Human Immunodeficiency Virus (HIV) is the causative agent of AIDS, a disease which affects over 24 million people globally and for which there is neither curative treatment nor vaccine available. As an intracellular pathogen that encodes only 15 proteins HIV-1 is highly dependent upon its host's cellular machinery in order to complete its life cycle. Host-directed therapy thus represents a potentially lucrative strategy for the development of novel anti-HIV therapies. microRNAs (miRNAs) are short noncoding RNA molecules that function as part of the endogenous RNA interference system which governs post transcriptional gene regulation. Current knowledge has placed miRNAs at the crux of HIV-host interactions, yet the functional relevance of the majority of the human miRNAome with regards to HIV replication has remained unknown. A microscopy-based high content screening (HCS) approach was thus developed to systematically evaluate the significance of augmenting or inhibiting the function of individual host miRNAs on the replication dynamics of HIV. A bespoke image analysis and data mining pipeline recovered 56 host miRNAs associated with suppressed HIV replication and 28 host miRNAs associated with enhanced HIV replication. Notably, the HIV-modulating potential of 80 of these miRNAs was previously unknown. Furthermore, HCS also uncovered a novel role for the miR-200 family in the modulation of HIV replication. In silico miRNA target identification and pathway enrichment analysis identified 24 pathways associated exclusively with suppressed HIV replication, 10 pathways associated exclusively with enhanced HIV replication and 38 functional pathways enriched for both enhanced and suppressed viral replication. These included a number of pathways previously implicated in HIV replication such as the PI3K, MAPK, TNF and WNT signalling pathways but also revealed novel functional associations including that of the Hippo signalling pathway. Intriguingly pathway analysis revealed an enrichment for host factors associated with viral carcinogenesis and a convergence on host processes and functional targets classically associated with chemotherapy including host DNA damage repair, cell cycle and tyrosine kinase receptor-mediated signalling. Experimental validation confirmed that HIV replication induced an aberrant cell survival phenotype in response to chemically induced DNA damage but this effect was reversed when DNA damage was induced prior to HIV exposure. A series of compound-based validation screens were thus undertaken in order to verify the functional associations recovered by miRNA screening. A targeted collection of 293 small molecule inhibitors, including a number of FDA-approved chemotherapeutics, were screened for HIV modulating activity. Novel anti-HIV activity was recovered for over 40 compounds including a number of FDA-approved therapies. Compound-target enrichment analysis revealed a strong concordance with functional associations initially described by miRNA-based HCS including EGFR-mediated signalling and DNA damage repair. Concordant HIV-suppressive activity was also recovered for miRNAs and compounds with common functional targets. The outcomes of this study thus represent a significant and novel contribution to current knowledge on HIV-host interactions. Furthermore, these findings have characterised novel miRNA and small molecule candidates for the treatment of HIV and have successfully demonstrated the utility of miRNA-based HCS for novel-drug discovery and drug repositioning.

