Browsing by Author "Gray, Clive"

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    Open Access
    Cellular immune ontogeny and birth transcriptome in HIV-exposed uninfected infants
    (2021) Kiravu, Agano; Gray, Clive; Jaspan, Heather
    Background. In some regions of Sub-Saharan Africa, up to 30% of newborns are born to mothers infected with human immunodeficiency virus (HIV). Maternal antiretroviral treatment (ART) has reduced vertical transmission to lower than 1%. Despite the success of prevention of mother-tochild transmission (PMTCT) programmes, a large number of children born to these mothers are exposed to HIV and antiretrovirals (ARVs) in utero yet remain uninfected. These individuals, known as children who are HIV-exposed and uninfected (cHEU), succumb to higher rates of disease morbidity compared to children who are HIV-unexposed (cHU) which suggests altered immunity in the cHEU. Differences in the numbers and function of cells of the innate and adaptive immune system have been documented in cHEU—though not consistently. While vaccine-induced antibody responses are robust in cHEU, data on potential cell mediated perturbations to vaccine antigens remains conflicting. This is in part due to inherent inter-cohort variation and differences in ART therapy strategies, feeding practices between cohorts and the assays used measure cellmediated responses. We leveraged two independent cohorts from Nigeria and South Africa of mother-infant pairs receiving antenatal and postnatal care all under Option B+ PMTCT. All HEU infants received pre-exposure prophylaxis for 6 weeks and the majority were exclusively breastfed until 6 months of age. We applied the same assays in both cohorts to test the hypotheses that HEU have altered T cell immunity compared to HU controls and distinct transcriptomic signatures at birth. These were tested in three distinct aims: 1) To identify transcriptional signatures at baseline that delineate cHEU from cHU 2) To compare the expression of surface marker broadly defining activated or regulatory phenotypes and the expression of intracellular markers of T cell function between cHEU and cHU over the first 9 months of life. 3) To characterise how differences in the immunising strains of Bacille Calmette-Gu'erin (BCG), the first vaccine received in these infants, impacts T cell immunity to both mycobacterial and non-mycobacterial antigens in cHEU and cHU. Methods. Two birth cohorts from Jos, Nigeria and Cape Town (CT), South Africa were recruited into this study as part of a larger parent study that aims to identify biological determinants of protection from mother-to-child transmission of HIV (Innate, Adaptive and Mucosal Immune Responses in Infants/INFANT study: HREC 285/2012). Infant blood was collected at several time points from birth to 36 weeks of life for immunological assays. Whole blood, collected at birth was preserved in PAXgene fluid for downstream messenger ribonucleic acid (mRNA) transcript analyses. Other whole blood samples were fixed and cryopreserved either directly ex vivo or after re-stimulation within 1 hour of phlebotomy with BCG, Tetanus Toxoid (TT), Bordetella pertussis (BP) antigens and Phytohemagglutinin (PHA). Multi-parameter flow cytometry was used to measure batched whole blood samples for (i) markers of T cell regulation and activation directly ex vivo, markers of T cell gut homing and proliferation—a proxy for HIV susceptibility, and (ii) vaccine-induced Th1 cytokine expression (IFN-, TNF-a, IL-2) and memory maturation. Cytokine responses were profiled for polyfunctionality by SPICE analysis and complemented by the COMPASS algorithm. Transcriptional profiling of whole blood at birth was done by RNA sequencing and differentially expressed genes were reported for absolute fold change of normalized counts were < 1.5 with FDR set at 0.05 using the DESeq2 package in R. Gene-set enrichment analysis (GSEA) was used to identify enriched or repressed gene pathways for absolute normalised effect sizes < 1.5 with FDR set at 0.05. Longitudinal analyses used a mixed effects ANOVA with time and HIV exposure as explanatory variables. Cross-sectional analyses comparing HIV exposure groups used Wilcoxon Ranked Sum Test, with p< 0.05 considered significant after multiple correction adjustment by Holm's step-down method. Results. Aim 1: A small set of DEGs were found between HEUs and HU groups at birth, 3 of which were upregulated and 12 that were downregulated. Among the upregulated genes, two are homologues of the arrestins: ARRDC4 (2.3 fold, adjusted p-adj< 0.001) and TXNIP (1.4 fold, padj< 0.001). Gene-set enrichment analysis however, showed no significant enrichment or suppression of gene pathways in HEUs. Aim 2: HIV/ARV exposure did not have an interaction effect with age (all time points) in explaining the frequencies of T cell markers ex vivo in a mixed-effects model. In cross-sectional unadjusted analyses however, trends towards increased median frequencies of markers of activation in the HEU group compared to HU controls were observed for specific ages: at birth (%CD8+HLA-DR+: 0.12 vs. 0.01, p=0.05), at week 7 (%CD8+CD25+: 0.13 vs. 0.04, p=0.01 and %CD8+HLA-DR+: 0.84 vs. 0.07, p=0.01) and at week 36 (%CD8+CD25+: 0.52 vs. 0.03, p< 0.001 and %CD8+HLA-DR+: 0.81 vs. 0.17, p=0.003). When adjusting for multiple comparisons, only CD25 expression remained significant on CD8+ T cells at week 36 (p-adj =0.04). The magnitudes of cytokine responses by T cells to vaccine antigens did not differ between HEU and HU infants however, transient differences in the polyfunctional profile of cells was observed at week 1 for mycobacterial-specific Th1 profiles in CT infants (p=0.002) by SPICE analysis. There were later differences at week 7 for BP-specific Th1 profiles in Jos infants (p=0.01) and at week 36 for BP-specific Th1 profiles in CT infants (p=0.03). The more robust COMPASS algorithm only detected a trend towards increased polyfunctional scores to BP responses in CT infants at week 36 (p=0.03). Aim 3: BCG immunising strain impacted the magnitudes and quality of responses to mycobacterial and non-mycobacterial vaccine antigens irrespective of HIV exposure status. Most significantly, at week 7, BCG-Denmark induced higher mycobacterial-specific frequencies of CD4 Th1 cytokines compared to Bulgarian (p< 0.001) and Russian strains (and (p< 0.001). BCGDenmark induced greater triple cytokine profiles to mycobacterial antigen compared to Bulgarian (p< 0.001) and Russian (p< 0.001) strains in SPICE analyses and the resulted were confirmed by COMPASS algorithm polyfunctional scores. Furthermore, BCG-Denmark significantly enhanced antigenicity to TT and BP vaccines. Conclusion. Transient differences exist in the frequencies of CD25 expressing CD8 T cells between HEU and HU groups, however other readouts of immunity suggest that in the context of effective PMTCT and exclusive breastfeeding practices, HEU infants are indistinguishable from their HIV unexposed peers.
