Doctoral Degrees (Virology)

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Acquired and transmitted drug resistance in HIV-1 subtype C : implications of novel mutations on replication capacity, cleavage and drug susceptibility.
(2015) Singh, Urisha.; Gordon, Michelle Lucille.
Introduction Large scale roll-out of combination antiretroviral therapy (cART) has been successful in improving the quality of life of HIV-1 infected individuals in South Africa (SA). However the development and transmission of drug resistance threatens the future success and longevity of cART in the country. Studies have shown that resistance to Protease inhibitors (PI’s), in the absence of mutations in Protease (PR), is increasing in SA. Whilst some studies attribute this to poor treatment adherence, others have shown that mutations in Gag contribute to PI resistance. The majority of these studies however have been conducted on HIV-1 subtype B, despite HIV-1 subtype C being the most prevalent subtype globally. Given that Gag is highly polymorphic between subtypes, studies focusing on HIV-1 subtype C are required. Despite the high rate of virologic failure of patients on PI inclusive treatment regimens, no transmitted drug resistance (TDR) studies have identified PI associated TDR mutations. This could be due to the high fitness cost associated with PR mutations which would result in rapid reversion or low frequency of mutations within the viral quasispecies. Most TDR studies in SA, as in other resource limited settings, have used recently infected cohorts to measure TDR. It is however unlikely that rapidly reverting mutations would be detected in recent infection. Furthermore, these studies have all used Sanger sequencing which only detects mutations at frequencies >15-20%. With recent studies showing that low frequency mutations present at frequencies as low as 1% impact treatment outcomes, the elucidation of these mutations using deep-sequencing techniques is necessary. For a true measure of TDR, studies employing acute infection cohorts and deep-sequencing techniques are required. The current study aimed to identify mutations in Gag-Protease associated with PI resistance/exposure, and to determine their impact on replication capacity and drug susceptibility. The prevalence of low frequency TDR mutations in an HIV-1 subtype C acute infection cohort was also investigated. Methods A cohort of 80 HIV-1 subtype C infected participants failing a PI inclusive treatment regimen (i.e. PCS cohort) from 2009–2013 in Durban, South Africa was used to assess the role of Gag in PI resistance. Gag mutations were divided into three groups: PI exposure associated Gag mutations; resistance associated Gag mutations (rGag) and novel Gag mutations (nGag). Frequencies of each of these mutations were compared amongst: 80 PCS cohort sequences, 2,481 HIV-1 subtype B treatment naïve sequences, 954 HIV-1 subtype C treatment naïve sequences and 54 HIV-1 subtype C sequences from acutely infected individuals, in order to identify PI associated mutations and natural polymorphisms. Next, recombinant viruses for all 80 participants were generated by co-transfection of a CEM derived T-cell line (i.e. GXR cells) with an NL43-deleted-gag-protease (NL43Δgag-protease) backbone and patient derived Gag-Protease amplicons. Thereafter, the replication capacity of each virus was assessed using a replication assay that employed a green fluorescent protein reporter cell line and flow cytometry. Associations between replication capacity and Gag-Protease mutations were established. Eighteen viruses with mutations of interest were then selected for use in drug susceptibility assays, where the impact of mutations on susceptibility to lopinavir (LPV) and darunavir (DRV) was assessed in a luciferase based assay. Lastly, the impact of novel Gag mutations on replication capacity and drug susceptibility was validated by generating site-directed mutant viruses with mutations of interest and using these mutant viruses in replication