2017 HPP Graduates

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• Ikanyeng Dolly Seipone, PhD

  Study Title: Investigation of viral characteristics and immune microenvironment between the blood and the central nervous system in patients with HIV associated neurological disorders or concomitant Mycobacterium tuberculosis infection. The Southern Africa region has the highest burden of HIV that is further exacerbated by co-infection with TB hence the interest to embark on HIV/TB coinfection research. This study investigated HIV replication characteristics and in the cerebrospinal fluid versus blood of individuals with meningitis including tuberculous meningitis and explored immunological biomarkers for tuberculous meningitis. Tuberculous meningitis was associated with high cerebrospinal fluid viral load and there was evidence of distinct viral strains in the cerebrospinal fluid compared to blood in some patients. Markers that may distinguish tuberculous meningitis from other meningitides were identified. This study has improved our understanding and enhanced our knowledge of HIV/TB co-infection of the central nervous system and forms a basis for further research on pathogenesis and diagnosis. It offers significant insight on Subtype C HIV-1 evolution and adaptation to the CNS during TB co-infection and might be valuable in the prevention of HIV-neurological disorders and thus managing HIV/TB co-infection. Dolly is currently a Post-doctoral Research Fellow at the University of KwaZulu-Natal’s Discipline of Public Health Medicine. Her future plans are to be one of the leading medical research scientists and amongst the health policy makers at a global level, making a difference in Africa through established organisations, such as the World Health Organization or UNAIDS. Dolly would like to extend her gratitude to her mentor and PhD supervisor, Prof Thumbi Ndung’u, for giving her the opportunity to pursue her PhD, and for his guidance. She also wishes to thank the staff of HPP who she says it was a pleasure to work with. “Let us continue to strive to end HIV/AIDS”, says Dolly. “Together we can”.

• Marion Kiguoya, PhD

  Study Title: Functional and clinical consequences of Gag-protease sequence variation in HIV-1 subtypes A, C, D and intersubtype recombinants Marion’s study sought to investigate whether there were subtype-specific replication capacity differences in recombinant viruses possessing patient-derived gag-protease genes from subtypes A, C, D and inter-subtype recombinants which predominate in the HIV-1 epidemic in East Africa. The study further sought to investigate whether viral replication capacity was associated with markers of disease progression and whether viral replication capacities differed according to host HLA class I molecules. Finally, the study sought to identify amino acids associated with differences in replication capacity within different subtypes. The data generated from this study show a hierarchy of Gag-protease driven replicative fitness where subtypes A/C are less fit than D, which is also less fit than inter-subtype recombinants. The data are consistent with reported subtype-specific differences in disease progression in East Africa, suggesting that Gag-protease-driven replication capacity is a determinant of differences in disease progression between subtypes. Furthermore, amino acids associated with altered replication capacity in subtype A viruses were identified and this study therefore extended previous studies that described fitness associations in Gag-protease from subtypes B and C. These data may have implications for the differences observed in the rate of disease progression and for the uneven spread and expansion of HIV-1 subtypes in the global epidemic. Marion is currently a postdoctoral fellow at the University of Nairobi. The focus of her current research is to pursue the fitness landscape assessing the impact of host HLA on HIV-1 Gag-Protease (Gag-Pro) function in early and acute HIV infection in East Africa.

• Bongiwe Ndlovu, PhD

  Study Title: Evolution of humoral immune responses in acute and early HIV-1 subtype C infection

• Qiniso Mkhize, MMedSci

  Study title: Effects of HLA associated mutations in HIV-1 Nef protein on Nef function. The HIV-1 Nef protein has been discovered to play a major role in the ability of HIV to cause disease. Major functions of this protein in the virus include the ability to downregulate CD4 and HLA-I molecules on the surface of the cells therefore inhibiting recognition by the CTL responses and further enhancing successive replication. Studies have shown that mutations in certain regions of the protein render the protein defective in its function. This study’s aim was to investigate some of the mutations that were previously associated with decreased ability of the protein to downregulate CD4 and HLA-I on cells surfaces. None of the mutations that were expected to downregulate these surface molecules resulted in a significant downregulation effect when tested and none of them affected viral replication. As much as this was the case the literature showed that combinations of mutations produced a much better effect than mutations in isolation, which was in line with our data. Qiniso says that his research project provided him with the opportunity to broaden his knowledge of how HIV has become the biggest epidemic the world is facing to date. When Qiniso joined HPP he acknowledges that he had very little knowledge of the HIV/AIDS research field. He therefore worked as hard as he could to gain as much knowledge and understanding of every concept that he could. Even though his project is at an end he continues to learn as much as he can. Qiniso is currently working as a medical technologist in the HPP laboratory. He is interested in pursuing a PhD degree in the future to further his knowledge of the HIV research field.

2016 HPP Graduates

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• Urisha Singh, PhD

  Study Title: Acquired and transmitted drug resistance in HIV-1 subtype C: implications of novel mutations on replication capacity, cleavage and drug susceptibility. As part of her research project Urisha investigated the impact of Gag-Protease mutations on viral replication and drug susceptibility in a cohort of HIV-1 subtype C infected patients receiving a Protease Inhibitor inclusive treatment regimen. The results showed that some mutations in Gag can confer resistance to Protease Inhibitors whilst other mutations in Gag act as a compensatory mechanism to rescue viral replication capacity in the presence of Gag-Protease mutations without impacting on drug susceptibility. Additionally, Urisha investigated the prevalence of minority variant (i.e. low frequency) transmitted drug resistance mutations in a cohort of patients acutely infected with HIV-1 subtype C and determined their impact on clinical outcomes using next-generation sequencing. Here we showed a 57% prevalence of minority variant transmitted drug resistant mutations with a rapid reversion of mutations in protease, reverse transcriptase and integrase. Overall this research advocates for the inclusion of Gag mutations into drug resistance algorithms. Furthermore, it shows that all patients should receive genotypic resistance testing prior to treatment initiation in order to ensure the prescription of an effective/efficient treatment regimen. This study was the first of its kind performed on HIV-1 subtype C samples, the most prevalent subtype globally. It was the first study to identify four novel mutations in Gag and show that a mutation in the amino-terminal of Gag was associated with the rescue of viral replication in the presence of other Gag mutations which confer resistance to Protease Inhibitors, highlighting the important role of Gag in drug resistance to Protease Inhibitors. Additionally, this is the first study to investigate minority variant mutations in HIV hyperacute infection and to show a prevalence of 57% of transmitted drug resistance, thereby highlighting the importance of genotypic resistance testing prior to treatment initiation. Urisha is currently a Post-Doctoral Research Fellow at the African Health Research Institute, formerly known as KwaZulu-Natal Research Institute for TB and HIV, K-RITH. She plans to continue her research in the field of HIV and TB combining her interest in translational medicine and public health to lead cutting-edge research projects.

