Thesis (Ph.D.)--University of the Witwatersrand, Faculty of Health Sciences, 2012. / The development of an effective HIV-1 vaccine is critical in mitigating the global HIV epidemic. Understanding the interplay between host immune functions, such as cellular memory differentiation, activation, inflammatory cytokine production and the virus, may provide key insight into anti-HIV immunity that can inform vaccine development. This PhD aims at understanding and identifying T cell memory, functional profiles and the effect of immune activation on in vivo HIV-1 control during primary/early infection. Furthermore, this study aims to examine and understand the potential mechanisms related to immune activation during primary HIV-1 infection. Use was made of a unique cohort of individuals recruited during primary HIV-1 infection and using a battery of assays to characterize and identify properties and mechanisms of T cell reactivity and activation. Multiparameter flow cytometry was used to measure memory differentiation (CD27 and CD45RO), activation (CD38, HLA-DR), proliferation (Ki67), and multiple cellular functions (CD107, IFNγ, IL-2, MIP-1β and TNFα) of total and antigen-specific CD4+ and CD8+ T cells from 15 HIV-1 and CMV-coinfected individuals followed over 15 months of HIV-1 infection. Plasma samples were used to measure markers associated with intestinal permeability (LBP, sCD14, I-FABP and IgM EndoCAb) and inflammation (IL-1β, IL-6, IL-7, IL-10, IL-12p70, TNFα and MCP-1). The differentiation profile of HIV-Gag specific memory CD4+ and CD8+ T cells was found to be mainly characterized by an early differentiated (ED) memory phenotype relative to CMV- specific CD4+ and CD8+ T cells. Moreover, the proportion of HIV-specific ED-memory CD4+ T cells inversely associated with viraemia, suggesting that HIV-1 antigen burden could be shaping the differentiation of HIV-specific memory CD4+ T cells during primary infection. Primary HIV-1 infection was also characterized by significantly elevated levels of activated and proliferating total and HIV-specific memory CD4+ and CD8+ T cells, which positively correlated with viraemia. Furthermore, upon sorting of total activated memory CD4+ T cells, these cells harboured more gag provirus DNA than non-activated memory cells, suggesting that activated memory CD4+ T cells support ongoing HIV-1 replication. When examining the relationship between memory differentiation and activation markers, the level of T cell activation was equally expanded across the different memory CD4+ T cell subpopulations, suggesting that memory differentiation of CD4+ T cells was unlikely driven per se by the level of T cell activation. In addition, when teasing out events that may result in T cell activation during primary HIV-1 infection using statistical models, plasma markers of microbial translocation and inflammation were found to correlate with immune activation. The lack of these associations in HIV-uninfected controls suggests that microbial translocation and inflammation were unlikely causative. Analysis of the polyfunctional profile of memory T cells during primary HIV-1 infection showed that HIV-specific CD4+ and CD8+ T cell responses are less polyfunctional relative to CMV-specific memory CD4+ and CD8+ T cell responses. Furthermore, the polyfunctional status of HIV-specific CD4+ T cells significantly correlated with viraemia at 3 months post-infection, indicating that the polyfunctionality of memory CD4+ T cells is likely driven by HIV-1 antigenemia. Overall, these observations suggest that HIV-1 antigenic burden appears to be a central driver of memory differentiation, activation/inflammation and polyfunctionality of T cells. Given the impact of HIV-1 viraemia on immune activation and memory T cell dysfunction (as measured by limited polyfunctional HIV-specific responses), preventing high levels of viral replication, with a vaccine or other early interventions may serve as an important strategy for delaying HIV-1 disease progression.

