of treatment initiation. Changes in toll-like receptor (TLR) responsiveness were analyzed following stimulation with the following toll-like receptor (TLR) ligands: TLR4 ligand Lipopolysaccharide, TLR7/8 ligand CL097, and the TLR9 ligand ODN2216/ CpG. Multiparameter flow cytometry was used to analyze the cytokine production upon TLR stimulation as well as the level of immune activation by analyzing phenotypic characteristics of antigen-presenting cells including monocytes subsets. In addition, multiplex analysis was used to determine levels of plasma pro-inflammatory cytokines (IFN-ɣ, IFN-α, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, TNF-α, IL-12p70, GM-CSF, MIP-1β and IP-10), in males and females at all time-points. Our results show that the expression of immune activation markers (measured by HLA-DR+ and CD38+ on T cells) did not significantly differ between males and females, although females showed elevated levels of both activated CD8+ and CD4+ T cells at baseline, the activation levels decreased upon ART initiation. Furthermore, our results demonstrated an increase in the proportion of classical monocytes (CD14++CD16-) in females compared to males at baseline (p=0.03). We did not observe any significant differences in the percentage of intermediate (CD14++CD16+) and non-classical monocytes (CD14+CD16++) between males and females at any time point analyzed. Similarly, no sex differences were evident after TLR ligand stimulation on monocytes in terms of cytokines measured (IFN-α, TNF-α, MIP-1β and IL-12). Interestingly, upon TLR9 stimulation, a significantly higher percentage of pDCs from females produced IFN-α (p=0.001, TP03), MIP-1β (p=0.001, at TP02) and TNF-α (p˂0.01, p˂0.001, TP02 and TP03 respectively) during ART compared to males. In addition, females had increased IFN-α (p=0.01) and TNF-α (p=0.004) production on pDCs during ART compared to baseline following TLR9 stimulation. Taken together, our data suggest sex-specific differences in the level of immune reconstitution during ART, as females show signs of elevated immune response and inflammation compared to males Therefore, these findings may provide a basis for future studies in larger cohorts aimed at adapting ART therapy based on sex differences in disease progression rates in men and women.Item Development and optimization of real-time PCR assays to detect anti-microbial immune factors and their response to type I and II interferons.(2016) Kieswetter, Nathan Scott.; Ndung'u, Peter Thumbi.; Wong, Emily.Abstract available in PDF file.Item Analysis of viral inhibitory activity of cytotoxic T. Lymphocytes targeting identical epitopes restricted by different class 1 HLA alleles from the same HLA supertype.(2015) Ogunshola, Funsho Japhet.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.Human leukocyte antigen (HLA) polymorphism and the genetic diversity of human immunodeficiency virus (HIV) are the major obstacles for designing an effective HIV Cytotoxic T Lymphocytes (CTLs) based vaccine. Interestingly, recent studies have demonstrated that multiple class I alleles can recognize common epitopes “supertopes” due to the homology of amino acids within the major binding pockets of the peptide binding cleft. The implications of this for vaccine design is that a vaccine containing a small number of highly promiscuous supertopes can confer protection against a wide range of HIV variants. This notion makes supertopes immunogen design an attractive option. However, it is not clear whether supertopes presented in the context of different class I HLA alleles would induce functional equivalent CTL responses. In this study, we investigated the inhibitory activity of CTLs targeting identical epitopes presented by class I HLA alleles from the same superfamily. The viral inhibitory activity was measured using a newly developed CEM-GFP reporter T-cell line (GXR-cell) as target cell. We first compared the inhibitory activity of CTLs from 8 subjects targeting TPQDLNTML (Gag p24 residue 180-188-TL9) epitope presented by HLA-B*81:01 or B*42:01 alleles. We then assessed the inhibitory activity of the 8 subjects’ CTLs when presented with in-vivo occurring mutant (Q182S)-TL9 epitope by HLA-B*81:01 or B*42:01 alleles. Furthermore, we compared the inhibitory activity of CTLs from 4 subjects targeting ISPRTLNAW (Gag p24 residue 147-155-IW9) epitope presented by HLA-B*57:03 or B*58:01 alleles. Comparative analysis of the inhibitory activity of the 8 subjects’ CTLs showed no statistical significant difference when TL9 epitope was presented by HLA-B*81:01 or B*42:01 alleles (1:1; p-value = 0.8785, paired t test), even at low target to effector ratio (1:8; p-value = 0.4418). No statistical significant difference was observed in the inhibitory activity of the 8 subjects’ CTLs when mutant (Q182S)-TL9 epitope was presented by HLA-B*81:01 or B*42:01 alleles (1:1; p-value = 0.8042), same