• Catherine Kegakilwe Koofhethile, PhD

  Study Title: Protective HLA allesles: investigation of viral control and lack of control in chronic HIV-1 subtype C infection Catherine studied a subset of HIV infected people with a genetic make up associated with strong and protective immune responses to HIV and these individuals naturally controlled the virus without any need for antiretroviral treatment. Catherine attempted to understand why some people control the virus while others progress to AIDS. She characterised longitudinally the immunological and virologic features that may explain the differences between those that are able to control the virus and those that are not able to control the virus. This study enabled better understanding of the mechanisms of HIV control during the chronic stage of infection and has also opened up new research questions, which are of great interest for the future vaccine design studies. Catherine is currently a Postdoctoral fellow/Fogarty Fellow at the Havard T H Chan School of Public Health in the USA. She is still interested in HIV research, especially cure studies. Once she has completed her fellowship at Harvard, she plans to go back to Africa and continue working on HIV research.

• Avashna Singh, MMedSc (Cum Laude)

  Study Title: Detecing minority variants in HIV-1 subtype C infected patients failing Darunavir, using Ultra Deep Sequencing and Computational analysis in KZN Despite the success of combination antiretroviral therapy for HIV-1 infection, a number of patients still fail treatment in South Africa. We studied the contribution of Protease inhibitor (PI) resistance to treatment failure. Amongst 156 second-line failures we found only 24% presenting with PI drug resistance. Within a sub-set of these patients that switched to a third-line Darunavir (DRV) regimen, we looked for low frequency variants that may influence failure. However, we only found an accumulation of DRV mutations and no low frequency variants. Our results suggest that medication non-adherence is a primary factor contributing to treatment failure. We also performed a structural analysis of HIV-1 protease, which showed that distinct structural differences were evident between LPV and DRV failure, which largely reduced drug-binding efficacy. This work will inform the clinical management of patients that are failing current antiretroviral therapy, and may assist in the design of more optimal salvage regimens. Avashna is currently a Research Assistant at the HIV Pathogenesis Programme. However, she intends on pursuing further research in HIV drug-design, using computational analysis, for the design of new and improved inhibitors for HIV-1 subtype C.

• Funsho Ogunshola, MMedSc (Summa Cum Laude)

  Study Title: Analysis of viral inhibition activity of CD8 T cells targeting identical epitopes but restricted by different HLA Class I alleles from the same HLA supertype The aim of Funsho’s study was to conduct a systematic analysis of viral inhibitory activity of HIV specific CD8 T cells targeting identical epitopes but restricted by different class I HLA molecules from the same HLA supertype. He hypothesized that CD8 T cells targeting identical epitopes restricted by distinct class I HLA molecules are functionally equivalent. He used the viral inhibition assay (VIA) to address his study question. In this assay, a cell line is engineered to express a single class I HLA molecule and also express green fluorescent protein (GFP) upon HIV infection. This approach allowed him to accurately delineate the role of HLA restriction on CD8 T cells’ inhibitory activity. Funsho was able to discover that some T cell receptors (TCRs) of HIV specific CD8 T cells can cross recognize identical epitopes restricted by closely related HLA molecules. This finding has important implications for HIV vaccine design to induce broad cross reactive CD8 T cell responses. Funsho is currently a PhD student at the HIV Pathogenesis Programme under the mentorship of Dr. Zaza Ndhlovu. The focus of his PhD study is on investigating the induction of adaptive immunity against HIV in the lymphoid tissues. The goal of the study is to identify immune parameters that can be harnessed to sustain CD8 T cell immunity against HIV infection, which could have implications for the development of immunotherapeutic interventions. Funsho is a recipeint of a SANTHE (Sub-Saharan African Network for TB/HIV) scholarship.

• Erasha Rajkoomar, MMedSc

  Study Title: In vitro testing of the predicted viral fitness landscape for the HIV-1 Nef protein
  

2015 HPP Graduates

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• Mopo Radebe, PhD

  Study Title: Characterization of Human Immunodeficiency Virus-specific CD8 T cell responses and their immunodominance patterns following acute HIV-1 clade C infection Cytotoxic T cells play an important role in HIV immune control. This study identified the earliest cytotoxic T cell responses generated following HIV infection. The study found that responses directed at the HIV Gag protein are associated with viral control but show delayed appearance kinetics and do not always persist. Furthermore, most immune responses generated during acute HIV infection did not cause harmful mutations in HIV. This study provides a rational basis for the design of HIV vaccines.

• Faatima Laher, Master of Medical Science (Cum Laude)

  Study title: Mapping immunodominant patterns of HIV-specific CD4 T cell responses in acute and chronic HIV-1 subtype C infection The focus of Faatima’s Master’s study was on the role of CD4+ T helper cells in the immune response to clade C HIV-1 infection. She tested the hypothesis that HIV-1-specific CD4+ T cell responses and protective class II HLA alleles are important determinants of effective immunological control of HIV-1 infection. The data identified immunodominant regions of HIV-specific CD4+ T cell responses and their association with viral control during clade C infection, highlighting a dominance of Gag and Nef targeting. In addition, high levels of epitope promiscuity was observed among class II HLA molecules, with the HLA-DRB1*13:01 allele variant in particular being associated with the highest frequency of responders in our cohort and restricting the highest number of HIV-specific peptides. Together, this information will be critical to vaccine efforts designed to induce these responses, as well as potential therapeutic manipulation of immunity in persons with acute and chronic HIV-1 infection. Faatima is currently a PhD student under the supervision of Dr Zaza Ndhlovu working on HIV-specific CD4 T cell responses in natural HIV infection. She joined the HIV Pathogenesis Programme after having completed her undergraduate and Honours degrees in Biological Sciences at UKZN (Pietermaritzburg). She has always had a keen interest in both human diseases and the impact they have on society. She decided to apply to HPP as it allowed her to bring these interests together. She is enthusiastic about translational medicine and would like to pursue this aspect of biomedical and public health research in the future. Inspired by the quote of Nelson Mandela that “Education is the most powerful weapon which you can use to change the world”, she would like to use her experience as a young researcher to teach and educate future aspiring scientists.

