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.

ABSTRACT Since its discovery as the etiological agent of AIDS in 1983, HIV-1 has been the focus of unrelenting research into an effective vaccine to control viral infection. Neutralizing antibodies constitute a correlate of immune protection for most available vaccines, but the induction of these antibodies against HIV-1 has become a major challenge. The HIV-1 envelope glycoprotein has evolved to evade neutralizing antibodies in an extraordinary way, yet a vaccine that can stimulate such antibodies remains the best hope to provide sterilizing immunity. The existence of a group of monoclonal antibodies, such as IgG1b12, 2G12, 2F5 and 4E10, capable of neutralizing a broad range of primary isolates signals vulnerable areas on the envelope glycoprotein. Furthermore, passive transfer of these antibodies can completely protect against viral challenge in animal models. The epitopes recognized by these antibodies are being intensely pursued as vaccine targets, in the hope of inducing such specificities. This thesis encompasses a series of studies on characterizing the epitopes recognized by these broadly cross-reactive monoclonal antibodies in the context of subtype C viruses. HIV-1 subtype C is responsible for the vast majority of infections worldwide, however, until recently, little research has been done on these viruses in contrast to the well characterized subtype B strains. Chapter Two describes the characterization of paediatric subtype C viruses for their sensitivity to IgG1b12, 2G12, 2F5 and 4E10. This study was done because of a planned clinical trial of some of these antibodies as post-exposure prophylaxis to prevent mother-to-child HIV-1 subtype C transmission. Only the MAb 4E10 was able to neutralize all the viruses tested, while IgG1b12 was only partially effective. 2F5 and 2G12 did not neutralize any of the viruses. The conclusion was that only 4E10 and IgG1b12 would be suitable for use as prophylactic agents in a population where HIV-1 subtype C is prevalent. Given that subtype C viruses were found to be largely insensitive to 2G12 neutralization, the commonly absent glycan at iv position 295 was introduced into envelope glycoproteins from this clade. The The work presented in Chapter Three explores the requirements of the 2G12 epitope on the envelopes of subtype C viruses. However, this antibody binding site was not readily reconstituted, suggesting structural differences from other HIV-1 subtypes in which the 2G12 epitope is naturally expressed. Chapter Four describes the study of 4E10 resistant virus quasispecies isolated from a seven year old perinatally HIV-1 infected child, in whom anti-MPER antibodies were found. Determinants of 4E10 neutralization were mapped to the epitope of this antibody in the MPER, as well as to the cytoplasmic tail, in particular, to four amino acids in the LLP-2 region. The role of neutralizing antibodies in natural HIV-1 subtype C infection was examined in Chapter Five by following the development of autologous and heterologous neutralizing antibodies in 14 patients during the first year of infection. Potent but relatively strain-specific neutralizing antibody responses were detected within 3-12 months of infection. The magnitude of the responses was associated with shorter V1-to-V5 envelope length and fewer glycosylation sites, in particular in the V1-V2 region. Furthermore, anti-MPER and anti-CD4i neutralizing antibodies were detected in some individuals; however, they were not associated with neutralization breadth. Finally, in Chapter Six these results are analyzed collectively, in the context of the latest findings in the field, and suggestions for further research are discussed.

