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Doctoral Degrees (Immunology)

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    Molecular mechanisms of CD8+ T cell mediated control of HIV-1 infection in peripheral blood and lymphoid tissues.
    (2019) Ogunshola, Funsho Japhet.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.
    Naturally induced CD8+ T cells do not clear human immunodeficiency virus (HIV) infection, partly because the virus rapidly escapes CD8+ T cell responses and the effector cells are excluded from HIV reservoirs sites. However, optimizing CD8+ T cell responses could potentially be leveraged in HIV vaccine or cure efforts if epitope escape and barriers to effector CD8+ T cells infiltrating the sites of HIV reservoirs are overcome. In our first study, we described a potential mechanism of HIV-1 control by CD8+ T cells targeting different variants in individuals infected with HIV-1. Our second study focused on describing the molecular regulation of CXCR5 expression in human CD8+ T cells. Study 1 HLA-B*81 is associated with control of HIV-1 subtype C infection, while the closely related allele B*42 is not. Interestingly, both alleles present the immunodominant Gag TL9 epitope, and the magnitude of this response correlates negatively with viral load. To examine the role of T cell receptor (TCR) in this process, we characterized the sequence and function of TL9-specific CD8+ TCR in B*81 and B*42 individuals. TL9-specific CD8+ T cells were identified and isolated using B*81 and/or B*42 TL9 tetramers. TCR beta genes were amplified from single sorted cells and sequenced. Paired alpha genes were identified for selected clones. TCR function was tested using a reporter cell assay where TCR+ Jurkat cells were co-cultured with peptide-pulsed or HIV-1 infected B*81 or B*42 target cells, and signalling quantified by luminescence. TCR recognition was assessed against all single amino acid TL9 variants and results were compared to HIV-1 subtype C sequences. A population of dual-reactive T cells was detected by both B*81- and B*42-TL9 tetramers in 7/9 (78%) B*81 and 4/11 (36%) B*42 individuals; and this population was associated with lower viremia. Mono- and dual-reactive TCR beta sequences were collected from six individuals. In B*81 individuals, all TCRs were highly restricted to TRBV12-3. In B*42 individuals, mono-reactive TCRs encoded a variety of V beta genes, while dual-reactive TCRs were restricted to TRBV12-3 and enriched for public clones. Functional analyses indicated that B*81 TCRs (1 mono, 2 dual) and a dualreactive public B*42 TCR displayed similar TL9 cross-reactivity profiles and enhanced capacity to recognize HIV-1 escape mutations compared to mono-reactive B*42 TCRs. This work highlights the impact of TCR promiscuity on T cell-mediated control of HIV-1. Study 2 HIV-1 infection is difficult to cure even with effective antiretroviral therapy (ART) because of persistent viral replication in immune privileged sites such as the B cell follicles of secondary lymphoid tissues. CD8+ T cells are generally excluded from B cell follicles, partially due to a lack of expression of the follicular homing receptor CXCR5. Recent murine studies have identified CXCR5+ CD8+ T cells, referred to as follicular CD8+ T cells (fCD8s), that localize in B cell follicles. However, the mechanisms governing expression of CXCR5 on human CD8+ T cells are not known. We investigated the epigenetic and transcriptional mechanisms involved in the regulation of CXCR5 expression in human CD8+ T cells. We FACS-sorted CXCR5+CD8+ (fCD8s), CXCR5-CD8+ (non-fCD8s), naïve CD8+ T cells and germinal center T follicular helper cells (GCTfh) from the lymph node of HIV- 1 infected individuals and performed RNA-sequencing (RNA-Seq), DNA methylation assays and the assay for transposase-accessible chromatin using sequencing (ATACSeq). RNA-Seq was used to quantify the expressed genes in FACS-sorted subsets and to determine transcriptional modules governing CXCR5 expression in CD8+ T cells. ATAC-Seq was used to quantify accessible genes, identify the transcriptional factors footprinting and determine epigenetic modules governing CXCR5 expression. DNA methylation, a major epigenetic gene silencing mechanism, was used to profile methylation pattern of the CXCR5 gene region in the sorted subsets. We observed hypermethylation of DNA around the transcriptional start site (TSS) of the CXCR5 gene in non-fCD8s but not in fCD8s. ATAC-Seq analysis revealed a closed chromatin conformation at the TSS in non-fCD8s, but not in fCD8s. Our gene expression data revealed significant differences in the CXCR5 associated factors between GCTfh and fCD8s. Computational analysis further revealed the presence ofa nucleosome at the TSS of fCD8s, which could be a plausible explanation for lower expression of CXCR5 in fCD8s as compared to GCTfh. Together, we identified epigenetic regulations involved in CXCR5 expression in human CD8+ T cells and propose that DNA methylation, chromatin structure and nucleosome positioning cooperatively regulate the expression of CXCR5 in CD8+ T cells. Our data open up the possibility of using epigenetic manipulation as a novel strategy for redirecting CD8+ T cells to B cell follicles where they are needed to eradicate HIV-1 infected cells.