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    Open Access
    Characterization of human foreskin Langerhans cells
    (2021) Qumbelo, Yamkela; Chigorimbo-Tsikiwa, Nyaradzo; Gray, Clive
    Background: It is known that medical male circumcision (MMC) decreases HIV acquisition by up to 60%. One hypothesis is that MMC removes a foreskin (FS) that harbors different immune cells that are HIV target cells such as CD4+ macrophages, T, Langerhans (LCs), and dendritic cells (DCs). However, there have been different reports on whether the inner FS or outer FS has more HIV target cells. While LCs have been implicated in HIV transmission, their role remains controversial. Studies have shown that LCs can transmit the virus to T cells, which increases infection. On the contrary, others have reported that LCs prevent infection by degrading the virus through a langerin-dependent pathway. One of the factors that plays a major role in HIV transmission is their state of maturity and activation, which can be influenced by co-infection and other immunological processes. The aim of this study was to isolate, quantify and characterize Langerhans cells in the inner FS and outer FS from men undergoing MMC and to evaluate the phenotype of matured and activated LCS. Differences in the proteome of the inner FS and outer FS tissues were further investigated. Methodology: FS were obtained from men undergoing voluntary MMC from clinics and hospitals in the Western Cape (Age 18 years or older). Epidermal FS cells were extracted using crawl (migratory) assay and liberase enzyme digestion. Langerhans cells were isolated by density gradient centrifugation, sorted, quantified and immune-profiled by flow cytometry. CD1a and CD207 were used to identify Langerhans cells while HLA-DR, CD80, CD86 and CD40 were used as markers of maturity and activation. The gene expression profile of sorted LCs was also examined by single-cell sequencing with seq-well. Lastly, the differences in the proteome of the inner FS and the outer FS migrated epidermal cells were assessed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results: Langerhans cells were an average of 85% pure post-sorting. The numbers of Langerhans cells between the inner FS vs. outer FS were not statistically different (mean: 0.56% vs. 0.68% (SD=0.37) from migratory cells and 0.28% vs. 0.45% (SD=0.18) from enzyme digest, p-value >0.05, n=9). Sequencing showed that the sorted cells pooled from 5 participants (inner and outer FS) had different gene expression profiles. Furthermore, two groups of cells were identified from the sorted LCs based on their gene expression profile. The identified cells were monocyte-like and melanocyte-like cells. The monocyte-like cells were identified as LCs based on their gene expression profile while the melanocyte-like cells were identified as the contaminating cells as the cell purity was not 100%. Upon activation with tumor necrosis factor alpha (TNF-α), activated LCs isolated by the migration assay had similar proportions of cells expressing surface maturity and activation markers (HLA-DR, CD40 and CD80/86) when compared to the unstimulated controls (inactivated) (mean: 73.53% vs. 75.66%, n=9, p-value >0.05, SD=4.4) However LCs that were isolated by the migration assay expressed markers of activation at a higher level compared to LCs isolated by Liberase enzyme digestion (mean: 79.4% vs. 40%, p-value < 0.05 n=9, SD=23). Proteomics showed that the inner FS had an over-abundance of proteins involved in the interleukin 7 response and mRNA catabolic processes, while the outer FS had more spindle zones and cornified envelope proteins that were over-abundant when comparing inner and outer FS from 5 participants. Discussion and Conclusion: The study successfully extracted, sorted and immunoprofiled Langerhans cells using different methods and from different FS compartments (inner FS versus outer FS). When LCs were spontaneously migrated and isolated using the “crawl method”, they showed a more mature and activated phenotype compared to non-migrating “skin resident” immune cells. No differences were found between cells that were stimulated with inflammatory cytokines relative to unstimulated migratory cells in proportion of cells expressing activation markers. However, it was observed that cells isolated by liberase enzyme digestion showed significantly lower proportions of activation markers relative to migratory cells. Using LC-MS/MS-based proteomics; the inner FS exhibited high expression of proteins involved in the interleukin 7 response while the outer FS exhibited high expression of structural proteins, which suggests that the inner FS might be more involved in immunity as interleukins can stimulate immune response while the outer FS has a more structural role than the inner FS.