result was observed at low target to effector ratio (1:8; p-value = 0.9396). Comparative analysis of the inhibitory activity of the 4 subjects’ CTLs targeting identical IW9 epitopes presented by HLA-B*57:03 or B*58:01 alleles showed a trend towards significance at target to effector ratio 1:1 (1:1; p-value = 0.0924), but at low target to effector ratio, no significance difference was observed (1:8; p-value = 0.1496). In conclusion, we have demonstrated that there is no observable significant difference in the inhibitory activity of CTLs targeting wildtype TL9 or mutant (Q182S)-TL9 epitopes presented in the context of HLA-B*81:01 or B*42:01 alleles. Thus, TL9 epitope could be immunogenic for individuals expressing HLA-B*81:01 or B*42:01 alleles. We have also shown that the inhibitory activity of CTLs targeting identical IW9 epitopes presented by HLAB* 57:03 or B*58:01 alleles is comparable. Indicating that IW9 epitope could be included in immunogen design for individuals expressing HLA-B*57:03 or B*58:01 alleles. These findings are relevant for HIV vaccine approach that seeks to identify immunogenic supertopes that can be cross-presented in a broadly cross-reactive T cell based vaccine design.Item Mapping immunodominant patterns and HLA class II restriction characteristics of HIV-specific CD4+ T cell responses in acute and chronic HIV-1 subtype C infection.(2014) Laher, Faatima.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.Increasing evidence suggests that virus-specific CD4+ T cells contribute to immune-mediated control of HIV-1 infection. However, precise details of CD4+ T cell contribution to immune protection against HIV have not been adequately defined and most of the existing data was predominantly generated in clade B HIV-1 infection. Understanding the contribution of CD4+ T cell responses in clade C infection is important for developing vaccines that would be efficacious in sub-Saharan Africa which carries the highest burden of the HIV epidemic in the world. Therefore this study focused on the role of CD4+ T helper cells in the immune response to clade C HIV-1 infection. We tested the hypothesis that HIV-1-specific CD4+ T cell responses and protective class II HLA alleles are important determinants of effective immunological control of HIV-1 infection. Firstly, CD8 depleted PBMCs were used in an IFN-γ ELISPOT assay to conduct a comprehensive analysis of virus-specific CD4+ T cell responses in acute and chronic HIV-1 clade C infection. Thereafter the host genetic effects of class II HLA-DRB1 alleles on HIV viremia were assessed using the HLA-DRB1 restriction assay, where HLA class II-restriction characteristics of detectable responses were defined. Lastly, functional differences of HIV-specific CD4+ T cells were further characterized using flow cytometric analysis. In our study, Gag and Pol regions of the HIV proteome were found to be the most frequently targeted in acute HIV-1 infection (69% of total responses), with CD4+ T cell targeting across the proteome remaining relatively stable over time. In chronic HIV-1 clade C infection, dominant HIV-1-specific CD4+ T cell responses were detectable against a limited number of epitopes. Epitopes in the Gag region were the most targeted by CD4+ T cells (30/40 peptides), with OLP 41 in the Gag p24 region being the most dominant epitope targeted (15% of responses). There were no significant differences observed between total or Gag-specific CD4+ T cell responses and contemporaneous viral load. Interestingly, responses rarely targeted the envelope region in clade C infection, in contrast to multiple epitopes targeted in this protein in previous clade B studies. Functional analysis demonstrated that IFN-γ, IL-2 and TNFα were the most secreted cytokines by HIV-specific CD4+ T cells in 18/25 individuals, with IFN-γ being the most dominant response in individual subjects. The HLA class II DRB1 restriction in clade C HIV infected individuals showed epitope promiscuity, consistent with previous studies in clade B infection. The HLA-DRB1*13:01 allele variant was associated with the highest frequency of responders (22%) in our cohort and restricted the highest number of HIV-specific peptides (9/15). Together, our data identify immunodominant regions of HIV-specific CD4+ T cell responses and their association with viral control during clade C infection. Furthermore, our findings will inform studies aimed at elucidating the underlying mechanism by which CD4+ T cells modulate effective CD8+ T cell and B cell responses. Additionally, these data suggest that epitope promiscuity among class II HLA molecules should be taken into account for vaccines designed to induce CD4+ T cell responses. This information will be critical to vaccine efforts designed to induce these responses, as well as potential therapeutic manipulation of immunity in persons with acute and chronic HIV-1 infection.