A Thesis Submitted to the School of Public Health, Faculty of Health Sciences University of the Witwatersrand, Johannesburg, South Africa, in fulfilment of the requirements for the Degree of Doctor of Philosophy 15 June 2017 / Background HIV infection has remained a major global health burden since its discovery in 1983 and Sub-Saharan Africa remains the region hardest hit by the HIV/AIDS pandemic. The HIV pandemic continues to ravage most parts of Southern African countries, current prevalence between 10-20%. Individuals worldwide differ in their degree of susceptibility to HIV infection and genetic polymorphisms play a major role. Mannose Binding Lectin (MBL) is one such immunological factor found in serum/plasma, it is a normal liver-derived protein and is a key component of the innate immune defence system. MBL deficiency, due to mutations in the MBL2 gene and promoter region, leading to decreased plasma/serum MBL concentration, characterised by defective opsonisation activities of the innate immune system and increased susceptibility to infections including HIV-1 and schistosomiasis. Rationale While there is a lot of advancement in HIV prevention and treatment in Southern African countries, there is still need to investigate host genetic molecules in adults and mother-baby pairs that could be playing a role in HIV-1 transmission/acquisition, disease progression and survival. It was imperative to carry out this study because of the need to quantify the burden of MBL deficiency in this Zimbabwean adult and PMTCT study populations. Alsoto contribute to the knowledge gap on the role of MBL deficiency in HIV-1 transmission, disease progression and survival in African populations in adults and children. The available literature shows that the majority of studies on the association of MBL deficiency and HIV-1 infection in adults and children have been done on populations outside the African continent. There is dearth of information on the role of MBL in this era when access to ART has greatly improved even in developing countries like Zimbabwe. This will be the second study that will assess MBL2 genes and promoter typing in mother-infant pairs in HIV vertical transmission/acquisition. This study aimed to identify and explore potential biomarkers for susceptibility to HIV infection and disease progression. We assessed role of MBL deficiency in HIV-1 and schistosoma infections in Zimbabwean adults enrolled in the Mupfure Schistosomiasis and HIV Cohort (MUSH Cohort) (Paper 1).We also assessed the role of MBL deficiency on HIV progression and survival in this African adult population. We hypothesized that MBL deficiency has a role to play in HIV infection by increasing HIV disease progression and decreasing survival (Paper 2). We also determined prevalence of MBL deficiency, as estimated by MBL2 haplotypes among Zimbabwean mothers and their children aged 9-18 months old as well as its association with risk of HIV-1 infection and vertical transmission from their HIV positive mothers (Paper 3). Main Aim The broad objective of this study was to determine the relationship between MBL deficiency and HIV infection in an adult population of males and females and among mother-infant pairs in Zimbabwe. Study Specific Objectives 1. To determine the prevalence of MBL deficiency among the Zimbabwean adult population. 2. To determine the relationship of MBL deficiency with HIV infection among the Zimbabwean adult population. 3. To determine the effect of MBL deficiency on disease progression and survival among the Zimbabwean adult population. 4. To determine prevalence of MBL deficiency among mothers and their infants in a Zimbabwean population. 5. To determine the relationship between MBL deficiency and HIV transmission from mother to child in a Zimbabwean population. Methods DNA and plasma samples for MBL and HIV analysis were collected from the 379 adult males and females from the MUSH cohort and stored dried blood samples from 622 mother infant pairs from a national PMTCT survey. HIV-1, S. haematobium and S. mansoni infections were determined at baseline using HIV commercial kits and parasitologically respectively. Plasma MBL concentration was measured by ELISA and MBL2 genotypes determined by PCR. We calculated and compared the proportions of plasma MBL deficiency, MBL2 structural variant alleles B (codon 54A>G), C (codon 57A>G), and D (codon 52T>C) as well as MBL2 promoter variants -550(H/L), -221(X/Y) and +4(P/Q) between HIV-1 and schistosoma co-infection and control groups using Chi Square test (Paper 1). We also assessed the role of MBL deficiency on HIV disease progression and survival inthe adult (MUSH) cohort.We analysed blood samples for MBL levels, MBL2 genotypes, HIV-1 status, viral load and CD4+ T cell counts (Paper 1). Participants were followed up for 3 years wherein the endpoints were measured at baseline, 6 weeks, 3, 6, 12, 24 and 36 months. Disease progression was measured as the rate of decline in CD4+ T cell counts and the rate of increase in HIV viral load (Paper 2). Generalised Estimating Equations (GEE) models were used to compare rates of change of the CD4+ T cell count and viral load measurements over the three-year follow-up period. The role of plasma MBL deficiency and MBL2 genetic variants on survival over the 3-year period were estimated using the Cox proportional hazard models. Regression analysis was used to test for interaction and confounding between MBL deficiency, MBL2 genetic variance, age and sex. We used the Wald Chi-square statistic to choose between full and nested models. We also assessed MBL2 polymorphisms in Zimbabwean HIV positive mothers and their children enrolled in a national PMTCT survey carried out in 2012. MBL deficiency was defined as presence of A/O and O/O genotypes in the mothers and their children. We extracted DNA from two dried blood spots for 622 mothers and infant pairs using the Gene Extract and Amp kit reagents. MBL2 Exon 1 genotypes and promoter region alleles -221(X/Y) and -550(H/L) SNP were detected by pyrosequencing. Differences in distribution frequency between HIV infected and uninfected children, of the MBL2 genotypes, promoter region variants and MBL2 haplotypes, were determined by the Chi square test or Fisher’s exact tests (Paper 3). Key findings For specific objective number 1, we assessed 379 adults, 80% females, median age (IQR) 30 (17-41) years. HIV-1, S. haematobium and S. mansoni prevalence were 26%, 43% and 18% respectively in the MUSH baseline survey. Median (IQR) plasma MBL concentration was 800μg/L (192-1936μg/L). Prevalence of plasma MBL deficiency was 18% with high frequency of the C (codon 57G>A) mutant allele (20%). For specific objective number 2, we found no significant difference in median plasma MBL levels between HIV negative (912μg/L) and HIV positive (688μg/L), p=0.066. However plasma MBL levels at the assay detection limit of 20μg/L were more frequent among the HIV-1 infected (p=0.007). S. haematobium andS. mansoni infected participants had significantly higher MBL levels than uninfected. All MBL2 variants were not associated with HIV-1 infection but promoter variants LY and LL were significantly associated with S. haematobium infection (Paper 1). For specific objective number 3, we assessed 197 HIV positive adults where 83% (164) were women with a median age of 31 years old. Prevalence of plasma MBL deficiency (less than 100μg/L) and MBL2 deficient genetic variants (A/O and O/O genotypes) was 21% (42 out of 197) and 39% (74 out of 190), respectively. We did not observe a significant role to explain individual variation in mortality, change of CD4+ T cell count and viral load by MBL plasma deficiency or MBL2 genetic variants from baseline to 3 years follow up period in this adult population (Paper 2). For specific objective number 4, from the PMTCT study, the median age (IQR) of the mothers was 30(26 - 34) years and the children mean age (IQR) was 12 (11-15) months old at the time of enrolment. All 622 mothers were HIV-1 infected, 574 babies were HIV negative and 48 were HIV-1 positive babies. MBL2 normal structural allele A and variants B (codon 5A>G), C (codon 57 A>G) and promoter region SNPs -550(H/L) and -221(X/Y) were detected. Prevalence of MBL deficiency was 34% among the mothers and 32% among the children. For specific objective number 5, we found no association between maternal MBL2 deficiency and HIV-1 transmission to their children. We found no difference in the distribution of HIV-1 infected and uninfected children between the MBL2 genotypes of the mothers and those of the children (Paper 3). Conclusions The results from our study indicate high prevalence of MBL deficiency but we found no evidence of association between MBL deficiency and HIV-1 infection. However, lower plasma MBL levels were associated with reduced prevalence of both S. haematobium and S. mansoni infections and MBL2 promoter and variants LY and LL were associated with increased susceptibility to S. haematobium infection (Paper 1). Our findings attest to the large between-population variability in a host of factors that can predispose individuals susceptible to HIV progression and mortality. We therefore cannot recommend at this time the use of plasma MBL levels or MBL2 genetic variants as a prognostic marker in HIV infection, disease progression and survival in this adult population in Africa (Paper 2). MBL deficiency was not associated with HIV-1 infection among the children nor was it associated with HIV-1 vertical transmission in this study population (Paper 3). / MT2017