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    T follicular helper cell dynamics during acute HIV-1 subtype C infection and relevance for T-helper cell-dependent responses.
    (2018) Baiyegunhi, Omolara Olujimi.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.
    Abstract available in pdf.
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    Immunological effects of asymptomatic vibrio cholerae, salmonella enterica serovar typhi and entamoeba histolytica enteric pathogens during HIV infection and exposure.
    (2017) Nhidza, Agness Farai.; Mduluza, Takafira.; Naicker, Thajasvarie.
    Background Asymptomatic infection by enteric pathogens has been attributed to causing several outbreaks in the world. Common pathogens in this category are Vibrio cholerae, Salmonella typhi and Entamoeba histolytica infection of pregnant and lactating mothers. Exposure of the infant and the immune response during the early developmental years requires investigation. In the era of high HIV burden and the introduction of HIV prevention of mother to child transmission (PMTCT), many infants are exposed to these pathogens yet remain uninfected, however still face the challenge of high morbidity and mortality as compared to their unexposed and uninfected counterparts. The cause of such an anomaly has not been fully investigated. Aim The aim of this study is to investigate the burden and the immunological effect of asymptomatic enteric pathogen carriage in pregnant mothers, both HIV-infected and HIV-uninfected, and their infants in a high HIV burdened population of Harare, Zimbabwe. Methodology The study was a purposive cross-sectional sub-study in a birth cohort established at the University of Zimbabwe, College Of Health Sciences (UZ-CHS Birth Cohort). Participants were from the highdensity suburbs of Glenview, Kuwadzana and Dzivarasekwa. These suburbs were previously affected by the 2008 cholera epidemic as well as the 2012 typhoid outbreak that hit Harare, Zimbabwe. HIV rapid testing was performed at enrolment for mothers unaware of their HIV status or those who wanted HIV re-testing for various personal reasons. Those HIV infected with documented HIV status were asked to provide the evidence and were not re-tested. The HIV status of neonates was ascertained using nucleic acid-based testing at the National Early Infant Diagnostic (EID) department based at the National Microbiology Reference Laboratory in Harare, Zimbabwe. The test made use of the Dried Blood Spot (DBS) samples collected from the infants at various time points. A total of 417 pregnant mothers were recruited based on their consent to provide stool samples, blood and urine for both the mother at recruitment and the infant upon delivery. A questionnaire to collect information on socio-demographic, clinical, delivery, water and sanitation, diarrheal history including antibiotic use, concurrent TB infections, ART regimen, planned baby feeding method, diet as well as nutritional data on probiotic uptake was administered to the mothers. Of the 417 mothers, only 204 mothers and 308 infants provided stool samples, which were cultured for enteric pathogens targeting Salmonella entrica serovar Typhi (S. typhi) and Vibrio cholerae, although in the process any non-lactose fermenter identified was considered a potential pathogen hence were further identified using an Analytical Profile Index (API; Biomerieux: France) and included in the study. The stool samples were also used for antigen ELISA targeting Entamoeba histolytica (E. histolytica) infection. The microbial results were used to calculate the prevalence of S. typhi, V. cholerae and E. histolytica in the study population. Data was analysed using STATA 13 analytical software. For further analysis on immunological factors in mother-infant pairs (MIPs), only paired mothers were included based on stool as well as plasma availability and the outcome of culture and ELISA results. Multiplex immunoassay utilised a 27-plex (IL-1β, IL-1r, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL12p70, IL-13, IL-15, IL-17a, Eotaxin, basic PDGF, G-CSF, GM-CSF, IFN-, IP-10, MCP1(MCAF), MIP-1α, MIP-1β , PDGF-BB, RANTES, TNF- α