capacity and drug susceptibility assays. Furthermore the cleavage profile of each site-directed mutant virus was established by western blotting. Samples available from 47 HIV-1 subtype C acutely infected individuals collected from 2007-2014 in Durban, South Africa, was used to assess low frequency TDR mutations in HIV-1 subtype C acute infection. Firstly the RT and PR region of each virus was genotyped using the Viroseq HIV-1 genotyping system in order to identify the prevalence of TDR in the cohort. Thereafter 14 participant samples were selected, based on the availability of plasma at one week after onset of plasma viremia (OPV), for sequencing by ultra-deep pyrosequencing (UDPS). This served to identify low frequency mutations. Comparisons in TDR prevalence was made between Sanger sequencing and UDPS. Thereafter, the impact of low frequency TDR mutations on treatment outcomes was assessed by comparing time to virologic suppression for two participants with low frequency mutations to that of four participants without low frequency mutations. Results Protease resistance associated mutations (RAMs) occurred in 34/80 (42.5%) participants, whilst Gag mutations associated with PI resistance in subtype B were detected in 67/80 (84%) participants. Overall, 12 Gag mutations associated with PI exposure (i.e. E12K, V35I, G62R, V370A/M, S373P/Q/T, A374P, T375N, I376V, G381S, I389T, I401T and H219Q), eight rGag mutations (i.e. R76K, Y79F, V128I, A431V, K436R, L449F, R452K and P453L) and four nGag mutations (i.e. Q69K, S111C/I, T239A/S and I256V) were identified in the PCS cohort. The E12K, V370A/M, T375N, G381S, R76K and Y79F mutations all occurred as natural polymorphism in HIV-1 subtype C. The A431V, K436R, L449F, R452K, P453L, Q69K, S111C/I, T239A/S and I256V mutations were all associated with PI resistance/exposure. Interestingly all viruses with PR RAMs harboured rGag and nGag mutations, however rGag and nGag mutations were also found to occur without PR RAMs. Protease RAMs were associated with significantly reduced replication capacity. The K335R and A431V mutations were the only Gag mutations associated with significantly reduced replication capacity. Viruses with PR RAMs were associated with significantly reduced susceptibility to LPV (>15 FC in IC50) and DRV (>6 FC in IC50). Furthermore, the following combinations of rGag and nGag mutations were found to confer reduced susceptibility to LPV and DRV in the absence of PR RAMs: R76K+Y79F+K436R+L449P+I256V (5.2 fold increase in IC50 for DRV), R76K+R453L (23.88 fold increase in IC50 for LPV and a 6.73 fold increase in IC50 for DRV) and R76K+K436R+Q69K+S111C (7.40 fold increase in IC50 for LPV). Analysis of recombinant viruses showed that the Q69K nGag mutation rescued replication capacity of all viruses harbouring A431V+PR RAMs. This was validated by SDM, where Q69K rescued the replication capacity of site-directed mutant viruses harbouring A431V+V82A. The Q69K mutation was also associated with increasing polyprotein cleavage when found in conjunction with A431V+V82A. With regards to TDR, we demonstrated a prevalence of 57% of TDR mutations with UDPS and 2.2% with Sanger sequencing. Sanger sequencing identified the K103N non-nucleoside reverse transcriptase inhibitor (NNRTI)-associated TDR mutation. In addition to K103N (frequency: >99%), the following low frequency mutations were detected by UDPS: the K65R (1-1.5%) and D67N (3.88%) nucleotide reverse transcriptase inhibitor (NRTI)-associated TDR mutations, the F53L (17.6%) and M46L (6.3%) Protease inhibitor (PI)-associated TDR mutations, and the T97A (2.90%) integrase strand transfer inhibitor (InSTI)-associated TDR mutations. Participants with low frequency TDR mutations took 40 days longer to achieve viral suppression than participants without low frequency TDR mutations, when placed on fixed dose combination antiretroviral therapy.
Biochemical characterization of highly mutated South African HIV-1 subtype C protease.
(2021) Eche, Simeon.; Gordon, Michelle Lucille.
