Browsing by Author "Koor, Gemma Whitney"

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    Exploring the interplay of chemokine receptors ccr5 and cxcr6 in mechanisms of natural control in HIV-1- infected black South Africans
    (2024) Koor, Gemma Whitney
    In sub-Saharan Africa, HIV-1 is a significant cause of morbidity and mortality. However, research remains primarily focused on North American and European population groups, who have remarkably different genetic backgrounds to individuals from sub-Saharan Africa. HIV1 controllers represent a model of HIV-1 functional cure, with some individuals able to control viral replication, and some able to sustain immune function in the presence of high viral loads, both in the absence of antiretroviral therapy (ART). The chemokine receptors CCR5 and CXCR4 are the major coreceptors HIV-1 utilises to enter cells. The use of alternative coreceptors, such as the CXCR6 coreceptor, is thought to contribute to the lower pathogenicity exhibited by the HIV-2 and SIVsmm strains. Building on previous work conducted in our research unit on these two coreceptors in South African populations, this thesis firstly describes CCR5 genetic variants that associate with HIV-1 control or risk of progressive infection in black South Africans, and then explores constitutive expression levels of CCR5 and CXCR6 on various peripheral blood immune cell subsets in the absence of HIV-1 infection in ethnically divergent population groups. The effect of sex, age, and select CCR5 and CXCR6 single nucleotide polymorphisms (SNPs) on expression levels of these two receptors was also investigated. The CCR5 5’UTR and 3’UTR regions were PCR-amplified and sequenced from genomic DNA extracted from 145 ART-naive black South African individuals living with HIV-1 (71 HIV-1 controllers – 23 elite controllers, 37 viraemic controllers, 11 high viral load long-term nonprogressors and 74 progressors). Findings confirmed results from other studies in showing that the CCR5 HHE haplotype is deleterious for HIV-1 disease progression, and the HHA haplotype and HHA/HHC genotype associated with protection from HIV-1 disease progression. Novel haplotypes were characterised, both in the 3’UTR and spanning the CCR5 5’UTR and 3’UTR. Overall, findings suggest that two CCR5 promoter SNPs (-2459 G>A and -2135 T>C) and one CCR5 3’UTR SNP (+2919 T>G) may be key functional variants with regards to HIV-1 control in black South Africans. To gain further insight into the constitutive expression of CCR5 and CXCR6 on peripheral blood immune cells and explore the relationship between select genetic variants and expression, immunophenotyping by flow cytometry was conducted using whole blood from age- and sex-matched ethnically distinct South African HIV-uninfected individuals (17 black, 21 white). Expression levels of CCR5 and CXCR6 were assessed on CD4+ and CD8+ T cells, B cells, monocytes and NK cells, and their respective subsets. The effects of age and sex on expression levels of these two receptors was also investigated. Population-specific differences with regards to CCR5 expression on all cell types, except for B cells, were evident. Generally, black South Africans exhibited a lower expression level of CCR5 compared to white South Africans. CXCR6 expression only differed with regards to percentage of CXCR6-expressing cells, not CXCR6 density (numbers of cell surface receptors). Black individuals had a lower percentage of CXCR6-expressing CD8+ T cell subsets (naïve and effector memory) and a higher percentage of CXCR6-expressing CD14+CD16+ monocytes compared to white individuals. Overall, we found significant population-specific differences in expression levels of both CCR5 and CXCR6, multiple associations with cell activation (as measured by HLADR expression) and CCR5 and CXCR6 expression, and CCR5 and CXCR6 expression was positively significantly correlated on multiple cell subsets. Furthermore, both sex and age influenced CCR5 and CXCR6 expression, however results varied widely across the two population groups studied. Sex differences were only evident in white individuals; predominantly CXCR6 expression was increased in males compared to females. Age associations with CCR5 and CXCR6 expression were also primarily found in white individuals. Four CCR5-related SNPs that are associated with HIV-1 control in this or other studies (rs553615728 -4223 C>T SNP, rs1799987 −2459 G>A SNP, rs746492 +2919 T>G SNP and rs1015164 G>A SNP) were assessed for their potential association with CCR5 expression levels. The +2919 TG genotype significantly associated with a higher percentage of CCR5- expressing total CD8+ T cells, transitional memory and terminally differentiated CD8+ T cells compared to the GG genotype. The +2919 GG genotype associated with a lower percentage of CCR5-expressing B cells compared to the TT and TG+TT genotypes, however, only in white South Africans. The +2919 TG and TG+TT genotypes associated with significantly higher CCR5 density on all CD8+ T cell subsets, except for naïve CD8+ T cells, when compared to the GG genotype. When evaluating two CXCR6 genetic variants previously associated with HIV-1 viraemic control (rs2234355 G>A and rs2234358 G>T) in relation to CXCR6 expression, possession of the rs2234355 SNP GA genotype associated with lower CXCR6 expression on select CD4+ and CD8+ T cell subsets as well as on B cells, while possession of the rs2234358 SNP TT genotype associated with higher CXCR6 expression on multiple cell types, primarily in white South Africans. Possession of the -358TT/+355GA genotype combination associated with lower CXCR6 expression on select subsets of CD4+ T cells and monocytes. In summary, this study provides information on genetic variation in the CCR5 gene in a South African context, describes genetic variants associating with HIV-1 control in black South Africans, adds novel insight into constitutive CCR5 and CXCR6 expression levels on CD4+ and CD8+ T cells, B cells, monocytes and NK cells in HIV-1-uninfected black and white South Africans, and describes the potential associations of select genetic variants and expression. Black and white individuals differed in their baseline expression levels of CCR5 or CXCR6, which was partly driven by host genetic factors that were explored. This work highlights the importance of considering effects of ethnicity, age, and sex in any studies addressing any immune molecules in relation to differential HIV-1 outcomes of infection susceptibility/protection, disease progression, or HIV-1 virological control on antiretroviral therapy. Although conducted on small numbers of individuals, these variables clearly influenced constitutive expression of CCR5 and CXCR6, and further population-specific studies are warranted to gain further insights. Findings from this study have implications for risk of acquisition of HIV-1 infection and for disease progression in people living with HIV-1. Understanding the role of these molecules is important for informing strategies for both HIV1 prevention and HIV cure.