and VEGF) cytokine profile and a 6- plex isotyping (immunoglobulin: IgG1, IgG2, IgG3, IgG4, IgM and IgA) assay. Immunological assays were performed on the 39 MIPs purposively selected based on the culture outcomes as described, using BioRad Luminex machine according to manufacturer’s instruction Based on the cytokine and immunoglobulin concentrations, maternal characteristics influencing infant cytokine and immunoglobulin production were analysed. Results The presence of V. cholerae nor S. typhi was detected but E. histolytica prevalence in mothers was calculated at 5.4% while in infants it was 5.6%. There was no significant difference in the prevalence of E. histolytica between HIV-infected and HIV-uninfected mothers (p=0.318) as well as between HIV-exposed uninfected and HIV-unexposed uninfected infants (p=0.056). E. histolytica infection was only noted in 1 HIV-uninfected mother-neonate pair. The resistance to 3rd generation cephalosporines in the non-lactose fermenting bacterial isolates was detected. The cytokine groups of IL-1r, IL-4, IL-9, IL-12p70, IL-17a, G-CSF and PDGF-BB were significantly raised in E. histolytica infected compared to non-infected lactating mothers (p<0.05). In babies, E. histolytica carriage had no significant effect on the cytokine and immunoglobulin concentration. In this study maternal E. histolytica carriage was significantly (p=0.026) correlated with infant Interleukin-12p70 (IL-12p70; p<0.026), Fibroblast Growth Factor-basic (FGF-basic; p<0.026), Granulocyte Monocyte-Colony Stimulating Factor (GM-CSF; p<0.026) and Tumor Necrosis Factor (TNF; p<0.026)-α. The odds ratio of passing high levels of these cytokines to infants is reduced by 86% if the mother is a carrier of E. histolytica during pregnancy (OR: 0.14). Carriage of any form of enteric infection such as Non-lactose fermenters (NLFs) including E. histolytica significantly increased the levels of IL-1r, IL-4, IL-9, IL-10, IL-12p70, IL17a, G-CSF, GM-CSF, IFN-, PDGF-BB and TNF-α cytokines (p<0.05). However, no significant difference in immunoglobulin levels was noted amongst the mothers. IL13 and G-CSF levels were significantly raised in HIV-infected compared to HIV-uninfected lactating mothers (p=0.0389 and p=0.0324, respectively). IgA immunoglobulin levels were significantly raised among HIV-exposed uninfected than HIVunexposed uninfected infants. It has also been observed that maternal E. histolytica carriers are significantly more likely to have infants with low levels of IL-12p70, FGF-basic, GM-CSF and TNF-α cytokines and high levels of IgA immunoglobulin. HIV-infected mothers are significantly more likely to have infants with low levels of IgG2 and IgA immunoglobulins. Mothers giving birth at a mean age above 30.38 ±STD 6.09 years are more likely to have infants with low IgG4 levels (p=0.050). Maternal HIV infection influences infant IgG2 and IgA immunoglobulin levels. Also, a maternal age of >30years reduces the odds ratio of passing high IgG4 to the infant (p=0.050). Maternal HIV infection influences infant IgG2 and IgA immunoglobulin levels. Infants born to HIV positive mothers are significantly associated (p=0.049) with lower odds of having high IgG2 levels, ie., reduced by 76% in infants born to HIV-infected mothers compared to those born to HIV-uninfected mothers. Similarly, an infant born to a HIVinfected mother is significantly associated (p=0.035) with lower odds of having high IgA immunoglobulin levels compared to those born to HIV-uninfected mothers (OR: 0.22). Underweight/small to gestational age (SGA) babies (birth weight <2500g) were found to have significantly raised IL-7 cytokine levels compared to normal for gestational age (NGA) babies whose birth weight was >2500g (p=0.0149). Conclusion Asymptomatic enteric E. histolytica carriage is a reality in pregnant women and their neonates with the possibility of congenital transmission. We confirm that there is no V. cholerae carriage in Glenview, Dzivarasekwa and Kuwadzana, areas affected in the 2008 cholera epidemic in Zimbabwe. Moreover, there was no evidence of S. typhi carriage in these suburbs following the devastating 2012 typhoid outbreak. This