MSc (Med) Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand / In recent years there has been much progress in understanding and defining the key protein structure-function relationships that mediate Human Immunodeficiency Virus (HIV-1) entry into host CD4+ cells. This process involves fusion of the virus and host cell membranes, following engagement of corresponding viral (gp120) and target (CD4) receptor proteins. Binding of gp120 to CD4 triggers extensive conformational changes in gp120, exposing binding sites for the co-receptor proteins (CCR5 or CXCR4), and facilitating insertion of gp41, the viral fusion protein, into the target cell membrane. Following insertion of gp41, oligomerisation of fusogenic domains on gp41 is thought to drive the juxtaposition of the virus and host cell and fusion of their membranes. Recent reports suggest that detergent-resistant membrane domains, known as lipid rafts, play a crucial role in orientating the receptor molecules during this step of HIV-1 infection. Lipid rafts are typically rich in cholesterol, sphingolipids and GPI-anchored proteins, and are biophysically distinct from the glycerophosolipid bilayer, which constitutes the bulk of mammalian cell membranes. The role of lipid rafts in virus entry, however, is still controversial, and further experimentation is needed to define their importance in this regard. To provide insight into the role of lipid rafts during HIV-1 entry, we evaluated the natural distribution of the host receptor proteins in HIV-1 target cells (U87.CD4.CCR5/CXCR4). CD4 was detected in membrane samples fractionated by sucrose density gradient centrifugation using immunoblotting techniques, while CCR5 and CXCR4 were detected on whole cells by fluorescence microscopy. We then used a primary CCR5-utilising subtype C HIV-1 isolate (FV5) to characterise dynamic changes in the distribution of these receptors and gp41 during viral entry in real-time. Viral fusion assays were set up by inoculating v target cells with FV5 at 23 ºC, a temperature that allows HIV-1 attachment, but is nonpermissive for advancement of the fusion reaction. This prefusogenic form of the virus-host receptor complex is defined as the temperature-arrested state (TAS). We found that, under normal, uninfected conditions, CD4, CCR5 and CXCR4 are distributed throughout both raft and non-raft microdomains on the U87 cell surface, and there is little evidence for any significant redistribution of these receptors into lipid rafts during the HIV-mediated fusion reaction. Interestingly, we observed a change in the structure of CD4 during the fusion process, which could describe a functionally important event in HIV-1 entry, or be related to compromises in the integrity of the virally-infected membranes. Moreover, we discovered that gp41 is capable of membrane insertion and oligomerisation at TAS, in contrast to previous reports that suggest the fusion peptide is not capable of breaching the membrane at this temperature. Our results provide valuable novel insights into the HIV-1 subtype C entry process, and the involvement of lipid rafts in this stage of the viral lifecycle, that may be relevant to novel therapy and immunogen design.

PhD (Molecular Medicine and Haematology), Faculty of Health Sciences, University of the Witwatersrand / Background: The availability of highly active antiretroviral (ARV) treatment in the South African government sector has reduced the morbidity and mortality associated with HIV-1 infection. However, ARV drug resistance and toxicity are major obstacles to achieving and maintaining virus suppression, but there is no provision for ARV drug resistance testing in the public sector. To date, most studies of ARV drug resistance in HIV-1 reverse transcriptase (RT) and protease (PR), are based on sequence data from HIV-1 subtype B, whereas subtype C is the predominant circulating subtype in South Africa. Moreover, host genetic polymorphisms associated with ARV drug toxicity have not been investigated in South African populations. This study evaluated viral and host genetic factors associated with ARV treatment outcome in 812 ARV drug-naive South African AIDS participants enrolled on the CIPRA-SA study from Johannesburg and Cape Town. Methodology: An affordable in-house genotyping protocol (subtype C specific) was established and validated to monitor the emergence of ARV drug resistance. This assay was used to genotype all CIPRA-SA participants failing the first- and second-line ARV drug regimens. Allellic discrimination assays to identify the G1344A, A6986G, G516T and C3435T SNPs in CYP3A4, 3A5, 2B6 and MDR-1, respectively, associated with ARV metabolism and absorption were performed. Results: The in-house ARV drug resistance assay successfully genotyped 95% of patient samples, including non-C subtypes from 8 African sites. Treatment failure was experienced in 371 participants, mainly due to toxicity (n=134) or virological failure (n=83). Overall, CIPRA-SA participants with a lower CD4+ T-cell count at study onset were more likely to experience viral failure. Genotyping using the in-house assay revealed that 6 participants had ARV drug resistance mutations at study entry. Treatment failure of 58 participants was a result of ARV drug resistance mutations, whereas 19 had no known ARV drug resistance mutations. The most frequent mutations were M184V (67%) and K103N (50%). K65R was present (3%) and one participant harboured TAMs. Longitudinal genotypic analysis showed that NNRTI mutations accumulated at a rate of one per three months left on failing therapy. No PR mutations were detected amongst participants experiencing second-line failure. The four SNPs analysed occured in similar frequencies between a background and the CIPRA-SA cohort. Furthermore, no statistically significant association could be found between these four SNPs and viral failure and/or toxicity. Conclusion: Overall, HIV-1 subtype C-infected individuals receiving ARV therapy develop many of the known subtype B drug resistance mutations. However, the ARV drug resistance patterns in the closely monitored CIPRA-SA cohort were less complex compared to published data from the region, confirming that more frequent viral load monitoring, genotyping, and a virological failure cut-off value of 1000 RNA copies/ml ensure a better prognosis, and preserve future ARV treatment options.