Background : Host protein beta-2 microglobulin (β2m) is incorporated into the human immunodeficiency virus (HIV) -1 coat during budding. Antibodies directed to R7V, an epitope contained in β2m, increased with the duration of infection in long term non-progressor patients (LTNPs). Purified R7V antibodies neutralized HIV isolates and did not bind to human cells. These data suggested potential for R7V antibodies to be developed as therapeutic tools or prognostic markers and the R7V epitope as a vaccine candidate. However, the literature on R7V is still incomplete. For example, most published work on this epitope make no direct reference to HIV subtypes. The rationale for this study is the lack of information on whether all HIV-1 subtypes incorporate R7V and elicit immune responses to the same extent. In particular the response of HIV-1 subtype C infected individuals to R7V antigen is evaluated here. Methodology and results : A synthetic peptide of the R7V epitope of HIV-1 was synthesized and an “in-house” enzyme-linked immunosorbent assay (ELISA) developed. The peptide was able to detect antibodies generated during natural HIV-1 subtype C infection when used as antigen in the ELISA. This response was not as strong as that reported in the literature. A significantly lower ELISA response was observed for uninfected compared to infected sera (probability, p, value ≤ 0.000152), whereas no differences were noticed between antiretroviral (ARV) treated individuals compared to those who were treatment naïve or LTNPs compared to progressors. These data hold promise for the use of these antibodies as diagnostic rather than prognostic indicators. Polyclonal R7V antibodies produced in rabbits and recombinant R7V antibody fragments did not neutralize an HIV-1 subtype C isolate (Du151.2). However, the latter antibodies neutralized an HIV-1 subtype B strain (SF162), suggesting that the R7V epitope may be more exposed in this subtype. The recombinant R7V antibodies did not neutralize a vasicular stomatitis virus (VSV-G), indicating that no nonspecific neutralization occurred. Human immunodeficiency virus type 1 subtype C infected sera containing R7V antibodies (positive response in the R7V ELISA) neutralized Du151.2 while archive sera containing strong HIV-1 subtype C neutralizing antibodies did not recognize the R7V antigen ELISAs. The R7V peptide exogenously added to HIV-1 infected peripheral blood mononuclear cells (PBMCs) did not stimulate proliferation in vitro nor the production of interferon (IFN) gamma which if produced by CD8+ T-cells would have been indicative of a cellular immune response. The parent protein β2m could not initiate these responses either. Conclusion : Data collected here support a diagnostic rather than a prognostic application for R7V antibodies. R7V conjugated to keyhole limpet hemocyanin (KLH) induced non-neutralizing antibodies in rabbits, suggesting that other modifications (branching, lipid conjugation, etc.) may be needed before this epitope can be successfully utilized in vaccine studies. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

Human immuno deficiency virus (HIV) incorporates host cellular protein, beta-2-microglobulin (β2m), into its surface envelope during budding. β2m is a cellular protein that belongs to the major histocompatibility complex (MHC) Class I molecules. Studies have shown anti- β2m monoclonal antibodies (mAbs) has the ability of to neutralize the virus. An epitope consisting of seven amino acids of the β2m protein designated as R7V produces antibodies that protect HIV infected people from progressing to AIDS. These protective antibodies, called anti-R7V antibodies, were able to neutralize different HIV isolates, despite their genomic variations, various cellular targets and geographic origin. Anti-R7V antibodies in the format of single chain variable fragments (scFvs) were produced in our laboratory using the M13 phage display technology. These scFv antibody fragments were used during in vitro studies for the detection and neutralization of the R7V antigen by enzyme linked immune sorbent assay (ELISA). The scFv fragments produced against the R7V epitope showed interaction, however the antibody-antigen affinity was too weak for the virus neutralization assay. Hence, this project focused on the affinity maturation of the anti-R7V scFv fragments through random mutagenesis using the error prone (EP) PCR method. The EP PCR method generated two mutated anti-R7V scFvs. The mutated clones were subcloned into the pAK400 expression vector. The computer-based models, created using the Swiss PDB Deep Viewer 4.02 software, were used to predict the antigen-binding site and affinity analysis of both parent and mutated scFv’s. Mutated clone 1 failed to bind to the R7V epitope whereas mutated clone 2 had similar binding pattern as the parent scFv. Mutated clone 2 was predicted to have a higher binding affinity compared to the parent scFv. The results obtained demonstrate the efficacy of EP PCR to generate high affinity antibodies. Future experiments using high affinity anti-R7V scFv’s may lead to its potential use in diagnostics, therapeutics or vaccine development. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Biochemistry / unrestricted