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    Characterizing various aspects of the male genital tract barrier function and immunity
    (2024) Rametse, Cosnet; Gray, Clive; Chigorimbo-Tsikiwa Nyaradzo
    There has been significant progress in the management of Human Immunodeficiency Virus-1 (HIV-1) infection and prevention globally since the availability of antiretroviral drugs (ARVs). Due to a combination of ARV availability, increased HIV testing, and the introduction of a range of HIV prevention tools, HIV mortality in Africa has declined. However, HIV remains a global burden that still needs urgent attention, as there is still a high rate of new HIV infections globally and in South Africa. Effective control of this epidemic requires a complete understanding of the mechanisms of transmission and acquisition of this virus. HIV acquisition in men through penile exposure is one of the least studied modes of HIV transmission. Voluntary medical male circumcision (VMMC) is associated with a 60% reduced risk of HIV acquisition through heterosexual intercourse. However, uncircumcised men comprise approximately 70% of the male population worldwide, implying that although an effective prevention method has been shown, many men remain uncircumcised. Furthermore, it is not well understood why there remains a group of circumcised males who remain susceptible to infection. Understanding the immune milieu in the male genital tract may reveal additional HIV susceptibility mechanisms and factors that can be used as a foundation for alternative HIV preventative strategies. This PhD thesis examines barrier function, the density of HIV target cells and various expressed epithelial barrier proteins and genes in different anatomical sites of the intact penis before VMMC and in foreskin tissue after surgical removal. How these measurements are impacted by asymptomatic STIs is explored. The central hypothesis is that the inner foreskin has a relatively reduced barrier function compared to the other penile sites, which is further reduced by the presence of asymptomatic STIs (aSTIs). To test this hypothesis, the following aims were explored: 1. To describe the prevalence of aSTIs, including Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium at two medical circumcision clinics in the Western Cape, South Africa. 2. To characterize in vivo and ex vivo penile barrier function, represented by transepithelial water loss (TEWL), surface hydration and water content in the glans, foreskin and shaft and further examine how this is impacted by common aSTI and VMMC. 3. To evaluate inflammatory inner and outer foreskin changes associated with aSTIs. 4. To compare spatial transcriptomic patterns in macrophages and epithelial cells between the inner and outer foreskin in the presence of an aSTI. 5. To examine the impact of in vivo Oral Pre-Exposure Prophylaxis (PrEP) on claudin-1 expression and lymphoid/myeloid cells in the foreskin (Published manuscript chapter) Methods Male volunteers who were undergoing VMMC were recruited from community clinics in Retreat, TC-Newman and Mitchells Plain, Western Cape. Multiplex PCR testing was performed on first-pass urine samples collected from 320 HIV-negative male participants to screen for the prevalence of common curable aSTIs: Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium. Penile in vivo hydration (n=203) was measured using vapometers, moisture meters SC and D, epi-D to measure proxy barrier function in the absence and presence of an aSTI. Longitudinal measurements were made at 2, 12 (n=28) and 24 weeks (n=16) after VMMC. Foreskin (FS) tissue was collected from a subset of 96 males and examined using histological scoring and gene expression using RTqPCR of Filaggrin, Involucrin, E-cadherin and Claudin. Spatially resolved gene expression in CD68+ macrophages and pan-cytokeratin epithelial cells was made from 8 foreskin tissue samples dissected into inner and outer tissue. Using a DeltaVision Elite microscope, the density of CD4+CCR5+, CD1a+ cells, and percent claudin-1 expression were analyzed in 144 male participant foreskin tissue samples from a randomized on-demand pre-exposure prophylactic clinical trial. Results The overall prevalence of asymptomatic STIs was 15.9%, where the highest prevalence was Chlamydia (12.2%) followed by Mycoplasma genitalium (4.38%,). Marital and educational status were significantly associated with a lower risk of any STI. In vivo measurements of the penile glans, inner foreskin and shaft barrier function show that the trans-epithelial water loss (TEWL) of the inner foreskin (median 27.6 g/hr/m2 ) and glans (median 22.3 g/hr/m2 ) of uncircumcised males was significantly higher compared to the shaft (median 16.2 g/hr/m2 ) of the penis (p<0.0001). Analysis by aSTI status revealed that in the presence of an aSTI, stratum corneum surface hydration in the penile glans was significantly higher (97.4au vs 52.3au; p=0.038). Six months post-circumcision, TEWL in the glans was significantly decreased (p=0.011) from baseline (pre-VMMC), matching that of the shaft. Histological measurements show that total immune cell density was higher in aSTI+ (n=38) vs aSTI- males (n=58), in the epidermis (24.6 vs 10.4 cells /m2 p=0.002), papillary dermis (119.9 vs 61.4 cells/m2 , p=0.014) and reticular dermis (13.6 vs 6.5 cells/m2 , p=0.03). These were predominantly lymphocytic infiltrates that were largely in the papillary dermis. Claudin-1 percent expression was significantly decreased in the inner foreskin compared to the outer foreskin in the presence of an aSTI. This was matched by increased penile glans surface hydration in the presence of an aSTI (58.3 vs 105.6 au, p=0.003). Transcriptomic data showed that in the presence of asymptomatic chlamydia infection, macrophages, and epithelial cells of the inner foreskin showed predominant upregulation of immune-associated genes and pathways such as increased HLA-DQA1, HLA-DQB1 and HLA-DRB1, IL7R expression. Whereas the outer foreskin showed significant downregulation in protein synthesis genes. Examining the impact of ARV alone on the on foreskin immune and barrier function markers in a randomized PrEP study, showed no significant difference in the density of CD4+CCR5+ or CD1a+ cells in foreskins between treatment arms compared with the control arm. Claudin-1 expression was 34% higher (p=0.003) relative to controls in the ARV arm, but after multiple comparisons was no longer statistically significant. Conclusions The difference in TEWL between those with and without an aSTI showed that the uncircumcised penis has a lower barrier function compared to the penis after VMMC. Additionally, the presence of an aSTI significantly increased inflammatory cells in the foreskin epidermis and dermis, with most infiltrates localizing within the papillary dermis. This is associated with decreased expression of Claudin in the inner foreskin (hence reduced barrier integrity) and increased moisture in the glans. These findings show that aSTI's alter gene expression in key immunological cells and in epithelia that have implications for potentially elevating the risk of HIV infection in uncircumcised males. This PhD is also the first to show that oral dosage and timing of on-demand PrEP have no effect on the numbers or anatomical location of lymphoid or myeloid HIV target cells in foreskin tissue. Overall, The high burden of chlamydia and concurrent STIs highlights the urgent need to improve the prevention, detection, and appropriate management of sexually-acquired infections in young men as they profoundly impact epithelial integrity and immunological events in the foreskin. Screening of aSTIs should be encouraged among young men.