Thesis ((Ph.D. (Physiology))--North-West University, Potchefstroom Campus, 2010.

Thesis ((Ph.D. (Physiology))--North-West University, Potchefstroom Campus, 2010.

PhD / Department of Microbiology

Thesis (PhD)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: The HIV epidemic in the past couple of decades has spread at an alarming rate throughout Southern Africa. Today the region accounts for roughly one third of all HIV infections, while prevalence rates in other areas of sub-Saharan Africa remain low. In the following study, sampled sequences from Cape Town, spanning over a 21-year period were used to investigate the epidemic history of HIV, which was compared to epidemic trends across Southern Africa. Longitudinal sequence data sets were generated from stored patient samples from Cape Town through standard molecular techniques. Firstly, these sequences were used to estimate the date of origin of the HIV epidemic in Cape Town and to reconstruct a demographic history of the epidemic with advanced Bayesian inference methods. These analyses placed the estimated date of origin of the Cape Town epidemic around the mid 1960‟s with periods of strong epidemic growth observed during the mid 1980‟s and 1990‟s. Secondly, reference strains of HIV from Southern African countries were used to estimate the date of origin of the epidemic in the Southern African region. These analyses placed the date of origin of the epidemic in the Southern African region around the mid 1950‟s roughly ten years before the start of the epidemic in Cape Town/South Africa. These sequences were also used for the reconstruction of the demographic history of the epidemic in the region. A two phased growth in the HIV epidemic in the Southern African region was observed with exponential growth occurring in the mid 1980‟s and 1990‟s. Such findings are also supported by HIV prevalence estimates made by some of the leading HIV research centres and government health departments. Thirdly, a large number of homologous reference strains were used to establish the evolutionary relationship of HIV isolates from Cape Town with those from around the world. A close genetic relationship between Cape Town isolates with other South African and other Southern African isolates was observed in these analyses. Finally, large monophyletic clusters of Cape Town isolates, which was observed during the evolutionary inference, were further investigated. After detailed analyses it appears that these transmission clusters of HIV-1 have been in circulation amongst the infected population of Cape Town for several years or decades. / AFRIKAANSE OPSOMMING: Die MIV-epidemie het in die afgelope paar dekades teen ´n snelspoed deur Suider-Afrika versprei. Een derde van die globale MIV-infeksies kom hiér voor terwyl ander dele van Afrika aansienlik minder infeksies aantoon. Verskeie studies skryf dit toe aan onder andere: manlike besnydenis, seksuele losbandigheid, migrasie en verskeie politike faktore. Die MIV-epidemie in Suider-Afrika word deur ´n enkele subtipe van die virus oorheers (nl. MIV Subtipe C) terwyl ander subtipes sirkuleer deur die res van sub Sahara-Afrika. In die opeenvolgende studie word DNS-monsters uit Kaapstad (wat oor ´n 20 jaar tydperk strek) gebruik om die oorsprong en verloop van die epidemie te bestudeer. Die data van die Kaapstad epidemie word met die geskiedkundige verloop van die epidemie in Suider-Afrika vergelyk. Deur gestoorde bloedmonsters van Kaapstad te gebruik, was DNS-datastelle gegenereer deur middel van standaard molekulêre tegnieke. Die DNS-monsters was eerstens gebruik om die evolusionêre oorsprong en verloop van die epidemie in Kaapstad te bepaal deur Bayesiaanse Markov-ketting Monte Carlo steekproefneming. Volgense die resultate het die epidemie sy oorsprong in die 1960‟s. Klein periodes van epidemiese groei kon waargeneem word gedurende die 1980's en -90's. Die bevindings is toe vergelyk met die geskiedkundige verloop van die epidemie in Suider-Afrika. Die Suider-Afrika epidemie se oorsprong en verloop was afgelei van DNS monsters wat verkry is van publieke databasisse en die gebruik van soortgelyke Bayesiaanse metodes. Die resultate van die ondersoek het bevind dat die epidemie in Suider-Afrika in die 1950‟s ontstaan het. In vergelyking toon dit 'n stadiger liniêre groei met kort periodes van eksponensiële groei. Verder is ´n standard filogenetiese analise onderneem om die evolusionêre verwantskap van die Kaapstad-monsters te bepaal met ander MIV subtipe C isolate. Die filogenetiese steekproef toon dat die Kaapstad-monster baie nou verwant is aan ander isolate van Kaapstad, Suid-Afrika en Suider Afrika. Buiten hierdie bevindings was transmissie-bondels van MIV in Kaapstad ontdek. Na ´n deeglike verdere filogenetiese ondersoek blyk dit of die transmissie bondels al vir ´n paar dekades deur die geïnfekteerde populasie van Kaapstad sirkuleer. / Poliomyelitis Research Foundation (PRF) / Faculty of Medicine and Health of the University of Stellenbosch / National Research Foundation (NRF) of South Africa,