Highly active antiretroviral therapy has successfully limited HIV-1 disease progression to AIDS, but is consistently compromised by the emergence and transmission of HIV-1 drug resistant strains. As a result, a continued search for novel anti-HIV-1 agents with improved pharmacological profiles has become fundamental. Chrysotherapy has been in use since the early 1920s in treatment of rheumatoid arthritis, and has since been investigated for various ailments including HIV/AIDS. This study evaluated 45 synthetic gold compounds for drug like properties using theoretical and experimental techniques with the aim of generating sufficient data to considerably aid the rational design of new anti-HIV agents. Theoretical techniques applied included the Osiris Property Explorer and the Lipinski’s Rule of Five which assessed drug-likeness and bioavailability respectively. In vitro studies included aqueous solubility assays, cytotoxicity (PM1 cell lines and PBMCs) assays, antiviral assays in PBMCs, direct enzyme (RT and IN) inhibition, and the effect of serum protein binding and biological stability on antiviral efficacy. An overall low druglikeness score and an intermediate bioavailability were predicted by the Osiris Molecular Property Explorer. Low drug-likeness was suggested to be due to a high frequency of foreign fragments in the synthetic gold compounds, while their high molecular weight reduced bioavailability. In general gold compounds exhibited cytotoxicity properties and moderate aqueous solubility in vitro. Overall, the 45 synthetic gold compounds did not show activity against HIV-1 replication in vitro. Seven compounds (AB05-AB11) exhibited direct HIV-1 RT inhibition, and compounds AB39 and AB04 demonstrated moderate direct HIV-1 IN inhibition, but this activity was abrogated in PBMC inhibition assays. Serum binding, compound stability and cytotoxicity were all implicated in the lack of HIV-1 inhibition in PBMCs. To this end, data obtained was sufficient to aid in the future rational design of second generation HIV RT and IN inhibitors with acceptable pharmacological properties.

ABSTRACT Incidence estimation and calibration from cross-sectional data of acute infection HIV-1 seroconvertors. May 2007 Eustasius Musenge Masters in Medicine in the Field of Biostatistics and Epidemiology Supervised by: Mr E Marinda and Dr A Welte Background: The HIV-1 incidence (a very important measure used as a proxy for disease burden) can be estimated from a cross-sectional study. This incidence estimate has the advantage of reducing on costs and time, thus enabling more timely intervention; it is also ideal for developing nations. A common procedure used in making this estimate utilizes two antibody tests (Sensitive/Less sensitive tests). Due to the long window period of such tests (at least three months), persons classified as recently infected would have been infected more than three months prior to the test date. Detecting acute HIV-1 infection is very important since this is the most infectious stage of the disease. This research report explores a method of estimating incidence using an antibody test and a virological test, Polymerase Chain Reaction Ribonucleic Acid (PCR-RNA).The cross-sectional data used are from the Centre for the AIDS Programme of Research in South Africa (CAPRISA). Methods: Actual follow-up cohort data from CAPRISA acute infection cohort (AIC), comprised of 245 sex workers, were used to estimate the incidence of HIV-1 using a PCR-RNA ,virology test based, incidence formula. The result obtained was compared to the incidence estimate obtained by the classical method of estimating incidence the AIDS Programme of Research in South Africa (CAPRISA). Methods: Actual follow-up cohort data from CAPRISA acute infection cohort (AIC), comprised of 245 sex workers, were used to estimate the incidence of HIV-1 using a PCR-RNA ,virology test based, incidence formula. The result obtained was compared to the incidence estimate obtained by the classical method of estimating incidence (prospective cohort follow-up). As a measure to reduce costs inherent in virological tests (PCR-RNA), multistage pooling was discussed and several pooling strategies simulations were proposed with their uncertainties. Point estimates and interval estimates of the window period, window period prevalence and incidence from crosssectional study of the AIC cohort were computed. Findings: The mean window period was 6.6 days 95% CI: (2.7 – 13.0). The monthly window period prevalence was 0.09423 percent 95 % CI: (0.0193 – 0.1865)%. The incidence from the prospective cohort follow-up was 5.43 percent 95% CI: (3.9 – 9.2) %. The incidence estimate from cross-sectional formulae was 5.21 percent 95% CI: (4.1– 4.6). It was also shown by use of simulations that an optimum pool sample size is obtained when at least half the samples are removed on every run. Interpretation and recommendations: The PCR-RNA test is very sensitive at detecting acute HIV-1 infected persons. The incidence estimate from the crosssectional study formulae was very similar to that obtained from a follow-up study. The number of tests needed can be reduced and a good estimate of the incidence can still be obtained. The calibration was not accurate since the samples used were small and the window period duration was too short, hence, it was difficult to extrapolate to the whole population. Further work still needs to be done on the calibration of the proposed incidence formulae as it could be a very useful public health tool.