Introduction: Improvements in early detection of human immunodeficiency virus (HIV), linkage to treatment, and availability of antiretroviral therapy (ART) have contributed to increasing life expectancy for people living with HIV (PLHIV) in South Africa. These improvements have resulted in the decline of HIV cause-specific mortalities. In addition to existing tuberculosis burden in PLHIV, cases of chronic non-communicable diseases (NCDs) are increasing in the general population. Considering the ageing population of PLHIV in South Africa, it is important to understand their health needs, as well as identify potential drivers of comorbidities that may provide avenues for future interventions. This study aimed at exploring HIV and comorbidity profiles in a virtual cohort of a population of healthcare clients accessing care in public facilities in Khayelitsha, Cape Town. Methods: Routinely collected data for healthcare clients accessing care in public facilities in 2016/17 were obtained from the Western Cape Provincial Health Data Centre, and analysed to describe ascertained comorbidities, comparing the profiles of PLHIV and HIV-negative individuals. The risks of comorbidity occurrence in PLHIV, in the context of other comorbidities and HIV metrics such as ART duration, viral load and CD4 cell counts, including the contribution of comorbidities to unsuppressed viral load levels in PLHIV were explored. Findings: The findings show that accessing HIV care may lead to earlier ascertainment of common chronic NCDs – hypertension, diabetes, chronic kidney disease (CKD), cervical cancer in PLHIV, compared to HIV-negative clients. Analysis of routine health data suggests that ascertainment of comorbidities differs for healthcare clients due to sub-population differences including age, sex, HIV status and reasons for accessing care. Routine laboratory testing results for renal function reflect distinct healthcare experiences by age for healthcare clients with and without HIV. Analysis of routine data shows that presence of an existing comorbidity may contribute to the incidence of other comorbidities and unsuppressed viral load levels in PLHIV. Conclusion: From real life routine health data, this study has explored comorbidities profiles of PLHIV and HIV-negative clients and observed that routine health data could provide a better understanding of disease profiles, healthcare access and requirements for both PLHIV and HIV-negative clients.

Women face a number of physical, emotional and psychological challenges following an HIV positive diagnosis during pregnancy. Psychological challenges, such as maternal anxiety and low mood, have been associated with disruptions to mother-infant bonding in the general population. Despite significant numbers of women receiving an antenatal HIV diagnosis in the UK each year, there remains a limited understanding about the experiences of this group in bonding with their babies. This Grounded Theory study aimed to explore the experience of mothers in bonding with their baby following an HIV diagnosis during pregnancy. The study explored the perceived challenges to mother-infant bonding, and the factors mothers felt helped them to manage this process following diagnosis. Ten mothers diagnosed antenatally at a London sexual health service were interviewed about their experiences. Data analysis led to a theoretical model of mother-infant bonding following a maternal HIV positive maternal diagnosis. The model comprised four theoretical codes: facing barriers to bonding; feeling disconnected from the baby; developing a special bond; and strengthening and moving on. These codes were comprised of challenges to mother-infant bonding, as well as factors relating to maternal strength and resilience. The model used a chronological structure, with processes plotted from the point of antenatal diagnosis through to following the infant HIV testing process after birth. Challenges with bonding were experienced primarily during the early stages after birth, with maternal resilience and positivity about the future developing towards the end of infant testing. Circular relationships, in which positive and negative processes fed into and influenced each other, were highlighted throughout. The findings highlight important areas for development in clinical practice, including more targeted psychological support for women following an antenatal diagnosis, and the provision of timely information regarding mother-to-child transmission. Clinical implications from this study are discussed alongside suggestions for future research.

This researched aimed to explore the effects of the Human Immunodeficiency Virus (HIV) upon the visuospatial abilities of HIV-positive adolescents on antiretroviral treatment in South Africa. The literature suggests that the neurology responsible for visuospatial abilities (specifically various white-matter tracts in the brain) is very susceptible to the damaging effect that HIV has on the brain. The research sample consisted of vertically transmitted HIV-positive adolescents, on first line antiretroviral treatment, with a HIV-negative control group comparable on age and SES. The results indicated that there is a significant difference in the visuospatial abilities between adolescents with and without HIV. The expressions of these deficits were displayed differently between males and females, highlighting a differing developmental neurology, and the effect of HIV upon it. The viral strength and health of the immune system were also examined as variables and illuminated interesting results. Overall, the research illustrates the negative effect that HIV has upon developing neurology and the subsequent effects on visuospatial abilities.

