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.

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.

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.

A research report submitted to the Faculty of Health Sciences, School of Public Health, University of the Witwatersrand, Johannesburg In partial fulfillment for the requirement for the degree Master of Public Health 25 August 2014 / BACKGROUND Tuberculosis is the leading opportunistic infection among HIV-infected adults, including pregnant women, globally. Accurate screening tools are needed to identify those requiring further laboratory testing and to initiate isoniazid preventive therapy in a timely manner. This study determined the accuracy of symptom-based screening and in particular the performance of the WHO recommended TB symptom screening algorithm in HIV-infected pregnant women. METHODS A cross-sectional study was conducted among consenting HIV-infected pregnant women attending routine antenatal clinics in Matlosana, South Africa recruited >1 week after first HIV diagnosis between June 2010 and February 2011. Sputum was collected from all women followed by a systematic TB symptom screen. The performances of each symptom (cough, fever, weight loss and night sweats) alone and in combination were assessed with TB confirmed by sputa using microscopy and liquid culture (MGIT), as reference or gold standard. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio were calculated for each of the four symptoms (cough, fever, weight loss and night sweats) and their combination. Logistic regression was carried out to find associations between patient characteristics and TB. RESULTS Overall, Mycobacteria Growth Indicator Tube (MGIT) confirmed prevalence of TB was 2.4% (35/1456) in this sample group. Only 11/38 (29%) women with confirmed TB reported any symptoms. Cough, fever, weight-loss and night sweats, individually and in combination had sensitivities ranging from 2.7-27% and specificities ranging from 84-97%. The positive predictive and negative predictive values for any symptoms of cough, fever, night sweats, or weight loss were 4.2% and 98%, respectively. TB was associated with decreasing CD4 count, close TB contact, cough, and night sweats. DISCUSSION The remarkable number of asymptomatic TB in HIV-infected patients, including in the cohort included in this study highlights the limitation of symptom-based screening. The low sensitivity of the symptom screen would incorrectly stratify patients who are being considered for Isoniazid Preventive Therapy (IPT). However, one could argue that the high negative predictive value of the symptom screen would justify its use in resource-limited settings as the initial step in identifying patients who should receive IPT. Although household TB and the father of the baby having TB were found not to have statistically significant associations with active TB, they are of public health importance as they play a role in the spread of the infection. CONCLUSION The WHO 4-symptom screen had low sensitive among HIV-infected pregnant women but negative predictive value was high. Few women with TB disease reported symptoms on direct questioning; the high rate of subclinical/ asymptomatic TB is concerning. There is an urgent need for more sensitive screening tools for TB in HIV-infected pregnant women