M.Sc. (Med.), Faculty of Health Sciences, University of the Witwatersrand, 2011 / Human Immunodeficiency Virus type 1 (HIV-1) is the causal agent of AIDS, and currently infects 33.3 million individuals worldwide. The gradual depletion of CD4+ T lymphocytes is a characteristic feature of a progressive HIV-1 infection. During HIV-1 infection the number of infected CD4+ T lymphocytes is fewer than the number of CD4+ T lymphocytes that are depleted, therefore suggesting a role for HIV-mediated apoptosis of uninfected bystander CD4+ T lymphocytes. Apoptosis, also known as programmed cell death, is a highly regulated, normal physiological process that results in the disposal of unwanted or corrupted cells such as virally infected cells. Several HIV-1 proteins are involved in HIV-1-mediated apoptosis, of which the envelope (Env) glycoprotein gp120 subunit is the most effective. However, the true mechanism of gp120-mediated apoptosis of uninfected CD4+ T lymphocytes is not fully understood and remains controversial. Furthermore, research focusing on HIV-1 subtype C Env-mediated apoptosis is limited. This study compared the ability of CCR5-utilizing soluble monomeric HIV-1 subtype C gp120 (gp120ZM651), monomeric HIV-1 subtype B gp120 (gp120BaL) and trimeric/oligomeric HIV-1 subtype C gp140 (gp140AncC) to induce apoptosis of uninfected Jurkat T lymphocytes and activated CD4+ T lymphocytes via CD4 and CD4/CCR5 signalling, respectively. All three Env glycoproteins were expressed in HEK 293T cells, purified by lectin affinity chromatography and characterized by assessing their binding capabilities to monoclonal antibodies IgGb12, 17b (in the presence and absence of soluble CD4) and 2G12. Purified recombinant gp120BaL, gp120ZM651 and gp140AncC were found to v be functional and conformationally intact and subsequently added in different concentrations to uninfected Jurkat T lymphocytes and activated CD4+ T lymphocytes. Apoptosis was detected by flow cytometry using Annexin V/7-AAD staining for up to 48 hours post treatment, and further confirmed by TUNEL analysis at 65 hours post treatment. Recombinant gp120BaL, gp120ZM651 and gp140AncC induced low levels of apoptosis in the Jurkat T lymphocytes (6.1%, 5.0% and 6.8%, respectively) and higher levels of apoptosis in the activated CD4+ T lymphocytes (13.3%, 15.6% and 11.5%, respectively) via TUNEL analysis of chromosomal DNA fragmentation. Moreover, comparable levels of apoptosis were observed between the monomeric gp120 and trimeric/oligomeric gp140 forms in both cell types. Interestingly, the subtype C gp140AncC induced higher levels of apoptosis than subtype C gp120ZM651 and subtype B gp120BaL in activated CD4+ T lymphocytes during the Annexin V/7-AAD analysis while the subtype C gp120ZM651 induced higher levels of apoptosis than subtype C gp140AncC and subtype B gp120BaL in activated CD4+ T lymphocytes during the TUNEL analysis. Overall, these results suggest that soluble gp120 is able to mediate low levels of apoptosis via CD4 signalling only in Jurkat T lymphocytes, and these levels are enhanced in activated CD4+ T lymphocytes, possibly due to engagement of gp120 with CD4 and the CCR5 co-receptor on the surface of the target cell.