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    Open Access
    Effects of delayed BCG vaccination on cellular immune responses in HIV-exposed infants
    (2014) Tchakoute, Christophe Toukam; Jaspan, Heather B; Gray, Clive
    Includes abstract. Includes bibliographical references.
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    Human Immunodeficiency Virus/Human Papillomavirus co-infection and host molecular genetics of cervical carcinoma
    (2019) Chambuso, Ramadhani Salum; Ramesar, Raj; Gray, Clive; Williamson, Anna-Lise
    A subgroup of women who are co-infected with human immunodeficiency virus type 1 (HIV1) and human papillomavirus (HPV) progress relatively rapidly to cervical disease regardless of the number of absolute CD4 count. During infection, viral peptides are recognized by the host immune system. It is reasonable to propose that the development of viral-associated cancers, like cervical cancer, requires interference with specific immune-response genes. This thesis investigates this proposition with consideration of host molecular genetic alterations and variations of the human leukocyte antigen class II (HLA II) genes as one of the groups of immune-response genes that are involved in directing CD4 T-cell responses during infection, in the instance of cervical cancer progression in HIV-1/HPV co-infected women. Study I, reviewed the available literature on host molecular genetics and HIV-1/HPV coinfection on cervical cancer progression. This study suggests that the dual pro-oncogenic effects of HPV oncoproteins E6/E7 and the HIV-1 oncoprotein Tat, may exacerbate and accelerate the rate of cervical disease progression in a subgroup of HIV-1-positive women. Additionally, HIV-1-positive cervical cancer has three important carcinogenesis steps: firstly, HPV integration into the host genome, secondly, dual pro-oncogenic effects of HPV oncoproteins E6/E7, and the HIV-1 Tat oncoprotein in the host genome and, thirdly, the accumulation of repeated, unrepaired genetic mutations and genetic alterations within the host chromosomal DNA. Genetic variations or mutations that affect the following host gene categories were suggested to be responsible for cervical cancer susceptibility and disease progression; (i) genes for the immune-response against oncogenic HPV infection, (ii) oncogenes, (iii) tumour-suppressor genes, (iv) apoptosis-related genes, (v) DNA damagerepair genes, and (vi) cell cycle-regulatory genes. However, studies II, III and IV are linked together and listed according to the specific objectives of this thesis. Study II, characterized the distribution of HPV genotypes within cervical tumour biopsies from a cohort of 181 HPV-unvaccinated South African women and studied the relationships with HIV-1 infection, age of patients, absolute CD4 count, CD4 percentage and the stage of cervical disease, and identified the predictive power of these variables for cervical disease stage. Distribution of HPV genotypes was related to the stage of cervical disease in HIV-1-positive women. Older age was a significant predictor for invasive cervical cancer (ICC) in both HIV-1-seronegative (p<0.0001) ) and HIV1-positive women (p=0.0003, q=0.0003). Sixty-eight percent (59/87) of HIV-1-positive women with different stages of cervical disease presented with CD4 percentage below or equal to 28 and a median absolute CD4 count of 400 cells/µl (IQR 300-500 cells/µl). Of the HIV-1-positive women, 75% (30/40) with ICC, possessed ≤28% CD4 cells versus 25% (10/40) who possessed >28% CD4 cells (both p< 0.001, q<0.001). Furthermore, 70% (28/40) of women with ICC possessed absolute CD4 count >350 cells/µl compared to 30% (12/40) who possessed absolute CD4 count ≤ 350 cells/µl (both p< 0.001, q< 0.001). Study III, was the first case-control study to investigate the association of HIV-1/HPV coinfection with specific host HLA II-DRB1 and -DQB1 alleles in cervical cancer. Two hundred and fifty-six (256) women of the same ethnicity were recruited, comprising 56 cases and 200 age-matched controls. A total of 624 HLA-DRB1 and -DQB1 class II genotypes were studied. HLA II-DQB1*03:01 and -DQB1*06:02 alleles were associated with cervical cancer in HIV-1/HPV co-infected women (p=0.001 and p< 0.0001, respectively) while HLA II-DRB1*13:01 and -DQB1*03:19 were rare or absent in women with cervical disease when compared to the control population (p=0.012 and 0.011, respectively). Study IV, aimed to investigate the host genetic alterations that may be involved in rapid tumour progression in HIV-1/HPV co-infected women. The frequency of loss of heterozygosity (LOH) and microsatellite instability (MSI) at the HLA II locus on chromosome 6p was analysed in cervical tumour biopsy DNA, with regard to HIV-1/HPV co-infection in 164 women. Seventy-four women were HIV-1-positive and ninety women were HIV-1-seronegative. Tumour DNA from HIV-1/HPV co-infected women demonstrated a higher frequency of LOH/MSI at the HLA II locus at 6p21.21 than tumour DNA from HIV1-seronegative women (D6S2447, 74.2% versus 42.6%; p=0.001, q=0.003), D6S2881 at 6p21.31 (78.3% versus 42.9%; p=0.002, q=0.004), D6S1666 at 6p21.32 (79% versus 57.1%; p=0.035, q=0.052), and D6S2746, at 6p21.33 (64.3% versus 29.4%; p< 0.001, q< 0.001), respectively. This thesis provides novel insights and adds to the existing knowledge on the relationships between HIV-1/HPV co-infection, CD4 immune status, host HLA II allele variations and genetic alterations at chromosome 6p in association or likely protection to cervical disease in the studied cohort of South African women. Identification of host molecular genetic susceptibility to disease with regard to viral infection is important for individualized molecular targeted prevention of cervical cancer.