• Michael Zulu, Master of Medical Science

  Study title: Natural Killer (NK) cell dysregulation in chronic clade C HIV-1 infection Natural Killer (NK) cells are important mediators of the innate immune response against viruses and have been shown to play a very important role in the control of HIV-1 infection. However, HIV-1 evades NK cell antiviral functions by inducing a wide range of changes in NK cell phenotype and function. To assess mediators of NK cell dysfunction in HIV-1 chronic infection, Michael conducted a cross-sectional study examining the phenotype and function of NK cells based on Siglec-7, NKG2A, NKG2C and CD57 phenotypic expression using multicolour flow cytometry in HIV-1 subtype C chronically infected participants compared to healthy donors recruited from Durban, KwaZulu-Natal. Michael demonstrated that HIV-1 viremia induces the expansion of a pathologic CD56-CD16+ NK cell subset and also alters Siglec-7 and CD57 expression on NK cells of infected participants. These phenotypic changes induced by viremia may lead to functional dysregulation on NK cell subsets during chronic HIV-1 clade C infection and therefore viral escape from the NK cell antiviral functions. Michael first enrolled at UKZN in 2008 as a BSc (4-year augmented programme) student majoring in Biomedical Sciences. He completed this degree in 2011 and then went on to register for a BSc Honours degree in Biological Sciences in 2012 before joining the HIV Pathogenesis Programme as a Masters Student (MMedSc) in 2013 under the supervision of Dr Christina Thobakgale. Michael is currently a PhD student at the University of Cape Town, Faculty of Health Sciences, Institute of Infectious Diseases and Molecular Medicine, Division of Immunology under the supervision of Professor Clive Gray.

• Kelly Pillay, Master of Medical Science

  Study title: The impact of the p7/p1 cleavage site mutations on replication capacity and drug resistance in HIV subtype C Kelly’s Master’s study focused on the viral replication capacity and drug resistance in HIV-1 subtype C patients. The study had identified drug resistance mutations found in the gag region of patients failing 2nd line treatment regimen, in the absence of major protease mutations. Additionally, she had found the p7/p1 gag mutation, A431V, was found to significantly correlate with the protease mutations L10F, M46I, I54V, V82A and L76V. In her study she was able to demonstrate that the A431V mutation alone had a higher replication capacity to that of mutants containing protease mutations in a subtype C virus. Her study highlighted the significance of gag CS mutations in drug resistance and viral fitness, however she believes future studies will be required to improve our knowledge on the interaction between gag CS’s and PR during the development of drug resistance especially in HIV-1 subtype C. Kelly is currently a 1st year medical student at the University of KwaZulu-Natal, as well as a research intern.

• Phumzile Khumalo, Master of Medical Science (Cum Laude)

  Study title: Minority HIV-1 Drug Resistance Mutations in Patients Failing Highly Active Antiretroviral Therapy (HAART). The impact of minority variants related to drug resistance on clinical outcomes remains unresolved, although there is evidence that these variants can influence treatment efficacy. Phumzile’s Master’s study determined the minority variants in HIV-1 Pol that were not detected by standard genotyping in patients failing HAART. Her study illustrates the persistence of 1st line Non-Nucleoside Reverse Transcriptase inhibitors (NNRTIs) mutations even after the patients are switched to their 2nd line therapy, highlighting the importance of detecting minority variants in patients who have failed treatment to fully assess the spectrum of drug resistance mutations. Phumzile is currently working as a Research Assistant at HPP, however she plans to complete a PhD degree in the future.

• Shevani Maharaj, Master of Medical Science (Virology)

  Study title: Allele-specific Polymerase Chain Reaction (ASPCR) to detect resistance mutations in minor variants of HIV-1 subtype C in patients failing Highly Active Antiretroviral Therapy (HAART)

2014 HPP Graduates

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• Vanessa Naidoo, Master of Medical Science

  Study title: Sequence variation and Gag-Protease driven Fitness influence on HIV-1 Mother to Child Transmission and Paediatric Disease Progression This study sought to further understand the impact of viral fitness on HIV-1 mother to child transmission and pediatric disease progression. The replication capacities of 135 recombinant viruses encoding plasma derived Gag-Protease from 53 non-transmitter mothers, 44 transmitter mothers and their 38 infected infants were assayed in an HIV-1 inducible green fluorescent protein reporter cell line. All study participants were infected with HIV-1 subtype C. Viral replication capacity in infants was found to be significantly lower than that of their transmitter mothers. Analysis of gag-protease sequences from 39 transmitter mothers and 39 infants revealed a significant association between the H28Q Gag polymorphism and increased replication capacity. Interestingly, there is a trend that more mothers (8/39) than infants (2/39) had Q at this position. The difference in viral replication capacity between infants that were defined as slow progressors and rapid progressors was also determined to investigate the impact of viral fitness on pediatric disease progression. However, no significant difference was found. These data suggest that there is a Gag-protease fitness mediated bottleneck in HIV-1 mother to child transmission and other factors are involved in pediatric disease progression. There have been many studies that investigated the role of Gag-Protease-driven viral fitness transmission in adult cohorts and adult disease progression. However, the studies done to investigate the role of viral fitness in the context of mother to child transmission and pediatric disease progression are limited. This study shows that a transmission bottleneck does exist between each HIV infected mother/infant pair. Transmitter mothers are more likely to transmit less fit viruses to their infants out of their diverse quasispecies of virus. Differences in viral replication capacity between the transmitter mothers and infants could be partially explained by differences in the Gag H28Q polymorphism. However, further studies will be needed to determine if viral Gag-Protease-driven replication capacity immediately after birth is a predictor of HIV paediatric disease progression since the sample size in this study may have been too small to adequately address that question. Vanessa is currently a Research Assistant at the HPP. At some point she wishes to pursue a PhD degree, but at the moment loves where she is in her career where she is maintaining collaborations with other research facilities around the world.