Background: A novel, isothermal nucleic acid amplification method, RT-LAMP, presents potential for nucleic acid amplification-based diagnostics in resource-limited settings. Low-cost HIV-1 viral load monitoring will improve access to ART for HIV-1-infected individuals present in settings where on-site viral load testing is unavailable. Aim: The aim of this dissertation was to develop an RT-LAMP HIV-1 viral load assay by combining the RT-LAMP reaction with colorimetric amplification detection by hydroxy-naphthol blue dye. Methods: Different approaches for HIV RNA extraction from patient plasma and culture supernatant were studied to obtain template for RT-LAMP. Reaction products for 4 different RT-LAMP primer sets were analysed using agarose gel electrophoresis and restriction digestion. Results: The first 3 primers sets produced persistent off-target amplification. The fourth primer set, designed against culture supernatant DU179, produced a target-specific colour change from violet to blue after 1 hour, following optimisation of amounts of Mg2SO4 and AMV RT. Further studies showed HNB detection sensitivity to template copy number. Conclusions: Initial reaction conditions pertaining to an RT-LAMP based, colorimetric HIV-1 viral load assay were established. Further work is required to determine the reaction duration at which the colour change represents a viral load of ≥1000 copies HIV RNA per ml plasma.

HIV-associated sensory neuropathy (HIV-SN) is one of the most common neurological problems of HIV. It is frequently painful and reduces quality of life. HIV-SN can be caused both by HIV itself and by exposure to neurotoxic antiretrovirals such as stavudine. The South African Department of Health now recommends use of tenofovir in place of stavudine as first line treatment. However many people remain on stavudine and or live with the side effects. Stavudine is still prescribed in many other resource-poor countries. This thesis presents the first systematic study of clinical and genetic risk factors for the development of symptomatic HIV-SN in Black Southern Africans. I recruited 404 Black HIV-positive Africans from the Virology Clinic of the Charlotte Maxeke Academic Hospital, Johannesburg and assessed HIV-SN using the AIDS Clinical Trials Group (ACTG) Brief Peripheral Neuropathy Screen. HIV-SN was defined as present if the patient had both symptoms and signs of peripheral neuropathy. If present, the distribution and intensity of symptoms were recorded. Of those exposed to stavudine, 57% (226/395) had HIV-SN. Pain was the most common symptom and was experienced by 74% (172/226). Of these, 76% (128/172) reported their pain as moderate to severe. As in previous studies, increasing age and height were independently associated with risk of HIV-SN. However nadir and current CD4 T-cell counts and sex were not associated with SN. Patients donated blood for DNA extraction and single nucleotide polymorphisms (SNPs) were selected from the literature and genotyped using Illumina Golden GateTM technology. 