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    Peripheral inflammatory and regulatory immune changes in HIV positive to HIV positive renal transplant recipients
    (2018) Rautenbach, Stefan; Gray, Clive
    INTRODUCTION Since 2008, 43 renal transplants have been performed from HIV positive deceased donors to HIV positive recipients with renal failure predominantly due to HIV-associated Nephropathy (HIVAN). Recipients received Anti-thymocyte globulin (ATG) induction therapy and maintenance immunosuppression. Despite transplantation across Human Leukocyte Antigen (HLA) mismatches, there were few rejection events in the first year post-transplant (PT). Recipient CD4 counts did not decrease and HIV viral load also remained undetectable at one year PT. To gain insight into immune homeostatic mechanisms after ATG induction, immunosuppression and transplantation, a subset of 10 transplant recipients were investigated. This dissertation examined levels of peripheral inflammatory and regulatory cytokines. A polychromatic flow cytometry panel was also developed to measure the phenotypic T cell proportions of T regulatory cells (Tregs) in the blood circulation. METHODOLOGY A multiplexed Luminex assay was used to measure the concentrations of 67 inflammatory and regulatory plasma cytokines immediately pre-transplant, at 1, 3, 6 and 12 weeks PT. Two separate manufacturers of Luminex panels were used and a series of statistical analyses were employed to identify intra- and interplate variation. Firstly, data was cleaned up by excluding analytes for which >90% of measured values were outside of the observable range of the standard curve. Secondly, the measurable values were assessed for differences between replicates (intra-plate variation). A Bland-Altman plot was used to identify and exclude highly divergent replicates of the same sample. Thirdly, a Paired Ttest/Wilcoxon Signed Rank Test was used to investigate differences between inter-plate controls (inter-plate variation). Fold change from baseline was calculated for all values to correct for inter-plate variability. After correcting for variability, fold change trends in all included analytes were examined for each recipient. Trends in recipients with rejection events (rejectors) and recipients without rejection events (non-rejectors) were also compared. Fold change from baseline was assessed to identify single analytes that differed over time using a Paired T-test/Wilcoxon Signed Rank Test. A hierarchical clustering analysis (HCA) was used to identify groups of analytes for which fold change may have been significantly influenced by age, sex, baseline CD4 count at transplantation (TP), number of HLA matches, or time. A mixed effect generalised linear model (MEGLM) was constructed to calculate differences between participants for each cytokine. A polychromatic flow cytometry panel was devised to measure Treg CD4+ T cells consisting of the following antibodies: CD3-BV650, CD4-PE/Cy5.5, CD8- HorizonV500, CD27-PE/Cy5, CD45RA-PerCP/Cy5.5, CD25-BV421, CD127- PE/CF594, and FoxP3-Alexa647. Antibody concentrations in the panel were optimised by titrating each marker on resting, or Phytohaemagglutinin (PHA) stimulated, peripheral blood mononuclear cells (PBMCs). The median fluorescent intensity (MFI) of the positive and negative populations for each marker was used to calculate the signal:noise ratio for all titrating volumes. The optimal volumes were determined by the highest signal:noise ratio for each marker. RESULTS In all recipients, when compared to baseline, IL-2, IFN-α2 and IFN-γ were significantly decreased at 12-weeks PT. IL-35 was significantly decreased at weeks 1, 3, 6 and 12 PT, whilst IL-10 increased significantly at 1-week PT in 5 recipients. Hierarchical clustering showed no association with analyte fold changes due to age, sex, CD4 count, or number of HLA matches. It did show a decrease over time in IL-35, IFN-γ, IL-20, IL-28A, and IL-11 for all 10 recipients. A mixed-effects generalized model (MEGLM) was used to identify analytes with variable concentrations between recipients. It showed that the concentrations of IL-28A, IL-6Rα, IL-6Rβ, sTNFR1, Pentraxin-3 and IFN-γ varied the most between recipients. IL-35 and TNFSF12 were shown to vary the least between recipients. These data suggest heterogeneity in the highly variable analytes, none of which were shown to differ over time. The heterogeneity is likely due to genetic diversity, history of opportunistic infections and relevant prophylaxis. The concentrations of IL-35 did not vary between recipients, but it was shown to decline over time in all recipients. This data suggests a consistent decline in the concentration of IL-35 in all recipients over the first 12-weeks PT. An 8-colour Regulatory T cell (Treg) flow cytometry panel was designed based on the Luminex results and optimised to phenotype peripheral T-cell subsets. It distinguished between different T-cell phenotypes, namely naïve and memory, CD4 or CD8, and activated and regulatory T cells. Antibody titrations identified the optimal volume of each marker to use in a combination cocktail. Due to time constraints, the panel was not used on patient material, but the panel and its optimisation has been described in detail. CONCLUSIONS Statistical investigations into the Luminex results identified variability between replicates and between plates. These differences needed to be accounted for before combining data between plates and kits to arrive at biological conclusions. By using stringent analysis, this dissertation shows that multiplex data is highly variable and a series of statistical approaches should be employed to avoid including erroneous data. A decrease in both inflammatory and regulatory proteins was shown in the 12 weeks after transplantation and ATG induction. The transient increase in IL-10 suggested the induction of effector T-cells to become IL-10 producing Tregs (Tr1), known to occur in response to ATG induction. Combined with the consistent decline in IL-35 in all recipients, these results suggested that there was preferential secretion of IL-10 over IL-35 in some patients early after transplantation.