No abstract available. / Dissertation (LLM)--University of Pretoria, 2013. / gm2014 / Centre for Human Rights / unrestricted

Infection with the human immunodeficiency virus (HIV) in the adolescent/young adult population of the United States is a serious, growing problem. The current HIV risk-reduction strategies for adolescents have been less than effective in stemming the tide of infection. This ineffectiveness can be linked to failure of making developmentally appropriate risk-reduction informational material and reliance on condom-based interventions, which have an unacceptably high failure rate. A critical analysis of current models of HIV-risk reduction should be undertaken to create more developmentally appropriate and effective methods.

Magister Public Health - MPH / Background: Antiretroviral therapy (ART) was rolled out in South Africa in the public sector in 2004 and the treatment coverage has increased over the years to 56% in 2016. The increased treatment coverage has the potential to increase the level of HIV drug resistance. Drug resistance presents a major challenge to the management of HIV infection through antiretroviral therapy at the population level. The aim of this study was to determine the impact of the public sector antiretroviral therapy rollout on the prevalence of HIV drug resistance in South Africa and the factors associated with drug resistance. Methodology: A cross-sectional analytical study was used to determine the prevalence of drug resistance before and after ART rollout. The study population was HIV infected South Africans (infected between 1996 and 2011) who were not on antiretroviral therapy. The study sample was therapy naïve HIV infected South Africans who participated in published studies conducted between 1996 and 2011. HIV DNA sequences and associated data (participants’ age, gender, geographic location and estimated year of HIV infection) were accessed through the Los Alamos HIV Database. The database contains all HIV DNA sequences and associated data from all published studies and the data was freely accessible. A descriptive analysis was carried out on the data to determine characteristics of the study sample. Drug resistance mutations were detected using Calibrated Population Resistance Program on the Stanford University HIV Drug Resistance database. The output from the Calibrated Population Resistance Program analysis were used to determine the prevalence of drug resistance mutations. Results: There were 1701 DNA sequences obtained from the Los Alamos HIV Database for the three gene regions targeted by ART (reverse transcriptase, protease and integrase). Of these, 604 (35,5%) were for reverse transcriptase, 794 (46,7%) were for protease and 303 (17,8%) were for integrase. There was overrepresentation of DNA sequences from female participants (91%). There was no significant difference in the prevalence of drug resistance mutations between 1996-2004 (before ART rollout) and 2005-2011 (after ART rollout) in all the drug classes. There was also no association between drug resistance and age as well as gender. Conclusion: The data from this study suggest that the public sector rollout of ART did not result in an increase in the prevalence of drug resistance mutations in therapy naïve HIVinfected South Africans. There is need for further studies, which have a wider coverage of the South African population.