2013 HPP Graduates

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• Dshanta Naicker, Phd (Immunology)

  Study title: Effects and Mechanisms of Interleukin-10 Promoter Polymorphisms on HIV-1 Susceptibility and Pathogenesis My Master’s research on the role of mutations in the promoter region of the IL-10 gene showed an association with HIV susceptibility. In this study, we expanded our research and cohort groups, looking at more polymorphisms in the IL-10 promoter, mechanistic studies of IL-10 mutations in acute and chronic infection. The role of IL-10 in HIV infection and pathogenesis is complex. IL-10-promoter variants may influence the rate of HIV-1 disease progression by regulating IL-10 levels and the breadth of CD8 T-cell immune responses, as well as certain markers of CD4, CD8 and B cell activation. This area of research is novel, and has not been previously studied in an African setting of HIV infection. Data from this research contributes to the knowledge of HIV/AIDS and possibly identifies new avenues of research and therapy. Dshanta says that she is proud to have been involved in this area of research, which will contribute to the HIV cause. Dshanta is currently a postdoctoral fellow at the AIDS Research Unit at the National Institute for Communicable Diseases (NICD) in Johannesburg.

• Pamla Govender, Master of Medical Science Cum Laude (Virology)

  Study Title: Characterisation of the CD161+ CD4+ T cell population in HIV and MTB infected individuals HIV infection is characterized by immune dysfunction that predisposes infected individuals to opportunistic infections such as Mycobacterium Tuberculosis (MTB). The result of this is an exacerbation of HIV-TB related deaths annually. Therefore there is an imperative need for HIV-TB focused research that aims to identify immunological factors that are involved in the control of MTB and HIV in both mono- and co-infected individuals. The CD161+ CD4+ T cell subset is linked to a distinct phenotypic and functional profile. Importantly, these CD161+ T cells may act as an important component of immunological defense and provide protection in infected tissues. The aim of this study was to assess the frequency, phenotype and functional characteristics of CD161+ CD4+ T cells in HIV and MTB infected versus uninfected individuals. There is little known regarding the impact of HIV and MTB infection on the CD161+ CD4+ subset. This study therefore contributes to knowledge on the impact that these infections have on this important cell subset, through characterization of the subset and assessment of its phenotypic and functional profile in healthy versus HIV and MTB infected individuals. Pamla aspires to one day become involved in science education. She feels it is important to educate South African youth on the importance of science and research, and hopes to be able to impart the knowledge she has gained on HIV and MTB through her experience in these areas.

• Ravesh Singh, Phd (Virology)

  Study Title: Regulation of TRIM E3 Ligases and Cyclophilin A and the Impact on HIV-1 Replication and Pathogenesis Type 1 interferons (IFN-1) induce the expression of the Tri-partite interaction motif (TRIM) family of E3 ligases but the contribution of these antiviral factors to HIV pathogenesis is not completely understood.  TRIM5α restricts HIV-1 by promoting premature disassembly of HIV-1 capsid while cyclophilin A (CypA), a human protein that is incorporated into the HIV-1 virion increases viral infectivity by facilitating proper uncoating of HIV-1 capsid.  TRIM E3 ligases play an important role in HIV-1 replication and pathogenesis.  Illustration of the mechanism of their regulation can identify novel anti-HIV targets. Ravesh hypothesized that increased expression of specific IFN-1 and TRIM isoforms is associated with significantly lower likelihood of HIV-1 acquisition and viral control during primary HIV-1 infection.  Ravesh demonstrated in this study that TRIM22 could be a candidate for novel therapeutics against HIV-1 in numerous stages of disease progression. Further studies to pursue this potential antiviral strategy are warranted. He has also demonstrated that CypA, a HIV-1 cellular cofactor plays a part in HIV-1 replication and speculates that blocking CypA may be advantageous as an antiviral strategy. Ravesh is currently a postdoctoral fellow at the HPP. He hopes to become a senior researcher in this field and ultimately, one of the leaders in this field of research.

• Ngomu 'Akeem' Akilimali, Master of Medical Science (Immunology)

  Study Title: The role of Leukocyte Immunoglobulin (Ig)-Like Receptors (LILRs) in Mycobacterium tuberculosis (Mtb)/HIV co-infection While Leukocyte Immunoglobulin (Ig)-Like Receptors (LILRs) have been reported to express on immune cell subtypes that are important in both Tuberculosis (TB) and HIV infection, a wealth of research has focused on the action of LILRs in the areas of transplantation, cancers and allergies. To date there is no data on LILR expression in Mtb infection or Mtb/HIV co-infection. We investigated the expression profile of LILRs on different immune cell subtypes (T cells, Monocytes and DCs) from TB and HIV uninfected individuals. We then compared the LILRs expression profile on these cell subsets in uninfected individuals and Mtb/HIV mono- and co-infected individuals with both suspected latent TB infection and active TB disease. The result of this study demonstrated that HIV-induced upregulation of LILRs is abrogated in individuals co-infected with Mtb. This research is important because it shows that LILRs are expressed on immune cells important in the Mtb immune response, and while HIV infection is linked to increased levels of LILRs expression, this is reduced in Mtb infection and Mtb/HIV co-infection. This study received 2nd Prize for the Best Research Paper Presentation awarded at the 3rd National TB Conference held at the ICC in Durban in June 2012.  Akeem is currently completing his PhD under the supervision of Professor Ndung’u at KwaZulu-Natal Research Institute for TB and HIV (K-RITH). Upon completing his PhD he hopes to pursue a post-doctoral fellowship in Bio-medical engineering and Biotechnology-related research in order to acquire relevant experience that he can use to grow this capacity in Africa.

  
  

• Eshia Moodley-Govender, Phd (Immunology)

  Study title: Cellular immunity, immune activation and regulation in HIV-1 infected mother-child pairs: What are the determinants of protective immunity?