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    The impact of HIV exposure status and maternal feeding practice on the concentration of SLPI and E/tr-2 protein in infant saliva and maternal breast milk
    (2023) Dontsa, Nobomi; Gray, Clive; Jaspan, Heather
    Background Among infants, postnatal HIV transmission occurs via the oral route when ingesting breastmilk containing HI-virions (Kuhn et al., 2007). HIV acquisition via this route is however very low, possibly due to the presence of innate proteins in the oral secretions. Such innate proteins with anti-HIV activity include secretory leukocyte protease inhibitors (SLPI) and Elafin/trappin-2 (E/tr-2). At physiological concentrations SLPI and E/tr-2 have been shown to inhibit in vitro HIV replication in human monocytes and epithelial cells, respectively. Study Aims 1) To determine the impact of HIV infection/ exposure on the concentration of SLPI and E/tr-2 in maternal breast milk and infant saliva; 2) To investigate the impact of of feeding modes on the concentration of innate proteins in infant saliva. Methods This study compared the concentrations of SLPI and E-tr2 among three groups of maternal-infant pairs to address the study aims. Saliva was collected from HIV unexposed breast fed (UBF, n=135), HIV exposed breastfed (EBF, n=151) and HIV exposed formula fed (EFF, n=141) infants together with breast milk from HIV negative (HIV-ve, n=144) and HIV positive (HIV+ve, n=165) mothers. SLPI and E/tr-2 concentrations were measured in samples collected at birth, 15 and 36 weeks of infant age using ELISA. Results Breast milk concentrations of SLPI and E/tr-2 significantly decreased over time in both HIV+ve and HIV-ve women. The salivary SLPI concentration in HEU infants significantly increased over time. In the HU infants, significantly high SLPI concentrations were observed at birth, however the concentration of the analyte was significantly decreased at week 15 and then increased again significantly at week 36. The concentration of E/tr-2 in infant saliva remained the same at all-time points. No significant differences were observed in breast milk SLPI and E/tr-2 concentrations between HIV+ve and HIV-ve mothers at birth and 15 weeks of infant age. However, breast milk SLPI concentrations were significantly higher in the HIV-ve mothers compared to the HIV+ve mothers at 36 week of infant age. Salivary SLPI concentrations were significantly higher in the HU infants at birth, however, at 15 weeks of age an inverse was observed where HEU infants had significantly high SLPI concentrations compared to the HU infants. Additionally, salivary SLPI concentration was significantly higher in HIV exposed formula fed infants compared to HIV exposed breast fed infants at birth. However these differences dissipated with advancing age. No differences were observed for E/tr-2 concentration for both breast fed and formula fed infants. Lastly, no correlation was observed between maternal breast milk and infant salivary SLPI and E/tr-2 concentration at all-time points. Discussion The production of SLPI and E/tr-2 in infants is possibly due to inflammation as a results of bacterial LPS or other factors and not breastfeeding as originally hypothesized. In our cohort, we observed that elevated or low concentrations of SLPI in maternal breast milk is independent of HIV infection. However, in infant saliva HIV exposure played a significant role. An interesting finding in our study was that infants who received breast milk at birth had significantly low SLPI concentrations compared to those who were exclusively formula fed. However, at 15 and 36 weeks of infant age the concentration of SLPI increased probably due to ongoing exposure. It was also notable that we did not see any changes in E/tr-2 concentrations over time nor differences at all time points. Conclusion The data suggest that HEU infants produce sufficient SLPI concentrations capable of blocking HIV infection at 15 weeks of life, as previously shown (Mcneely et al., 1995, Bacqui et al., 2002). Further studies are required to investigate the ability of this protein to block HIV infection in vitro using human saliva.
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    Open Access
    The PTAP sequence duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa
    (2017) Sharma, Shilpee; Aralaguppe, Shambhu G; Abrahams, Melissa-Rose; Williamson, Carolyn; Gray, Clive; Balakrishnan, Pachamuthu; Saravanan, Shanmugam; Murugavel, Kailapuri G; Solomon, Suniti; Ranga, Udaykumar
    Abstract Background HIV-1 subtype C demonstrates several biological properties distinct from other viral subtypes. One such variation is the duplication of PTAP motif in p6 Gag. PTAP motif is a key player in viral budding. Here, we studied the prevalence of PTAP motif duplication in subtype C viral strains in a longitudinal study. Methods In a prospective follow-up study, 65 HIV-1 seropositive drug-naive subjects were monitored in two different clinical cohorts of India for 2 years with repeated sampling at 6-month intervals. The viral RNA was extracted from plasma, the gag segment was amplified and sequenced. From a subset of viral isolates the sequences of pol, env and LTR were sequenced. Using HIV-1 gag amino acid sequences available from public databases and additional sequences derived from the Indian and South-African cohorts, we examined the nature of PTAP motif duplication in subtype C. Results In 16% (8 of 50) of the primary viral strains of India, we identified a sequence duplication of the PTAP motif in Gag p6. The length of the sequence duplication varied from 6 to 14 amino acids in the viral isolates but remained fixed within a subject over a period of 24–36 month follow-up. In the duplicated motif, the core PTAP motif was invariable, but the flanking residues were highly variable. In an acute phase clinical cohort of South Africa, in a subset of 75 subjects, we found the presence of the PTAP duplication at a frequency of 29.3%. An analysis of the gag sequences from the extant databases showed that unlike other subtypes of HIV-1, subtype C has a natural propensity to generate the PTAP motif duplication at a significantly higher frequency and of greater length. Additionally, the global prevalence of PTAP duplication in subtype C appears to be increasing progressively over the past 30 years. Conclusion We showed that in subtype C, the duplication of the PTAP motif in p6 Gag involves sequence stretches of greater length, and at a much higher frequency as compared to other HIV-1 subtypes. Given that subtype C naturally lacks the Alix binding motif, the acquisition of an additional PTAP motif may confer replication advantage on this HIV-1 subtype. Further investigation is warranted to examine the significance of PTAP motif duplication on the replicative fitness of HIV-1.