342 individuals were assessed for genetic associations with HIV-SN and a subset of 159 positive for HIV-SN were assessed for associations with painful HIV-SN. I completed four genetic analyses: SNPs and haplotypes from TNF and adjacent genes from the major histocompatability complex on chromosome six were assessed for association with HIV-SN. I found no association with TNF-1031, even though this had associated with risk of HIV-SN in Caucasian, Chinese and Malay cohorts. Novel associations were identified between HIV-SN protection and 5 other SNPs (BAT1 rs3130059, rs2523504; ATP6V1G2 rs2071594; NFKBIL1 rs2071592, rs2071591). Associations were also found with haplotypes: FV15-23 weakly associated with risk and FV30-31 associated with protection against HIV-SN in this cohort. Analysis of 8 SNPs not previously assessed produced two novel associations with LTA SNPs (rs1041981, rs909253), where the minor alleles conferred protection against HIV-SN. Analysis of linkage disequilibrium (LD) suggests that there is linkage disequilibrium within the TNF block, that it differs between ethnicities and that TNF-1031 is unlikely to be a causative SNP for risk of HIV-SN. SNPs from other cytokines and chemokines implicated in the pathogenesis of HIVSN and the associated pain were assessed in Chapter 5. The major allele of the antiinflammatory gene IL4 (rs2243250) associated with risk of HIV-SN. This allele has been associated with higher CD4 T-cell counts, so I have proposed a role for high IL- 4 in early stage HIV-SN. A 3-SNP haplotype of IL10 associated with protection against HIV-SN whilst another IL10 haplotype showed a trend for risk of painful HIVSN. These data and the involvement of TNF haplotype (Chapter 4) suggest an inflammatory etiology for HIV-SN. Polymorphisms of UCP2 (rs659366) and UCP3 (rs1800849) have previously associated with risk of diabetic neuropathy. These SNPs encode uncoupling proteins 2 and 3 which regulate reactive oxygen species and may affect development of neuropathy via the effects of oxidative stress and mitochondrial dysfunction. Alleles of these SNPs did not associate with HIV-SN in this cohort. Patterns of linkage disequilibrium may differ between the two ethnicities or UCP2 and UCP3 may associate with a mechanism particular to diabetic neuropathy. I also assessed a ‘pain protective haplotype’ and SNPs of GCH1 which have been associated with decreased pain intensity in radicular pain following lumbar discectomy. Associations of the 3-SNP ‘pain protective’ haplotype (rs10483639*C, rs3783641*A and rs8007267*T) and a 6-SNP haplotype containing this motif with protection against pain were significant but dependent on age, sex and CD4 T-cell count. Association of another 3-SNP haplotype (rs10483639*G, rs3783641*T and rs8007267*C) with increased risk of pain in HIV-SN was also not independent of age, sex and CD4 T-cell count. The weaker associations here compared to Caucasian cohorts may be a result of differing LD between ethnicities or demonstrate different pain mechanisms between HIV-SN and radicular pain following lumbar discectomy. My results highlight the prevalence of HIV-SN and frequency of pain in this Southern African cohort. The genetic studies identify a likely inflammatory component and identify genes worthy of further investigation both in HIV-SN and the associated pain.

Degree of Doctor of Philosophy in Medicine by research only A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree of Doctor of Philosophy in Medicine. Johannesburg, 2014 / Introduction Patients with HIV infection frequently encounter oral candidiasis, caused by Candida species. However, factors responsible for Candida colonisation and development of oral candidiasis in these patients are controversial. This study investigated the effect of social and clinical factors on oral Candida colonisation in HIV positive women. In addition, virulence of these organisms during clinical infection, the role of non-albicans Candida and reinfections with C. albicans were investigated.