  

• Nasreen Ismail, Master of Medical Science (Immunology)

  Study Title: Characterization of HLA-B*4201 Restricted Responses in Clade-C HIV Infection Human leukocyte antigen (HLA)-B*4201 is a common allele in the Zulu/Xhosa population in South Africa and has been associated with relative control of HIV-1 viral load in some studies. However, the specific epitopes presented by this HLA protein have not all been identified. Additionally, the mechanisms by which protective HLA class I molecules such as HLA-B*4201 influence HIV-1 viral load are poorly understood and this has important implications for rational vaccine design. Here, we optimally defined seven HLA-B*4201- restricted cytotoxic T lymphocyte epitopes from an analysis of 34 HLA-B*4201 positive, antiretroviral therapy naïve, clade C infected subjects using epitope-specific T-cell lines and HLA-mismatched B-cell lines by interferon-gamma (IFN-γ) intracellular cytokine staining. Analysis of 464 chronically infected HIV-1 positive patients revealed that HLA-B*4201-positive individuals were more likely to make IFN-γ ELISPOT responses to the Gag overlapping peptide 25 (OLP 25) than HLA-B*4201 negative patients (p-value < 0.0001, Fisher‟s exact test). HLA-B*4201 positive patients were more likely to have immune-driven mutations in this peptide (p <0.0001, Fisher‟s exact test). The optimally defined peptide found within OLP 25 is TPQDLNTML (TL9). HLA-B*4201 positive individuals with an immune response to this peptide had higher median CD4 count (p=0.0138) and lower median viral load (p=0.0002) than non-responders. This study optimally defined 7 novel epitopes targeted by HLA-B*4201 which may assist in studies of HLA-driven HIV pathogenesis and vaccine design. We have demonstrated that HLA-B*4201 selects for escape mutations within Gag OLP 25 and have characterized immune escape pathways associated with HLA-B*4201. Nasreen is currently the Laboratory Manager at the HPP.

• Serron Wilson, Master of Medical Science - Summa Cum Laude

  Study Title: Impact of p2/NC Cleavage Site Polymorphisms on HIV-1 Subtype C Viral Fitness

  

• Sinaye Ngcapu, Master of Medical Science

  Study title: Nucleoside Reverse Transcriptase Inhibitors-associated Mutations in the RNase H Region of HIV-1 isolates in South African Adults and Children failing Highly Active Antiretroviral Therapy This project focused on mutations in the RNase H region of HIV-1 that occur in patients treated with a specific class of antiretroviral (ARV) drugs, the nucleoside reverse transcriptase inhibitors (NRTIs). One of the short-comings of ARV therapy is the development of resistance to these drugs, which is usually caused by mutations in the regions of the virus that the drugs target. Currently, very little data exist on the effects of changes in the RNase H region on development of resistance to the NRTIs, particularly for subtype C, the subtype that is dominant in Southern Africa. What has already been shown is that some mutations in this region can enhance NRTI resistance in patients infected with subtype B, the subtype most common in the US and Europe. Most ARV regimens that are available in resource-poor regions, including South Africa, include the NRTIs in both first and second-line regimens. Therefore, mutations relating to resistance to these ARVs have important public health implications. The findings of this project will assist policy-makers to develop treatment guidelines that will ensure that drugs are used effectively, and that the virus is suppressed for as long as possible on that regimen. Sinaye says that this achievement is both an exciting and emotional one. As he was forced to work to support himself through his studies, Sinaye did not have the luxury of being a full time student, and yet he consistently aimed for excellence under difficult conditions. “A sincere thank you to Prof. Ndung’u and Dr. M. Gordon for believing in me when I was too weak and exhausted to believe in myself”, he says. Sinaye is currently a senior research associate and PhD candidate under the supervision of Professor Jo-Ann Passmore (UCT) and Professor Quarraisha Abdool Karim (UKZN-CAPRISA) in the HIV Mucosal Immunology Laboratory at CAPRISA. His current project focuses on the influence of injectable hormone contraceptive (DMPA) use on susceptibility to HIV infection in the context of a heterosexual epidemic and the potentially confounding effect of DMPA use in prevention research.

• Vinod Patel, MD, PhD

  Study Title: Utility of newer and novel technologies for the diagnosis of tuberculosis meningitis The diagnostic confirmation or exclusion of tuberculous meningitis (TBM) is challenging. In resource-limited settings, fuelled by the HIV co-infection, there is a burgeoning tuberculosis epidemic and hence increase in the number of cases of TBM. In HIV-infected immunocompromised patients the results of CSF examination are variable and non-specific, culture and microscopy yields are low and polymerase chain reaction (PCR) is often unavailable. This study evaluated several novel or newer diagnostic technologies including measurement of early secreted antigenic target six (ESAT-6), culture filtrate protein ten (CFP-10), lipoarabinomannan (LAM) antigen and unstimulated interferon gamma (IFN-γ) levels in the cerebrospinal fluid (CSF) for the diagnosis of TBM. These tests are novel and have now been evaluated such that their utility in appropriate clinical application for patient diagnosis and management is possible. Dr. Patel is the Clinical Head of the Neurology unit at Inkosi Albert Luthuli Central Hospital.

2012 HPP Graduates

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• Derseree Archary, Phd (Virology)

  Study title: Neutralizing antibody responses and viral evolution in a longitudinal cohort of HIV subtype C infected antiretroviral-naïve individuals    HIV-1 envelope (Env) diversity is arguably the most significant challenge for the development of an efficacious vaccine. An ideal vaccine would elicit the production of broadly neutralizing antibodies, capable of retaining potent activity against a diverse panel of viral isolates. The evolutionary forces that shape the diversity of envelope and ensuing neutralizing antibody responses are incompletely understood in HIV-1 subtype C infection, the dominant subtype globally. Therefore there is an urgent need to define the patterns of envelope diversity, determine the correlates of immune protection and to discover subtype C immunogens in order to develop a globally relevant vaccine. Our data suggest that separate regions of Env are under differential selective forces, and the heterogeneity of env diversity and evolution differ with HIV-1 disease course. Analysis of circulating viruses in slow progressors and progressors indicate that Env diversity, length polymorphisms, sites under positive selection pressure, and potential N-linked glycosylation sites (PNGs) consistently map to specific regions in Env.  Cross-reactive neutralizing antibodies targeting key regions suggest that neutralizing antibody breadth may be dictated by a limited number of Env regions. The potencies of autologous antibody responses were directly affected by certain key Env gp160 genotypic characteristics.  Candidate vaccines may have to include multiple subtype immunogens to elicit the production of potent, broad cross neutralizing antibodies with high binding affinity. Overall, the data suggest that continuous evolution of neutralizing antibodies was a potential marker of HIV-1 disease progression. Further studies on larger cohorts to identify people with potent neutralizing antibodies to identify specific targets are needed.  Although neutralizing antibodies do not contribute to disease attenuation, antibodies found in chronic disease are known to have breadth. Vaccine immunogens based on these antibody targets may be important for the development of potent, broadly cross-neutralizing antibodies that may block HIV-1 infection. While Dr. Archary says that she feels extremely honoured to have been awarded her degree, she is also relieved that this challenging journey has come to an end. “I have had the honour of working with Thumbi Ndung’u who continues to inspire me”, she goes on to say. “Thanks Prof for all your encouragement and the faith you have had in me”. Dr. Archary currently occupies a post-doctoral research associate position at the CAPRISA Immunology Research Unit and she is working on B cell immunology and correlates of HIV-1 acquisition/protection in the cervico-vaginal compartment of the female genital tract and establishing both male and female models of HIV-1 infection.