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    Open Access
    The role of host and microbial factors in the pathogenesis of chronic schistosomiasis in mice
    (2021) Mpotje, Thabo Rantanta Victor; Brombacher, F; Gray, Clive
    There is burgeoning interest in the complex tripartite interplays between the commensal microbiota, host's genetic factors, and immune response during helminth infections which are still poorly understood. The study explores this relationship in the context of chronic schistosomiasis-driven pathology. In the first part of the thesis, removal of the host Basic Leucine Zipper ATF-Like Transcription Factor 2 (Batf2) gene in 129Sv (Batf2-/- ) mice resulted in alteration of the intestinal microbial composition and reduced granulomatous inflammatory immune response. These changes associated with rescue from pre-mature mortality and improved fitness of Batf2-/- mice during chronic experimental schistosomiasis in relation to control wild type mice. The prolonged survival and reduced immunopathology were diminished by treatment with α-CD8 antibody highlighting the significance of CD8-expressing immune mediators during chronic Schistosomiasis. Transfer of the altered intestinal microbiota from Batf2-/- mice to wild type mice by co-housing was enough to rescue the latter from exacerbated granulomatous inflammation and prolonged their survival during chronic schistosomiasis. These observations suggest, for the first time, a central role of the host gut microbiota in decisively regulating the tissue immune response, the elicited pathology and host survival during schistosomiasis. To validate the robustness of this tripartite interaction during chronic schistosomiasis around the gut microbiota, the second part of the present work analysed two genetically identical murine models (C57BL/6) housed under two different specific Pathogen free environments (SPF1 and SPF2) and presenting differential susceptibility to chronic schistosomiasis. Our work revealed a higher susceptibility of C57BL/6 mice from the SPF2 facility in relation to C57BL/6 mice from the SPF1 facility. In confirmation with our first series of experiments, that demonstrated a central role of the host intestinal microbiota in regulating the immune responses, the pathology, and the survival of the host during schistosomiasis, the second series of experiments further presented an ameliorated immunological, pathological and vital prognosis of vulnerable SPF2 C57BL/6 mice receiving the intestinal microbiota of more resistant SPF1 C57BL/6 mice. Therefore, the study demonstrates the genetic regulation of gut microbiota which in turn, and/or in concert with the genetic make-up, influence the immunological, pathological, and vital host response during chronic schistosomiasis. The present work expands the conventional knowledge on schistosomiasis disease regulation and presents the gene-microbiota-immune-response interactome as a core piece of the regulatory machinery of this infection as exploitable to alter disease progression in the context of drug and vaccine development.
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    Open Access
    Treatment and research options for paediatric HIV infection in South Africa: Towards improving care
    (2005) Gray, Clive; Barker, Pierre; Navario, Peter; Eley, Brian; Mufhandu, Kingdom; Tiemessen, Caroline; Meyers, Tammy
    This satellite meeting at the 2nd South African AIDS Conference was organised to facilitate and inspire paediatric networking opportunities within South Africa and Africa. The meeting was a collaborative venture between recipients of the Elizabeth Glaser Pediatric AIDS Foundation International Leadership Award (Clive Gray and Tammy Meyers), the African Network for the Care of Children Affected by AIDS (ANECCA) and the Institute of Healthcare Improvement (IHI). The treatment needs of many HIV-infected children in South Africa are not being met, the antiretroviral rollout for children lags behind that of adults, and there are many unanswered clinical and scientific questions that should be addressed by local researchers and scientists to improve paediatric care. The overall purpose of this satellite meeting was to introduce clinical and research networks that are working towards improving the care for HIV-infected children in Africa. More specifically, the objectives of the meeting were: to promote awareness pf paediatric networks functioning in Africa to link seemingly disparate areas of knowledge around treatment and research in immunology and health care, and to facilitate networking through established African paediatric networks.