Understanding the underlying molecular mechanism of HIV-1 protease (PR) inhibition by HIV-1 protease inhibitors (PIs) is essential to gain mechanistic insight into the evolution of resistance to HIV-1 PIs. HIV-1 PIs have improved patient care management, but the accumulation of drug resistance mutations in the HIV-1 PR gene diminishes their inhibitory capacity. The current study investigated the kinetic and structural characteristics of highly mutated South African HIV-1 subtype C PR from clinical isolates obtained from individuals failing a lopinavir (LPV) inclusive regimen at the point of switch to darunavir (DRV) based therapy. In this study, enzyme activity and inhibition assays were used to determine the biochemical fitness of HIV-1 PR variants and the inhibitory constants of HIV-1 PIs for drug-resistant HIV-1 subtype C proteases. The mechanistic insight into the impact of the accumulated drug resistance mutations on the HIV-1 PR structure and its interaction with LPV and DRV was obtained using fluorescence spectroscopy and molecular dynamic simulation. The study showed that the unfavorable binding landscape caused by the accumulation of drug-resistance mutations resulting from LPV associated drug pressure would shape the outcome of DRV-based therapy after a switch in the treatment regimen. This is related to the distortion of the HIV-1 PR structure associated with increased solvent exposure and instability of the HIV-1 PR dimer caused by these mutations leading to a shorter lifetime of the enzyme-inhibitor complex. Analysis of the binding kinetics of LPV and DRV with the HIV-1 PR variants showed that the drug resistance mutations caused an imbalance between the association and dissociation rate constants favoring a fast dissociation rate. The latter resulted in a reduced inhibitor residence time. Our findings showed that LPV had a longer residence time than DRV when bound to the HIV-1 PR variants; this shows LPV can be a suitable platform for developing newer HIV-1 PIs with a longer residence time. However, the enzyme inhibition mechanism shows both LPV and DRV act via a two-step tight-binding mixed inhibition mechanism, suggesting the existence of a second binding site on HIV-1 PR for these inhibitors. The information provided in this thesis adds to existing knowledge about HIV-1 PI drug resistance and for the design of novel HIV-1 PIs with the potential to evade drug resistance mutations.
Coevolution of mutations in HIV-1 ENV and GAG-PR genes: implications for the development of protease inhibitors resistance.
(2023) Maphumulo, Ntombikhona Fortunate.; Gordon, Michelle Lucille.
Abstract available on PDF.
Defining HIV persistence and host immune responses in lymph nodes of combined antiretroviral therapy (cART) suppressed individuals and the determination of the impact of HIV infection on SARS-COV-2 specific t cell responses in South Africa.
(2023) Chasara, Caroline.; Ndhlovu, Zaza Mtine.
Abstract 1 People living with HIV (PLWH) who have unsuppressed HIV are at a greater risk of acquiring infectious diseases such as Coronavirus disease of 2019 (COVID-19). More recent data has shown that unsuppressed HIV is associated with severe COVID-19 symptoms, but the mechanisms underpinning this susceptibility are still unclear. In our study we used flow cytometry and culture T lymphocyte expansion to assess the impact of HIV infection on the quality and epitope specificity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cell responses in the first wave and second wave of the COVID-19 epidemic in South Africa. We observed that HIV-seronegative individuals had significantly greater CD4+ T cell responses against the Spike protein compared to the viremic individuals living with HIV. In addition, there was diminished T cell cross-recognition between the two waves, which was more pronounced in individuals with unsuppressed HIV infection. Importantly, we identified four mutations in the Beta variant that resulted in the abrogation of T cell recognition. These findings partly explain the increased susceptibility of PLWH to diseases such as COVID-19 and