PhD (Microbiology) / Department of Microbiology / High genetic diversity is a major contributory factor in the development of drug resistance, in addition to challenges in diagnosis and treatment monitoring in the therapeutics of human immunodeficiency virus (HIV) .Within the wide HIV-1 diversity, differences in mutational frequency, disease progression, drug response and transmission amongst HIV-1 subtypes have been shown. In spite HIV-1 subtype C (HIV-1C) being the most prevalent variant globally, none of the available drugs nor screening assays for inhibitory molecules have been developed targeting the genetics of this important subtype. This study therefore aimed to overexpress and biophysically characterize HIV-1C reverse transcriptase and protease to serve as reagents in the development of assays for routine screening of molecules inhibitory to HIV-1C. Heterologous expression of HIV-1C reverse transcriptase and protease isolates that are prevalent in South Africa was carried out in Escherichia coli (E. coli (BL21-DE3). The secondary and tertiary structures of the proteins were determined using, circular dichroism (CD) and fluorescence spectroscopy respectively. Thereafter, interaction studies to delineate interaction properties of natural products for possible inhibition of protease were conducted. Furthermore, in silico studies to determine binding interactions, further confirmed by in vitro binding assays of a pepsin inhibitor homolog (Bm-33) from Brugia malayi , against protease were also conducted. Expressed reverse transcriptase and protease from the globally prevalent HIV-1C were shown to be structurally and functionally intact for application in downstream HIV-1 inhibition assays. Interaction studies on the other hand revealed successful inhibition of the expressed HIV-1C PR with gallotanin. Furthermore, binding interactions of Bm-33 and HIV-1 PR revealed the first intermolecular interactions of the two molecules displaying possible inhibition of HIV-1 PR / NRF

