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Introduction: Interferon regulatory factor-7 (IRF-7), the “master regulator” of type 1 interferon, has shown to orchestrate anti-viral immune responses via fine-tuning expression of interferons and interferon-stimulated genes. Methods: IRF-7 levels were examined using multi-parametric flow-cytometry in HIV-uninfected Manitoban donors and in HIV-infected and HIV-uninfected Kenyan volunteers from a well-characterized Kenyan sex worker cohort. IRF-7 expression level was reduced by IRF-7 specific siRNA or shRNA encoded in lentivirus and administered into ex-vivo CD4+ T cells by transfection or transduction. Results: In unstimulated PBMC, IRF-7 was constitutively expressed at low levels in every defined subset we examined. We observed less HIV-infected cells (~10%) with IRF-7 knockdown, suggesting that IRF-7 may play a role in HIV infection. Conclusions: Unexpectedly, it was found that even though IRF-7 had been implicated in orchestrating antiviral events, reducing IRF-7 expression in ex vivo CD4+ T cells did not increase the cellular susceptibility to productive HIV infection. / February 2017

HIV-1, the virus that causes AIDS, is spreading at an alarming rate. Subtype C, which accounts for approximately 50% of infections worldwide, and 98% of infections in Southern Africa, is by far the most prevalent form of the virus. Most molecular and biochemical studies have been performed on HIV-1 subtype B isolates and products, however, and there is a relative scarcity of corresponding data on subtype C. It is therefore of crucial importance to study subtype C HIV-1 strains in order to understand their characteristic pathogenic effects and to develop effective treatment strategies. The aim of research in our laboratory is the development of novel treatment strategies, with particular focus on identifying novel Subtype C Env-binding peptide ligands. This necessitates the development of reagents for use in the discovery and testing of these compounds. In line with this, the aim of this project was the production and characterisation of recombinant Subtype C gp120s generated from a recently compiled HIV-1 virus cohort. To this end, the gp160-coding regions of 20 South African Subtype C HIV-1 strains isolated from AIDS patients presenting at the Johannesburg General hospital in 2005 were amplified by PCR and sequenced. The gp160 amplicons were used to amplify and clone the gp120-encoding regions of these isolates. Two clones, pTriEx- FV3 and pTriEx-FV5, originating from CXCR4- and CCR5-utilising strains respectively, were selected for further use. These clones were cotransfected into insect cells together with a baculoviral DNA backbone in order to generate gp120-expressing baculoviruses by homologous recombination. Recombinant baculoviruses were used to infect Sf9 insect cell cultures for expression of recombinant gp120, which was then purified using a combination of lectin affinity chromatography and ion exchange chromatography. In order to determine the functionality and conformational integrity of the recombinant gp120, the ability of these purified gp120s to bind CD4 and a panel of well-characterised monoclonal antibodies against various epitopes on gp120 (F425 A1g8, 2G12, F425 B4a1, F425 B4e8, 48d, 17b, IgG1 b12, 5F7, 4G10, 9301, ID6, Chessie 13-39.1, 654-30D and 670-30D) was assessed. Gp120 from the CXCR4-using isolate, FV3, appeared to have an intact, functional CD4 binding site as measured by its ability to bind to CD4 and the CD4 binding site antibody 654-30D. It showed low binding to the monoclonal antibody 654- 30D, moderate binding to 2G12, Chessie 13-39.1 and 9301, and high binding to ID6, but did not show binding to any of the other antibodies used in the recognition profile. Gp120 from the CCR5-using isolate, FV5, showed low binding to the monoclonal antibodies F425 B4a1 and Chessie 13-39.1, moderate binding to 2G12, and showed good binding to 9301and ID6. FV5 gp120 could not, however, bind to CD4. This is likely to be related to a D368G substitution, a mutation affecting a critical structural determinant of CD4 binding. The lack of CD4-binding activity of this gp120 highlights the importance of Asp368 for CD4 binding and hints at a region vulnerable for therapeutic targeting. Our results also highlight the challenges of developing broadly therapeutic drugs for HIV-1, as well as the importance of investigating the specific biochemical and pathogenic properties associated with subtype C HIV-1.

MSc (Med), Virology, Faculty of Health Sciences, University of the Witwatersrand / Introduction High resolution melting analysis (HRMA) accurately, rapidly and cost effectively detects single nucleotide polymorphisms by monitoring DNA dissociation kinetics. This technology was applied to HIV samples to assess whether it could be used to detect clinically relevant drug resistance mutations. Methods HRMA-PCR assays incorporating unlabeled probes were designed to genotype 12 mutation codons in the HIV-1 p66/p51 of engineered plasmids and 116 HIV-1 samples. Results HRMA correctly genotyped 63%-88% of the K103N, Y181C, M184V, Q151M and G190A mutations. Each assay had a 1.7%-3.4% discordance, most of which was due to the increased analytical sensitivity of HRMA (~5-20%). Only mutant K65R and V106M were correctly identified while the 41, 67, 70, 215 and 225 codons could not be genotyped. Assay modifications had some success in masking the affects of polymorphisms. Conclusion These assays can be used for genotyping selected major HIV-1 resistance mutations and should be further developed as a resistance surveillance tool.

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

Low affinity Fcγ receptors (FcγR) mediate key immune effector mechanisms through the engagement of the Fc portion of immunoglobulin G (IgG). These receptors are involved in multiple biological processes, including clearance of antigen/antibody immune complexes, enhancement of antigen presentation, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, regulation of antibody production, and activation of inflammatory cells. FcγR phenotypic variability modulates these processes through altering receptor IgG subclass binding affinity (FcγRIIa-H131R and FcγRIIIa-F158V), subcellular localization (FcγRIIb-I232T), post-translational modification (FcγRIIIb-HNA1a/b/c), expression of an otherwise pseudogene (FcγRIIc), and receptor surface density (gene copy number variability and promoter haplotypes). Accumulating data suggest that FcγR-mediated effector functions play a significant role in HIV-1 protective immunity, which is substantiated by the association of FcγR phenotypic variants with HIV-1 disease outcome. This study set out to characterize FcγR functional variability in the South African population, and to investigate the potential role thereof in HIV-1 transmission and disease progression in South African Black individuals. Since the only known determinant of FcγRIIIa surface density – FCGR3A gene copy number – is rare, this study investigated novel genetic determinants of FcγRIIIa expression by flow cytometry and nucleotide sequencing. FcγRIIIa expression on peripheral blood mononuclear cells was characterized for 32 South African Caucasian individuals and 22 South African Black individuals (Chapter 3). Significant differences in the proportion of FcγRIIIa-positive monocytes and FcγRIIIa expression levels on natural killer (NK) cells were observed between the population groups. A novel four-variant FCGR3A intragenic haplotype that associated with increased surface expression of FcγRIIIa on NK cells was detectable in Caucasian individuals, but not Black individuals and may account for the observed population differences. Further exploration of genetic diversity at the low affinity FCGR gene locus was extended to include all currently known functional variants of FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb using a commercial multiplex ligation-dependent probe amplification assay (Chapter 4). Thirty-two South African Caucasian individuals and 131 South African Black

M.Sc. (Nursing), Faculty of Health Sciences, University of the Witwatersrand, 2011

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