highlight their vulnerability to emerging SARS-CoV-2 variants of concern. Abstract 2 The major keys to developing an HIV cure is through understanding HIV reservoir dynamics. The role of tissue macrophages in HIV reservoirs is complex and not yet fully understood. However, their ability to support viral replication, longevity, localization in immune sanctuaries, and potential for viral latency all contribute to the persistence and resilience of HIV reservoirs in various tissues throughout the body. Understanding and targeting these reservoirs is a critical area of research in the quest for an HIV cure. To gain insight into the macrophage reservoir, we used a combination of flow cytometry and immunofluorescence microscopy to characterize and investigate HIV persistence in lymph node (LN) macrophages. We detected pro-inflammatory (CD68+ ) macrophages harboring HIV Gag p24 and HIV1 RNA in the germinal centers of HIV positive early and late treated individuals suggesting their potential role as an HIV reservoir. In contrast, anti-inflammatory (CD206+ ) macrophages were localized along lymphatic vessels and outside the germinal centers. Importantly, we show the presence of longlived CD4+ TIM-4+ macrophages in LNs. The data reported in this thesis will go a long way in furthering our understanding of macrophage HIV reservoirs in lymph node macrophages. ISIZULU ABSTRACTS Abstract 1 Abantu abaphila ne-HIV (PLWH) abane-HIV engacindezelwe basengozini enkulu yokuthola izifo ezithathelwanayo njenge-Coronavirus ka-2019 (COVID-19). Idatha yakamuva ibonise ukuthi i-HIV engacindezelwe ihlotshaniswa nezimpawu ezinzima ze-COVID-19, kodwa izindlela ezisekela lokhu kuba sengozini azikacaci. Lapha, siqale sasebenzisa i-flow cytometry kanye nokwandiswa okuthuthukisiwe ukuhlola umthelela wokutheleleka nge-HIV kukhwalithi nokucaciswa kwe-epitope ye severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cell izimpendulo kumagagasi okuqala kanye negagasi lesibili. Siqaphele ukuthi abantu abangenayo i-HIV babene-CD4+ T cell impendulo enkulu kakhulu ngokumelene ne-Spike protein uma kuqhathaniswa nabantu abane-HIV abaphila ne-HIV. Ukwengeza, kube nokuncipha kokuqashelwa kwe-T cell phakathi kwamagagasi amabili, okwakubonakala kakhulu kubantu abane-HIV engacindezelwanga. Okubalulekile, sihlonze izinguquko ezine kokuhlukile kwe-Beta okuholele ekuhoxisweni kokubonwa kweseli le-T. Lokhu okutholakele kuchaza ngokwengxenye ukwanda kwe-PLWH ezifweni ezinjenge-COVID-19 futhi kugqamisa nokuba sengozini kwezinhlobonhlobo ezisafufusa ze-SARS-CoV-2 zokukhathazeka. Abstract 2 Enye yezinkinobho ezinkulu zokuthuthukisa ikhambi le-HIV ngokusebenzisa ukuqonda amandla weHIV reservoir. Iqhaza lama-macrophage ezicubu kumithombo ye-HIV iyinkimbinkimbi futhi ayikaqondwa ngokugcwele. Kodwa-ke, ikhono labo lokusekela ukuphindaphinda kwegciwane, ukuphila isikhathi eside, ukwenziwa kwasendaweni ezindaweni ezivikela amasosha omzimba, kanye namandla ngokubambezeleka kwegciwane konke kunomthelela ekuphikeleleni nasekuqineni kwemithombo ye-HIV ezicutshini ezihlukahlukene emzimbeni wonke. Ukuqonda nokukhomba lezi zindawo zokugcina amanzi kuyingxenye ebalulekile yocwaningo ekufuneni ikhambi le-HIV. Ukuthola ukuqonda nge-macrophage reservoir, sisebenzise inhlanganisela yokugeleza kwe-cytometry, iimmunofluorescence microscopy, ne-RNAscope ukukhombisa nokuphenya ukuphikelela kwe-HIV kuma-macrophages e-lymph node (LN). Sithole i-pro-inflammatory (CD68+ ) ama-macrophages aphethe i-HIV-Gag p24 ne-HIV-1 RNA ezikhungweni zamagciwane ze-HIV e-ET nabantu be-LT abaphakamisa iqhaza labo njenge-HIV reservoir. Ngokuphambene nalokho, ama-macrophages alwa nokuvuvukala (CD206+ ) ama-macrophages enziwa endaweni ngemikhumbi ye-lymphatic nangaphandle kwezikhungo zamagciwane. Ngokubalulekile, sibonisa ukuba khona kwama-CD4+ TIM4+ ama-macrophages ama-LNs. Idatha ebikwe kule thesis izohamba ibanga elide ekuqhubekiseleni phambili ukuqonda kwethu amarezebe e-HIV e-macrophage kuma-lymph node macrophages
Functional and clinical consequences of immune-driven sequence variation of gag-protease in HIV-1 subtypes A, C, D and recombinants.