• Jaclyn K. Wright, PHD (Virology)

  Study title: Impact of Immune-Driven Sequence Variation in HIV-1 Subtype C Gag-Protease on Viral Fitness and Disease Progression   The main aim of this project was to investigate the impact of HIV-1 Gag mutations selected by the immune system on HIV-1 replication capacity, and the impact of this on HIV-1 disease progression. To investigate this, recombinant viruses encoding patient-derived HIV-1 Gag-protease were constructed from a large cohort of HIV-1-infected individuals and the replication capacities of these viruses were measured.  The relationship between these viral replication capacities and different host HLA proteins (host immune system proteins), HIV-1 Gag mutations that are selected by the HLA proteins and promote escape from HLA-restricted cytotoxic T cell immune responses, and markers of HIV-1 disease progression, were then investigated.  It was found that HLA proteins associated with slower HIV-1 disease progression selected for slower HIV-1 replication capacity through driving mutations in Gag. The results suggested that both the viral replication capacity and effectiveness of the cytotoxic T cell responses as influenced by mutations in Gag are important in influencing HIV-1 disease progression. It was also found that mutations in more conserved regions (regions that infrequently have mutations) of the Gag protein were the most strongly associated with lower viral replication capacity.  Overall, the data support the concept of an HIV-1 vaccine in which immune responses are directed towards several conserved regions of HIV-1 Gag, with the aim to constrain immune escape mutations (thereby maintaining effective cytotoxic T cell responses) and/or slow HIV-1 replication (in the event of partial escape), resulting in slower disease course and reduced HIV-1 transmission at the population level. This research ultimately contributes to a better understanding of the viral and host immune factors determining the rate of HIV-1 disease progression, which is relevant to HIV-1 vaccine design. Dr. Wright says that she is grateful to have had the opportunity to undertake this research project and achieve her PhD degree. “It has been a wonderful experience” she says, “allowing me to travel and contribute to science”. Dr. Wright is currently employed as a lecturer and researcher at the HPP at the University of KwaZulu-Natal. Her current research involves directly testing in vitro the effect on viral replication capacity of different combinations of Gag mutations predicted to be harmful to viral replication capacity. The relationship between HIV-1 Nef functions (e.g. CD4/HLA down regulation), immune-selected Nef mutations and HIV-1 disease progression will also be investigated.

• Danni Ramduth, PhD (Immunology)

  Study title: Characterization of CD4+ and CD8+ T Cell Responses in HIV-1 C-Clade Infection

   

  HIV-1 infection induces virus-specific immune cells (amongst them CD4+ and CD8+ T cells) to identify and destroy infected cells. C-Clade infection is dominant in developing countries so characterising the immune responses in adults and children in these countries is very important for designing vaccine candidates. We found that the HIV structural protein, Gag, was the dominant target of CD8+ and CD4+ T cells in both adults and infants, and in adults, responses to this protein were associated with control of HIV-1 infection.

  In addition, we investigated whether treating HIV infected at an early stage and then stopping treatment after one year would confer some long-term clinical benefit. We found that this strategy did not confer any protection against clinical progression. HIV specific CD4+ T cell responses were detected at very low frequencies in infected infants, indicating a dysfunctional immune response in untreated HIV infected infants.

  This study was ultimately able to identify specific HIV-1 epitopes targeted by CD4+ T cells that were associated with control of HIV viremia. These epitopes require further investigation as potential vaccine targets.

  Dr. Ramduth says that she was very proud to have been involved in such a research project, which she describes as being an educational and positive experience on various levels. She feels that it is important to acknowledge those individuals who made her research project possible, from the study’s Principal Investigators to the patients themselves.

  Dr. Ramduth currently works for a multinational company, where she is able to utilise the knowledge gained from her studies in assisting other researchers in the Bioscience field.

• Paradise Madlala, Phd (Genetics)

  Study title: Association of Genetic Polymorphisms in Select HIV-1 Replication Cofactors with Susceptibility to HIV-1 Infection and Disease Progression    People are not equally susceptible to HIV-1 infection and those that are infected with HIV-1 progress to AIDS at different rates. Genetic polymorphisms (variation) within human (host) genes that enhance HIV-1 replication (or infection) partially explain the observed differences in susceptibility to HIV-1 infection and disease progression. Dr. Madlala’s research focused on characterizing human genetic variation in a validated group of host HIV-1 replication co-factors (host proteins that enhance HIV-1 infection) and the role of such genetic variation in HIV pathogenesis in the South African population. Perturbing the replication cofactor-HIV-1 interaction could be an important and novel HIV-1 therapeutic strategy. Many of the genes involved in host immune response and HIV-1 replication co-factors that are required for completion of HIV-1 life cycle show differences in allele frequencies, as a result of historic selective pressures. Understanding the influence of human genetic variation on HIV-1 pathogenesis could provide clues on and guide the design of antiretroviral drugs and possibly the development of an HIV vaccine. Paradise’s research team were the first group to show that genetic variation in PSP1 gene, which codes for LEDGF/p75, plays a role in HIV pathogenesis. Paradise is currently a postdoctoral fellow training at the University of Leuven in Belgium. He hopes to continue to make a contribution towards the fight against HIV/AIDS.