La donnée de séquences nucléotidiques permet d'inférer des arbres phylogénétiques, ou phylogénies, qui décrivent leur lien de parenté au cours de l'évolution. Associer à ces séquences leur date de prélèvement ou leur pays de collecte, permet d'inférer la localisation temporelle ou spatiale de leurs ancêtres communs. Ces données et procédures sont très utilisées pour les séquences de virus et, notamment, celles du virus de l'immunodéficience humaine (VIH), afin d'en retracer l'histoire épidémique à la surface du globe et au cours du temps. L'utilisation de séquences échantillonnées à des moments différents (ou hétérochrones) sert aussi à estimer leur taux de substitution, qui caractérise la vitesse à laquelle elles évoluent.Les méthodes les plus couramment utilisées pour ces différentes tâches sont précises, mais lourdes en temps de calcul car basées sur des modèles complexes, et ne peuvent traiter que quelques centaines de séquences. Devant le nombre croissant de séquences disponibles dans les bases de données, souvent plusieurs milliers pour une étude donnée, le développement de méthodes rapides et efficaces devient indispensable. Nous présentons une méthode de distances, Ultrametric Least Squares, basée sur le principe des moindres carrés, souvent utilisé en phylogénie, qui permet d'estimer le taux de substitution d'un ensemble de séquences hétérochrones, dont on déduit ensuite facilement les dates des spéciations ancestrales. Nous montrons que le critère à optimiser est parabolique par morceaux et proposons un algorithme efficace pour trouver l'optimum global.L'utilisation de séquences échantillonnées en des lieux différents permet aussi de retracer les chaînes de transmission d'une épidémie. Dans ce cadre, nous utilisons la totalité des séquences disponibles (~3 500) du sous-type C du VIH-1 (VIH de type 1), responsable de près de 50% des infections mondiales au VIH-1, pour estimer ses principaux flux migratoires à l'échelle mondiale, ainsi que son origine géographique. Des outils novateurs, basés sur le principe de parcimonie combiné avec différents critères statistiques, sont utilisés afin de synthétiser et interpréter l'information contenue dans une grande phylogénie représentant l'ensemble des séquences étudiées. Enfin, l'origine géographique et temporelle de ce variant (VIH-1 C) au Sénégal est précisément explorée lors d'une seconde étude, portant notamment sur les hommes ayant des rapports sexuels avec des hommes. / Nucleotide sequences data enable the inference of phylogenetic trees, or phylogenies, describing their evolutionary re-lationships during evolution. Combining these sequences with their sampling date or country of origin, allows inferring the temporal or spatial localization of their common ancestors. These data and methods are widely used with viral sequences, and particularly with human immunodeficiency virus (HIV), to trace the viral epidemic history over time and throughout the globe. Using sequences sampled at different points in time (or heterochronous) is also a mean to estimate their substitution rate, which characterizes the speed of evolution. The most commonly used methods to achieve these tasks are accurate, but are computationally heavy since they are based on complex models, and can only handle few hundreds of sequences. With an increasing number of sequences avail-able in the databases, often several thousand for a given study, the development of fast and accurate methods becomes essential. Here, we present a new distance-based method, named Ultrametric Least Squares, which is based on the princi-ple of least squares (very popular in phylogenetics) to estimate the substitution rate of a set of heterochronous sequences and the dates of their most recent common ancestors. We demonstrate that the criterion to be optimized is piecewise parabolic, and provide an efficient algorithm to find the global minimum.Using sequences sampled at different locations also helps to trace transmission chains of an epidemic. In this respect, we used all available sequences (~3,500) of HIV-1 subtype C, responsible for nearly 50% of global HIV-1 infections, to estimate its major migratory flows on a worldwide scale and its geographic origin. Innovative tools, based on the principle of parsimony, combined with several statistical criteria were used to synthesize and interpret information in a large phylogeny representing all the studied sequences. Finally, the temporal and geographical origins of the HIV-1 subtype C in Senegal were further explored and more specifically for men who have sex with men.