Introduction: Migrant and mobile seasonal farm workers face multiple challenges in preventing sexual transmission of HIV. They also fall beyond direct reach of district health promotion services and national HIV prevention communication interventions. HIV prevalence rates in rural farming communities are significantly higher than provincial averages. An integrated health promotion intervention was initiated in 2005 on commercial farms in Hoedspruit, Limpopo province, through the International Organization for Migration. In terms of HIV prevention the Hlokomela project’s key innovation was to employ a local process of participatory communication, with and within the farm worker community, in order to create a local context enabling of health promotion and within which efforts to prevent HIV could be more effective. The research sought to explore the social processes and actions related to the on-going process of dialogue at the core of the participatory communication process. The objective was to describe and analyse the role of dialogue during regular purposive face to face interactions with farm worker change agents, in promoting health and addressing vulnerability to HIV. Method: The study population comprised Hlokomela coordinators, farm worker change agents (Nompilos and Gingirikani) and key farm stakeholders from the 59 partner farms. Research was conducted in Hoedspruit, at the Hlokomela Wellness Centre and on a partner farm. A grounded theory approach was used for sampling: participants were selected through purposive sampling for the initial study sample, and theoretical sampling for the balance. Data was gathered monthly, in three stages between August and November 2010, through: 10 semi-structured in-depth individual interviews; 5 focus group discussions, and observation of 2 monthly meetings and a special event organised by the change agents. Data was analysed using a grounded theory approach. Findings: Farm workers perceive and experience the process of on-going dialogue in face to face interactions as being intertwined with other aspects of the intervention, in particular identification and action to enable access to health services. Hlokomela Coordinators guide and support the process as a means to empower a corps of primary farm worker Change Agents (Nompilos). Nompilos, in turn, apply the system to benefit and empower a wider group of farm worker as second level change agents (Gingirikani). Through this system farm workers have found ways to negotiate HIV-related stigma and cultural taboos on speaking about sex, and to address interpersonal tensions and violence, often gender related, on farms. They have come to consider themselves leaders and role models. Individuals have been enabled to define for themselves appropriate HIV-protective behaviours, and new HIV protective social norms which enable protective behaviours, have gained local currency. These norms include placing value on the opportunity and ability to communicate, to learn from each other, to develop different views, and to attain or protect family, physical and spiritual wellness. Discussion: The process of engagement and regular dialogue, nested in processes related to the other elements of the projects, has positively altered the material, experiential and symbolic context on partner farms. It constitutes effective communication for social change, and has enabled health promotion, as described by the Ottawa Charter, to be realised. This demonstrates that an on-going, participatory process of local communication can create an enabling environment for health promotion. A community of communication practice has been developed in the farming community; this constitutes a reservoir of social capital and capacity to communicate and addresses the need for innovative communication in rural settings. A discursive space and public of discourse around wellness and HIV has been created, and new leaders and alternative narratives, which constitute self and collectively defined “AIDS competency” in a marginalised setting, are becoming visible, suggesting pathways for future interventions to enable equivalent responses in similar settings. Conclusion: An opportunity exists to make more effective use of the power of face to face communication in defined local settings, in order to enable disempowered individuals to claim their human and health rights, to protect themselves from HIV, and to help activate and realise synergies in health and development objectives such as the Millennium Development Goals.

Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg, 2012 / Introduction: Most new HIV-1 infections world-wide are caused by subtype C viruses. The C3V4 region, including the alpha2-helix and V4 loop, has been identified as a major target for autologous neutralising antibodies in subtype C infections. Factors associated with the immunogenicity of this region, and the mechanisms of escape from anti-C3V4 responses have not been described, although charge changes in the alpha2-helix have been proposed to mediate neutralisation escape. Methods: Seventeen HIV-1 subtype C infected individuals were classified as C3V4 responders or nonresponders using chimeric viruses in env-pseudotyped neutralisation assays. Longitudinal sequences obtained from C3V4 responders were used to identify putative neutralisation escape mutations. The role of these mutations in mediating escape was investigated using site-directed mutagenesis. Results: The C3V4 region was confirmed as a major target in HIV-1 subtype C infections. The development of an anti-C3V4 response was associated with shorter V4 loops and fewer potential N-linked glycans (PNGs) in the C3V4 region. Anti-C3V4 responses were associated with higher autologous neutralising titres. Neutralisation escape from an anti-C3V4 response was rarely mediated by charge changes in the alpha2-helix and generally occurred through mutations in other structurally proximal regions of the envelope. This study confirmed the use of glycan shuffling as a predominant escape pathway. In three individuals multiple mechanisms of escape were identified and in two other cases escape mutations within the C3V4 and structurally proximal regions clustered at opposite termini of the alpha2-helix, inconsistent with the surface area of a single epitope. Conclusion: A more exposed and accessible C3V4 region was more likely to elicit an anti-C3V4 response. The highly immunogenic nature of this region may contribute to the higher overall neutralisation titres in subtype C infections. Distinct clusters of mutations may suggest the existence of two “sub-epitopes” within the C3V4 domain that warrant further investigation. These findings emphasise the adaptability and plasticity of the C3V4 region in the context of viral evasion of host defences.