• Bongiwe Ndlovu, Master of Medical Science (Paediatrics and Child Health)

  Study Title: Innate Immune Mechanisms in Limiting HIV-1 pathogenesis among South African Mother-Infant pairs We evaluated the role of natural killer cell surface receptors, KIRs and their cognate HLA ligands in HIV-1 exposed infants. Using dried blood spots stored for 8 years, we evaluated 3 methods of genomic DNA extraction with and without WGA to characterize immune-related genes: IL-10, KIR and HLA class I. QIAamp DNA mini kit yielded the highest gDNA quality with sufficient yield for subsequent analyses and WGA was not reliable for SSP-PCR analysis of KIR genes or high resolution HLA genotyping.   In a cohort of 370 infants (124 HIV-1 infected, 120 exposed uninfected and 126 unexposed healthy infants), we characterized KIR and HLA genotypes. The frequency of HLA-Cw*04:01 allele was associated with increased susceptibility of mother-to-child acquisition of HIV-1. Moreover, HLA-A*23:01 was associated with decreased CD4 T cell count and HLA-B*81 was associated with higher CD4 T lymphocyte count in HIV-1 infected infants (p=0.04, ANOVA). KIR2DS1 and KIR2DL5 were both associated with faster HIV-1 disease progression. This study identified a robust method for DNA isolation from the stored DBS samples and demonstrated that whole genome amplification should be used with great caution for subsequent genetic analysis of KIR and HLA genotypes. We identified protective KIR/HLA genotypes which could be used to present viral epitopes to either NK cells via KIR genes or cytotoxic T cells and enhance immune activation with may promote resistance to HIV-1 infection. Bongiwe is currently a PhD student at the HPP, as well as a Developmental Lecturer in the School of Laboratory Medicine and Medical Sciences.

2011 HPP Graduates

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• Christina Thobakgale, Phd (Immunology)

  Study title: HIV specific CD8+ T cell responses in infants enrolled on a study of early highly active antiretroviral treatment (HAART) and supervised treatment interruption (STI)   Dr. Thobakgale’s study aimed to examine immunological factors that contribute to rapid disease progression in infants following HIV-1 acquisition through mother-to child-transmission when compared to older children or adults. Particularly, the study focused on the immune cells called CD8+ T cells, when they develop in infants, their contribution and function in fighting HIV and how the genetic make-up of the mother affects the ability of the child to fight the virus during early stages of life. The key findings of the study were that slow progression to disease in a few group of children was attributed to the genetic make-up of the mother or child, the acquisition of viruses that were less fit from the mother or crippling of the virus by the child’s own immune responses and also the functionality of the immune CD8+ T cells. The ability of infants to respond to HIV within early days of life has important implications for vaccines. “I’m delighted at the body of information a single study like this one has produced over the years and still has potential to answer so many more research questions”, Dr. Thobakgale had to say. “I’m humbled to have had the guidance to contribute to so much knowledge.” Dr. Thobakgale is currently a Senior Lecturer and Researcher at the HPP at the University of KwaZulu-Natal. “HPP is one of the best places to undertake a career in research”, Dr. Thobakgale believes. “The mentorship, support and opportunities are endless.”

• Veron Ramsuran, PhD (Genetics)

  Study Title: Genetic/epigenetic determinants in chemokines and chemokine receptor genes that influence HIV susceptibility in a cohort of high-risk women from South Africa. There are a lot of differences as to who gets HIV infected. Some individuals remain HIV negative despite repeated exposure to HIV. A person’s genetic make-up has explained some of these differences; however a lot is still unknown. Most studies are conducted in more developed countries, therefore little is known of the genetic impact on developing countries like South Africa. In Veron’s study, he examined female sex workers, a group that is exposed to HIV on a regular basis, but do not necessarily contract the virus. One gene that has gained much attention is the Chemokine Co-Receptor 5 (CCR5) gene. This receptor is needed for HIV to enter into a cell. A mutated form of this gene protects against HIV. However, this mutated form is mainly found in Caucasians. Therefore, other factors protecting the female sex workers exist. Veron began by examining the CCR5 gene and it was noticed that increased numbers of ligands (genes that bind onto CCR5) protect against HIV. Secondly, he showed that the CCR5 expression (amount of CCR5 found on surface of a cell) is affected by methylation (change in the DNA sequence) in the CCR5 promoter region. CCR5 methylation is lower in HIV+ subjects compared to HIV- subjects. He further explored another chemokine receptor, Duffy Antigen Receptor for Chemokines (DARC). In this result he was able to show that prior to acquiring HIV the neutrophil and platelet counts influence risk of HIV infection. A mutation in the DARC gene causes this gene to lose expression on the cell surface. Individuals that have this mutation and have lower neutrophil counts, have a higher likelihood of acquiring HIV. This study was the first to explore host genetic influence on South African female sex workers. New mechanisms that protect and influence HIV infection were identified. The high prevalence of African individuals with Duffy-null-associated low neutrophilmay contribute significantly to the dynamics of the HIV epidemic in Africa. This study has identified a few potential vaccine target sites. “Firstly to have successfully completed a project like this there has to be numerous people involved, I thank everyone especially my supervisors Prof. Thumbi Ndung’u and Dr. Sunil Ahuja for all help and support”, Veron had to say. Veron currently occupies a post-doctoral position at the National Cancer Institute of the National Institutes of Health (NIH), in Maryland, USA. He is training in Dr. Mary Carrington’s laboratory - a world renowned HIV host genetic scientist. He says his future plans are to obtain a faculty position in a prestigious research institute after he completes his post-doctorate training.

• Hannah O. Ajoge, Phd (Microbiology)

  Study title: Molecular epidemiology of HIV among pregnant women sampled in 2007 from North-Central Nigeria

  Ahmadu Bello University, Zaria, Nigeria

  

• Nompumelelo Mkhwanazi, MMedSc (Immunology)

  Study Title: The role of HLA-C restricted CD8+ T cell responses in the control of HIV replication Certain HLA-B restricted CD8+ T cell are associated with the control of viremia whereas HLA-C restricted responses, including Gag epitopes are associated with high viremia. In this study we were trying to understand the role of HLA-C restricted epitopes in the viral control. We quantify the HLA-C restricted CD8+ T cells using ELISPOT and intracellular cytokine staining. We studied the polyfunctionality of HLA-C and HLA-B restricted CD8+ T responses within the same patients. The results suggested that polyfunctionality of HIV-1 specific CD8+ T cells is associated with disease progression independent of restricting alleles, and that loss of these polyfunctional cells correlates with increased in the frequency of monofunctional virus-specific CD8+ T cells. In addition, sequence variation does not appear to significantly impact CD8+ T cell polyfunctionality in chronic infection. This information helps to understand the HIV-1 specific CD8+ T cells restricted with different HLAs. Vaccine designers will therefore know which component of the virus will be included and which one will be excluded. Mpume is currently completing her PhD, entitled “The phenotypic effect of mutation in the connection and RNase H domains of HIV-1 subtype C on the reverse transcriptase”, under the supervision of Dr Michelle Gordon. She is also a Developmental Lecturer at UKZN.