study reports an expansive pro-inflammatory cytokine and chemokine profile that is up-regulated in lactating mothers with asymptomatic enteric pathogens. HIV exposure had no effect on baby cytokine profiles. The importance of IL-7 in neonates requires further investigation as it might assist in reducing the prevalence of small for gestational age (SGA) babies. Significance and impact of study Our study culminates in the need to consider maternal characteristics such as age, HIV status and enteric pathogen carriage in pregnancy as these might impact on the infants’ immune responses. This will assist in the decisions on disease treatment and management of pregnant women and infants. The study findings also assist in developing effective immunotherapy in E. histolytica and HIV exposures and infections in an effort to promote maternal and child health.
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    Characterizing the role of CD4+ T cell immunoregulatory networks in peripheral blood and lymphoid tissue during HIV-1 clade C infection.
    (2018) Laher, Faatima.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.
    HIV eradication efforts have been unsuccessful due to virus persistence in cellular and tissue reservoirs. Recent evidence suggests that germinal centers (GCs) within lymph nodes (LN) contain a novel subset of regulatory T cells (TREGs), termed follicular regulatory T (TFR) cells. These cells control the magnitude and specificity of the GC response and like TREGs are essential for the maintenance of self-tolerance and immune homeostasis. However, the exact role of TFR cells in HIV infection and their contribution to viral control is not completely understood, possibly due to their low frequency, heterogeneity and more so, the difficulty in accessing human lymphoid tissue samples to fully study them. Thus, we set out to comprehensively investigate TFR cells in LN and peripheral blood (PB) samples, using a multifaceted approach including flow cytometry, MHC class II tetramers, immunofluorescence microscopy (IF), ELISA, digital droplet PCR and singlecell RNA sequencing (SeqWell), in HIV-1 clade C infection. Furthermore, we aimed to determine the effect of very early treatment on the frequency and function of this cell subset. Overall, our studies contributed various notable findings to the field. Firstly, we were able to develop MHC class II tetramers, specific in our HIV-1 clade C setting, as a more sensitive method of identifying very low cell frequency antigen-specific CD4+ T cells without relying on function. Tetramers eliminate the bias associated with in vitro stimulation required for functional assays and the limitation associated with only detecting subsets of cells capable of secreting a cytokine. Notably, we used class II tetramers to demonstrate that HIV-specific CD4+ T cell responses restricted to DRB1*11-Gag41 are associated with immune control of HIV-1 infection. We next focused on understanding the role of CD4+ regulatory cells during HIV-1 infection. Firstly, we showed that TFR cell frequencies were significantly higher in LN compared to PB samples. Secondly, TFR are a phenotypically and transcriptionally distinct subset compared to regulatory T cells (TREGs) and T Follicular Helper cells (TFH). Thirdly, we were able to detect HIV-specific TFR using our newly synthesized MHC class II tetramers, and showed higher frequencies observed in LNs during untreated HIV infection. Fourthly, as measured by both flow cytometry and IF, most of TFR localized outside of the GC, with very early ART initiators displaying larger proportions of TFR within the GC. Lastly, TFR cells exhibited a potential suppressive functional capacity as they produced IL-10, which is a canonical suppressive cytokine and they were also positively associated with gp41 IgG antibodies titers. Overall, the data presented in this thesis highlights the advantage of MHC class II tetramers in evaluating HIV-specific CD4 + T cell responses in natural infections. More so, the results give important insights into regulatory cells within lymph nodes; their biology, function and their role in the setting of very ART initiation.