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    Genetic variation and differential expression of p21 (WAF1/CIP1) in the context of HIV-1 control
    (2016) Koor, Gemma Whitney
    A recent study has shown variable p21 expression levels linked to individuals displaying different levels of HIV-1 control, with elite controllers (ECs) and viraemic controllers (VCs) exhibiting higher p21 expression when compared to both healthy HIV-1 negative individuals and HIV-1-infected progressors. The role of p21 in HIV-1 control in a sub-Saharan African population has not been established. Polymorphisms in the regulatory regions of p21, as well as in the microRNAs (miRNAs) that affect p21 regulation can contribute to differential p21 expression. In this study we developed real-time PCR assays to genotype the p21 exonic rs1801270 and 3‘UTR rs1059234 SNPs, in addition to the p21-associated miRNA (miR-106b) rs999885 SNP. We determined their allelic and genotypic frequencies in Black South African HIV-1 negative individuals (n=72), HIV-1 controllers (HICs) (n=52) further subdivided into ECs (n=11), VCs (n=30) and high viral load long term non-progressors (HVL LTNPs) (n=11), and HIV-1 infected progressors (n=74). We sequenced a region of the p21 5‘UTR and 3‘UTR in a subset of these individuals (HICs: n=52, progressors: n=44) to identify variants that may be modulating p21 expression. We compared levels of p21 mRNA, a marker for p21 expression, in a smaller group of individuals (n=50) with similar clinical phenotypes to determine if p21 upregulation was associated with natural control of HIV-1. Lastly, we developed a real-time PCR assay to genotype a p21 5‘UTR SNP, rs733590, that alone, and together with HLA-B*2705, was recently shown to directly impact on p21 expression in Caucasians. This SNP was genotyped and analysed in the individuals with p21 mRNA expression data. The p21 rs1801270 and rs1059234 SNPs were found to occur in partial linkage disequilibrium (LD) (r2=0.61). Although ECs had markedly less representation of the 3‘UTR rs1059234 mutant allele (T) and heterozygosity (CT) compared to progressors (T allele: 9.1% ECs vs. 25% progressors; CT genotype: 18.2% ECs vs. 42% progressors), this did not reach significance (p=0.11, OR=3.33; p=0.19, OR=3.49, respectively). Interestingly, HIV-1 controllers with <400 HIV-1 RNA copies/ml (<400 HICs) also had less representation of the CT genotype when compared to progressors (20% vs. 42%, respectively; p=0.11, OR=2.91). In silico analysis of this 3‘UTR SNP suggested that there are functional implications in terms of miRNA regulation, however when p21 mRNA expression was analysed with respect to this SNP, no effect was seen. The role of this 3‘UTR SNP on p21 expression and/or function and HIV-1 control requires further investigation. The p21 exonic rs1801270 SNP showed no difference in representation among the clinical phenotypic groups and no effect was seen on p21 mRNA expression. When comparing HIV-1 controllers with >400 HIV-1 RNA copies/ml (>400 HICs) to progressors, the >400 HICs had significantly lower representation of the minor allele (A) of the miR-106b rs999885 SNP (p=0.04, OR=2.28). In addition, heterozygosity for this SNP (GA) was found in a much lower frequency in >400 HICs when compared to progressors (p=0.05; OR=2.56). Stratification of individuals according to their miR-106b rs999885 SNP genotype and p21 mRNA expression revealed the GA genotype to be associated with a trend to higher p21 mRNA expression (p=0.066). A role for the miR-106b rs999885 SNP in HIV-1 control in individuals with higher viraemia needs to be validated in larger cohorts. Characterisation of the p21 regulatory regions, namely a region of the 5‘UTR and the 3‘UTR, identified 19 polymorphisms (18 SNPs and one indel) and 12 SNPs in the respective regions. A prevalent, previously uncharacterised 11-SNP haplotype (LD: r2=1) was detected in the p21 promoter region at a frequency of 39.42% in the HIV-1 controllers and 48.86% in the progressor cohort. In addition, a 2-SNP haplotype was identifed and was found to be in moderate LD with the 11-SNP haplotype (r2=0.67). The ECs were found to have a trend of less representation of the 2-SNP haplotype minor allele when compared to progressors (p=0.08, OR=2.83). Other than the rs1059234 SNP, no other SNPs in the 3‘UTR were differentially represented in any of our studied groups. p21 mRNA expression analysis showed significant correlations between p21 mRNA expression and markers of disease progression (HIV-1 viral load: r=0.69, p<0.0001 and CD4+ T cell count: r=-0.53, p=0.0005). In our study, ECs and VCs had significantly lower p21 mRNA expression compared to progressors (p=0.002 and p=0.001, respectively). Furthermore, in our Black South African population (n=50), the p21 5‘UTR rs733590 SNP CT and TT genotypes were not associated with higher p21 mRNA expression as has been shown in Caucasians. This, together with the absence of HLA-B*2705 in our Black South African population, points to host genetic differences as the likely contributors to the different results seen in our study with respect to p21 expression and HIV-1 control when compared to reported literature. Future work with larger sample sizes and varied population groups will be highly informative in determining the role of p21 and natural control of HIV-1 in the Black South African population.