• Reshmi Dayanand, MMedSc

  Study title: The association of early neonatal feeding with clinical outcomes and cytotoxic T lymphocyte (CTL) responses in HIV exposed low birth weight infants Sub-Saharan Africa remains to date at the forefront of the HIV/AIDS epidemic. Despite breastfeeding being a significant mode of postnatal HIV transmission it remains the main nutritional source and pillar of child survival for the majority of infants born in Africa. It is therefore not surprising that considerable research has centred on making breastfeeding safer in terms of HIV transmission. The flash heat treatment method (HTEBM) provides a unique opportunity to safely breastfeed infants but prevent mother-to-child transmission of HIV. Cytotoxic T lymphocyte (CTL) responses have been well documented in HIV-infected adults and children. However, there is a lack of literature on CTL responses in HIV exposed low birth weight infants. This pilot study attempted to examine the association of early neonatal feeding on the clinical outcomes and CTL responses in HIV exposed low birth weight infants. This research project provided further insight into the optimal feeding method for HIV exposed infants. The study showed that it is feasible for HIV infected mothers to heat-treat their expressed breastmilk during hospital admission. Furthermore, Reshmi was able to demonstrate in this cohort of patients that the clinical outcomes and growth parameters of infants fed HTEBM were similar to that of infants fed either formula or unheated breastmilk. She was unable, however, to demonstrate HIV-specific responses in the infected infants or the uninfected infants who had been exposed to heat inactivated virus in HTEBM. The findings indicate that this pilot study was limited in its ability to detect CTL responses in HIV exposed low birth weight infants and further studies are warranted. “This has truly been an opportunity of a life-time”, says Reshmi. “I was deeply humbled and grateful to work closely with both the clinical team and study participants from various socioeconomic backgrounds. I now have a much deeper understanding and appreciation for research.” Reshmi is currently pursuing her PhD at the Discipline of Pharmaceutical Sciences at UKZN. Her future ambition is to use such experiences and opportunities to strengthen her career as a scientist and to be at the forefront of research in Africa.

• Sahil Tulsi, Bachelor of Science (Honours), (Microbiology)

  Study title: Regulation of TRIM E3 Ligases by Pro- and Anti-Inflammatory Cytokines in Human Immune Cell Lines

2010 HPP Graduates

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• Ashika Singh, PhD (Virology)

  Study title: Coreceptor utilization and primary cell tropism by HIV-1 subtype C strain This research project was undertaken to better understand the functional and genotypic characteristics of dual tropic HIV-1C isolates, and to characterize drug resistance and coreceptor usage patterns in HAART-naïve versus HAART-failing HIV-1C infected patients. The study has important implications for HIV pathogenesis in HIV-1 subtype C infected individuals, for monitoring of persons on therapy and for future ARV treatment options for individuals failing HAART in resource-poor settings. The findings of Ashika’s research enhances our understanding of HIV-1 subtype C pathogenesis and the results have important implications for the use of coreceptor antagonists for the clinical management of HIV-1C infection. Obtaining a PhD degree, Ashika says, is achieved by commitment and hard work. “I am proud to know that I have fulfilled the opportunity of attaining this accomplishment. I am extremely grateful to all those who supported me throughout the completion of my PhD." Ashika currently works as a medical scientist at the National Institute for Communicable Diseases. Her current research involves antibiotic resistance in hospital acquired-infections.

• Shabashini Reddy, MMedSc (Immunology)

  Title: HIV-1 specific T-cell responses in chronic HIV infected children during continuous treatment and structured treatment interruptions (STI) This was a pilot study where we studied chronically HIV-1 infected, treatment naïve children (aged between 2-12 years). We examined their CD8+ T cell immune responses and compared these responses to those of children in acute infection or in continuous or structured treatment regimens.  HIV drug resistance testing was done for the children in case of treatment failure. CD4 counts and viral load measurements were done on all visits. Immune responses were monitored to see if there were any trends associated with time since infection or treatment status. The following were the main findings of the study: 1. It was learnt that structured treatment interruptions (STIs) cannot be performed in HIV-1 chronically infected children due to high treatment failure rate. 2. CD8+ T cell immune responses of children became broader and of higher magnitude as infection progressed to the chronic stage, resembling those in chronically infected adults. 3. HLA-B5801 was a good allele and children did well if they possessed this allele. 4. School teachers reported good progress on the children’s wellbeing and attitude towards school. Shabashini acknowledges that conducting her Master’s research project was a challenging experience which required a great deal of hard work, as she was employed full time at the time with a family to take care of. She was thus thrilled about her achievement when she was awarded her degree. Shabashini was a loyal HPP employee for 8 years. She says that she gained significant research experience whilst at HPP and is proud of her contributions to the organisation. She now works at the Medical Research Council’s HIV Prevention Research Unit (HPRU) in Westville.

• Jennifer Giandhari, MMedSc, Cum Laude (Virology)

  Title: The Role of the Protease Cleavage Sites in Viral Fitness and Drug Resistance in HIV-1 Subtype C

  

2009 HPP Graduates

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• Shamman Sewram, Master of Science (Genetics)

  Study title: Characterization of Human TRIM5α Expression and Association with Viral Control during Primary HIV-1 Infection

2008 HPP Graduates

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• Sivapragashini Danaviah, PhD (Virology)

  Study title: A Molecular Study of the Immunopathogenesis of Spinal TB in HIV Positive and Negative Patients

• Dshanta Naicker, Master of Science (Genetics)

  Study title: The Role of Interleukin-10 Promoter Polymorphisms in HIV-1 Susceptibility and Primary HIV-1 Pathogenesis The aim of this study was to determine if the cytokine IL-10 plays a role in HIV susceptibility and disease progression. Mutations in the promoter region of the gene have been previously shown to associate with HIV susceptibility and disease progression. In this study, Dshanta investigated the frequency of these mutations in a South African cohort, which had not been previously studied. She found that these mutations were present in our study population, and individuals that had mutations associated with lower IL-10 levels were more likely to become infected. However, the role during HIV infection was more complex and seemed to be dependent on the phase of infection. This research is novel, and had not been previously studied in an African setting, where the burden of disease is high. This data contributes to knowledge on HIV/AIDS as it provides new research. Dshanta says that she feels proud to have been involved in this novel area of research.