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    Protective HLA class I alleles : investigation of viral control and lack of control in chronic HIV-1 subtype C infection.
    (2015) Koofhethile, Catherine Kegakilwe.; Ndung'u, Peter Thumbi.; Thobakgale, Christina Fanesa.
    Abstract available in PDF file.
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    Patterns and features of HIV-1 specific CD8+ T-cell responses during acute HIV-1 infection and their association with viral control.
    (2015) Radebe, Mopo.; Ndung'u, Peter Thumbi.
    Evidence suggests that CD8+ T-­‐cells play a major role in the control of HIV-­‐1 viremia and apply significant immune pressure on HIV-­‐1 replication. However, the presence of virus-­‐specific CD8+ T-­‐cells in individuals with varying levels of viral control suggests that CD8+ T-­‐cells may differ in their antiviral function or efficacy. The mechanisms underlying differences in the control of viremia, particularly the reasons why particular individuals experience more effective acute viremia resolution, which is a good correlate of the subsequent rate of disease progression, are still not well understood. In order to uncover some of the features of CD8+ T-­‐cell subsets responsible for the control of HIV replication, particularly during the critical early infection phase, we investigated the patterns and features of HIV-­‐1-­‐specific CD8+ T-­‐ cell responses during acute and primary HIV-­‐1 infection and their association with viral control. We also sought to determine the impact of acute phase immune activation on the acute HIV-­‐1-­‐specific CD8+ T-­‐cell response and on disease progression. We hypothesized that protein-­‐specific and epitope-­‐specific immunodominance patterns during the first 12 weeks of HIV-­‐1 infection are associated with subsequent disease progression. Our data show the presence of HIV-­‐1 specific CD8+ T-­‐cells with limited breadth during acute HIV-­‐1 infection and also demonstrate that the magnitude and breadth of interferon gamma (IFN-­‐γ) ELISPOT assay responses measured within 12 weeks post-­‐infection are unrelated to the course of disease in the first year of infection. During the first weeks of infection Nef protein was most frequently recognized by T-­‐ xv cells and was the target for the earliest response. Although initially subdominant, there was a broadening of the Gag-­‐specific T-­‐cell immune response such that these responses became immunodominant by one year post infection. The broadening and preservation of early Gag–specific T-­‐cell responses during the follow up period was associated with better control of viremia and lower viral load set point. Although many of the acute/early HIV-­‐1-­‐specific IFN-­‐γ enzyme linked immunospot assay (ELISPOT) CD8+ T-­‐cell responses targeting Gag and Pol persisted, the majority of acute and early T-­‐cell responses targeting Env, Nef and other regulatory proteins waxed and waned over time and could not be detected at the last time point evaluated. Some of the early T-­‐cell responses which where no longer detectable when using overnight ELISPOT assay were detectable when PBMCs were stimulated with corresponding peptides and cultured for 10 days before measuring IFN-­‐γ secretion via the ELISPOT assay. The presence of these cultured ELISPOT central memory type T-­‐cell responses targeting epitopes in Pol, Env, Nef, Regulatory and Accessory proteins were not significantly associated with viral set point. However, cultured ELISPOT Gag-­‐specific responses correlated with low plasma viremia, thus further providing evidence for the favourable role of Gag-­‐specific T-­‐cell responses in the control of viral replication. We also show that three cytokines IL-­‐10, IP-­‐10 and IL-­‐ 12 were associated with changes in viral load set point and/or CD4+ T-­‐cell dynamics during the first year of HIV-­‐1 infection. Interestingly, the activation of the PD-­‐1 inhibitory pathway in acute HIV-­‐1 infection was associated with a slower disease progression.