Os pacientes infectados pelo vírus da imunodeficiência humana (HIV) freqüentemente apresentam alterações digestivas, sendo o esôfago um alvo comum de lesões estruturais. A etiologia infecciosa é a mais freqüente neste grupo de pacientes. Múltiplos agentes já foram implicados como causadores de lesões esofágicas. As infecções virais são uma das principais causas de tais lesões, sendo os vírus mais implicados o citomegalovirus (CMV) e o vírus herpes simples (HSV). Muitas lesões ulceradas permanecem sem diagnóstico etiológico, mesmo após exaustiva investigação, sendo denominadas úlceras idiopáticas ou aftosas. Os métodos de diagnóstico usuais são demorados e pouco sensíveis. Assim, nosso estudo tem como principal objetivo estudar o papel do método da reação em cadeia da polimerase (PCR) no diagnóstico destas lesões. Durante o período de outubro de 1996 a outubro de 1997, foram estudados 79 pacientes HIV-positivos, que foram submetidos ao exame de endoscopia digestiva alta por indicação clínica. Estes foram submetidos a 89 exames endoscópicos, sendo colhidas 96 biópsias, as quais foram armazenadas em nitrogênio líquido (50) ou em freezer a –70oC (46). O DNA foi extraído usando método baseado na lise hipotônica, digestão com proteinase K, extração com fenol-clorofórmio e precipitação em etanol. Uma quantidade fixa foi usada para amplificação em ciclador térmico, utilizando primers específicos para CMV, Herpesvirus, HPV, HIV, Haemophilus ducreyi, Treponema pallidum e as micobactérias M. tuberculosis, M. avium e M. intracellulare. O produto final foi submetido a uma eletroforese em gel de agarose e corado com brometo de etídeo. A endoscopia não revelou alterações esofágicas em 26 exames (29,2%). As alterações observadas foram monilíase esofágica em 33 exames (37,1%), úlceras em 22 (24,7%); esofagite em 10 (11,2%) e áreas lugol-negativas em 9 (10,1%). A PCR resultou positiva para o CMV em 19 amostras (19,8%), para o Herpes em 4 (4,2%), para o HPV em 17 (17,7%), para o HIV em 37 (38,5%) e para o H. ducreyi em 3 (3,1%). Nenhuma amostra foi positiva para o T. pallidum e para micobactérias. No estudo de 29 amostras de 22 úlceras esofágicas a PCR detectou o CMV em 9 amostras (31%), o Herpes em 3 (10,3%), o HPV em 6 (20,7%), o HIV em 19 (65,5%) e o H. ducreyi em 2 (6,9%) e em 8 (36,4%) não foi detectado nenhum agente. O CMV foi detectado com freqüência nas úlceras esofágicas, sendo difícil diferenciar se havia infecção ativa ou latente. O HIV teve uma incidência elevada nas biópsias de úlceras, o que pode sugerir um possível papel etiológico deste agente em tais lesões. O HPV foi o terceiro agente mais freqüente, mas não foi possível caracterizá-lo como causador de lesões esofágica ulceradas. A PCR apresentou potencial para tornar-se um método útil na investigação das lesões esofágicas em pacientes infectados pelo HIV. / Patients infected by Human Immunodeficiency Virus (HIV) usually present digestive abnormalities and the esophagus is a common target of structural lesions. Infections are the most frequent cause of esophageal lesions in these patients. Several agents were already implied in this process. Viral infections are one of the main causes of such lesions and cytomegalovirus (CMV) and herpes simplex virus (HSV) were the most involved agents. Many ulcerated lesions persist without etiologic diagnosis even after exhaustive investigation, being denominated idiopathic or aphthous ulcers. The usual diagnostic methods are difficult and have low sensitivity. Thus, the main objective of our study was to evaluate the role of the polimerase chain reaction (PCR) method in the diagnosis of these lesions. During the period of October of 1996 to October of 1997, 79 HIV-positive patients were studied. They were submitted to upper digestive endoscopies, which were indicated on clinical basis. These patients were submitted to 89 upper digestive endoscopies, being obtained 96 biopsies, which were stored in liquid nitrogen or in a 70oC freezer. DNA was extracted using a method based on hypotonic lyses, proteinase K digestion, extraction with phenol-chloroform and precipitation in ethanol. A fixed amount was used for amplification in thermal cycler, using specific primers for CMV, herpesvirus, human papillomavirus (HPV), HIV, Haemophilus ducreyi, Treponema pallidum, Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare. The final products were submitted to an electrophoresis in agarose gel and stained with ethidium bromide. The endoscopies did not reveal esophageal alterations in 26 exams(29,2%). The abnormalities observed were esophageal candidiasis in 33 exams (37,1%), ulcers in 22 (24,7%); esophagitis in 10 (11,2%) and lugol-negative areas in 9 (10,1%). The PCR was positive to CMV in 19 samples (19,8%), for Herpes in 4 (4,2%), for HPV in 17 (17,7%), for HIV in 37 (38,5%) and for the H. ducreyi in 3 (3,1%). No sample was positive for T. pallidum or micobacterium. In the study of the esophageal ulcers by PCR, CMV was detected in 9 samples (31%), Herpes in 3 (10,3%), HPV in 6 (20,7%), HIV in 19 (65,5%), H. ducreyi in 2 (6,9%) and any agent was detected in 8 samples (36,4%). CMV was frequently detected in esophageal ulcers, being difficult to differentiate between active and latent infections. The HIV had an elevated incidence in ulcer biopsies, which may suggest a possible etiologic role of this virus in such lesions. HPV was the third more frequent agent, but it was not possible to attribute the esophageal lesions to that virus. In conclusion, this study suggests that the PCR can be an useful method in the investigation of esophageal lesions in HIV infected patients.