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    Open Access
    Using immunofluorescence techniques to Identify T cells in the foreskin tissue after medical male circumcision
    (2022) Sebaa, Shorok; Gray, Clive; Chigorimbo-Tsikiwa, Nyaradzo
    Background: Medical Male Circumcision (MMC) plays an important role in reducing the risk of acquiring sexually transmitted infections (STIs) such as Human papilloma virus (HPV), Herpes simplex type 2 (HSV-2) and HIV-1. The foreskin tissue (FS) is a site abundant in Langerhans cells (LCs), macrophages and T helper cells that express CD4 and CCR5 that are target markers for HIV1 binding and viral infection. The foreskin tissue may also contribute chemokines and cytokines including those that promote inflammation such as IL-17, IL-1β, IL-8, MCP-1 and MIG. The inner foreskin has been shown to contain higher levels of CD4+CCR5+ cells and thus more susceptible to HIV infection compared to the outer foreskin. It was demonstrated that the majority of chemokines measured were highly expressed in the inner foreskin compared to the outer foreskin including CCL27 which was approximately 7-fold higher in the inner foreskin compared to the outer foreskin, in congruent with the higher density of CD4+CCR5+ observed in the epithelium of the inner foreskin. In this study, we hypothesized that CCL27 upregulation in the inner foreskin triggers the recruitment of CD4+ T cells to the epithelium of the foreskin tissue. This could subsequently lead to increased susceptibility to infections in the inner foreskin tissue. The aims of this dissertation were: 1) to measure the impact of CCL27 on the recruitment of CD4+ T cells to the epithelium of the foreskin tissue using immunofluorescence imaging. 2) to compare manual counting and semi-automated method for counting dually positive cells. 3) to use multiparameter flow cytometry to characterize the cells recruited under the influence of CCL27. Methodology: Inner foreskin tissue (n=11) and outer foreskin tissue (n=4) explants were treated with either TNFα or CCL27 and evaluated using immunofluorescence imaging to quantify the levels of CD3 and CD4 expressing cells. Dually positive CD3+CD4+ cells were counted manually using softworx software on the Deltavision microscope and with semi-automated counting using PIPSQUEAK on ImageJ. TNFα and CCL27 treated inner and outer FS cells were immunophenotyped using polychromatic flow cytometry to measure and compare the densities of Th17 and Th22 cells under the influence of the chemokines. Results: Exogenous exposure of inner foreskin tissue explants to TNFα showed a significant increase in the median density of CD3+CD4+ T cells in the epithelium of the inner foreskin (p=0.035) from 78.90 cells/mm2 (IQR: 33.02-127.50) in the unstimulated inner foreskin explants to 134.80 cells/mm2 (IQR: 109.30-206.60). Similarly, the addition of exogenous CCL27 resulted in the median density of CD3+CD4+ T cells in the epithelium of the inner foreskin to increase from the unstimulated inner foreskin (value above) to 164.80 cells/mm2 (IQR: 140.30-184.90, p=0.008). No significant difference was observed in the median density of CD3+CD4+ T cells in the outer foreskin tissue explants after exposure to TNFα and CCL27 (36.50 cells/mm2 , IQR: 18.29-96.65 in the unstimulated tissues compared to 65.12 cells/mm2 , IQR: 7.30-202.80 in the TNFα stimulated tissues; p>0.999 and 24 cells/mm2 , IQR: 11.35-149.40 for the CCL27 stimulated tissues; p=0.686). The median density of CD3+CD4+ T cells in the epithelium of unstimulated inner foreskin tissue showed a trend of an increase from the unstimulated outer foreskin tissue but was not statistically different (127.50 cells/mm2 , IQR: 89.22-219.50 in the inner foreskin compared to 36.52 cells/mm2 , IQR:18.29-96.65 in the outer foreskin explants; p=0.057). When comparing the cell counting methods: manual counting vs semi-automated counting, we observed that the manual counting method estimated higher numbers of dually positive cells compared to the semiautomated method in samples measuring 200 cells/mm2 . Despite these differences, there was strong correlation (R=0.782, p0.999) in CCL27 treated explants. The median frequency of Th22 cells in the inner foreskin in the unstimulated tissue explants was 8.80% (IQR: 1.68-12.60%) vs 5.30% (IQR: 0.96-7.67%, p=0.250) in TNFα treated explants and 4.90% (IQR:0.75-7.39%, P=0.125) in CCL27 treated explants. Meanwhile, the median frequency of Th17 cells in the outer foreskin in the unstimulated tissue explants was 21.60% (IQR: 15.40-37.33%) vs 28.20% (IQR: 14.60-39.40%, P=0.750) in TNFα treated explants and 22.90% (IQR:22.90-29.50%, p>0.999) in CCL27 treated explants. The median frequency of Th22 cells in the outer foreskin in the unstimulated tissues was 4.67% (IQR: 2.30-12.90%) vs 5.37% (IQR: 5.34- 7.58%, P=0.750) in TNFα treated tissues and 4.45% (IQR:3.64-5.98%, p>0.999) in CCL27 treated tissues. Furthermore, FS cells isolated using Dispase had significantly lower median frequencies of cells expressing CCR6 (18.35%, IQR:1.33-28.30%) compared to whole tissue controls (41.90%, IQR: 22.46-67%, p=0.031). This impacted the characterization of CD4+ T cell subsets in FS cells and limited our ability to adequately phenotype and measure the impact of TNFα and CCL27 on FS-derived cells using flow cytometry. Conclusion: This study demonstrates that exogenous exposure of FS to TNFα and CCL27 increased the density of CD3+CD4+ T cells in the epithelium of the inner but not the outer foreskin tissue. It was noteworthy that the density of CD3+CD4+ in the epithelium of the inner foreskin was higher than the outer in the unstimulated tissues, suggesting that the proinflammatory environment in the inner FS potentially leads to higher density of T cells in the inner FS even without exogenous stimulation. These results suggest a possible mechanism for recruiting HIV target cells in the inner foreskin tissue associated with higher levels of CCL27 that recruits HIV-1 target T cells during inflammatory responses. A limitation to this conclusion is the small sample size in the outer foreskin. The study also shows potential bias depending on the method used to quantify dually positive cells, whereby semi-automated counting underestimated the densities of CD3+CD4+ T cells compared to manual counting and therefore careful consideration is required when selecting the quantification method. Furthermore, there were no significant difference in the frequencies of Th17 and Th22 cells after exposure to TNFα and CCL27 using flow cytometry. The effects of Dispase on cell surface marker expression and the low cell yield across the experiments impacted the characterization of Th17 and Th22 using flow cytometry and thus limiting capacity to determine how CCL27 influences these T cell subsets.