(2016) Kiguoya, Marion Wangui.; Ndung'u, Peter Thumbi.; Mann, Jaclyn Kelly.
Abstract available in PDF file.
Gag-protease driven viral replication capacity among HIV-1 subtypes: Implications for disease progression, epidemic spread and vaccine design.
(2020) Farinre, Omotayo Oluwaremilekun.; Ndung'u, Peter Thumbi.; Mann, Jaclyn Kelly.
The HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. In this study, I tested the hypothesis that the subtypes prevalent in the East African epidemic, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower. Materials and methods: Gag-protease sequencing was performed on plasma samples from 213 and 160 antiretroviralnaïve participants from West and East Africa, respectively. Online bioinformatic tools were used to infer HIV-1 subtypes and recombination patterns. Replication capacities of patientderived gag-protease chimeric viruses from West (n=178) and East (n=114) Africa were determined using a green fluorescent protein reporter-based cell assay. Subtype and regional differences in viral replication capacity and amino acid variants impacting replication capacity were identified using appropriate statistical methods. Results: Subtypes identified in West Africa were CRF02_AG (65%, n=139), G (7%, n=15), A3 (5%, n=10), other CRFs (12%, n=26), various pure subtypes (9%, n=19) and A1G recombinants (2%, n=4). Subtypes A1 (64%, n=103), D (22%, n=35), AD (11%, n=17) and AC (3%, n=5) were identified in East Africa. Chimeric viruses from West Africa had significantly higher VRC compared to those from East Africa (p < 0.0001), with subtype-specific differences found among strains within West and East Africa (p < 0.0001). Recombination patterns showed a preference for subtypes D, G or J rather than subtype A in the p6 region of gag, with evidence that subtype-specific differences in this region impact viral replication capacity. Furthermore, the Gag A83V polymorphism was associated with reduced viral replication capacity in CRF02_AG (median < 0.86). HLA-A*23:01 (p = 0.0014) and HLA-C*07:01 (p = 0.002) were associated with significantly lower viral replication capacity in subtype A infected individuals from East Africa. Conclusion: Overall, the data showed that viruses from West Africa displayed higher replication capacity than those from East Africa, which is consistent with the hypothesis that lower viral replication capacity is associated with higher population prevalence.
GB Virus C / Hepatitis G Virus (GBV-C/HGV) infection in KwaZulu Natal, South Africa : its diagnosis, distribution and molecular epidemiology.
(2003) Sathar, Mahomed Aslam.; York, Denis Francis.
Recently a new Flavivirus, GB Virus C also referred to as Hepatitis G virus (GBV-C/HGV) was identified in humans with indeterminate hepatitis . Whilst in non-African countries this discovery led to an enormous enthusiasm to elucidate an association with liver disease, very little was known about the prevalence and pathogenicity of GBV-C/HGV infection in KwaZulu Natal, South Africa, where Hepatitis B Virus (HBV) infection is endemic and infection with the Human immunodeficiency virus (HIV) is a catastropic health problem. Sera from patients with liver disease (chronic liver disease [n = 98]; alcoholic liver disease [n = 50]); high risk groups (haemodialysis patients [n = 70]; HIV positive mothers and their babies [n = 75]) and control groups (alcoholics without liver disease [n = 35] and blood donors from the four racial groups [n = 232]) were screened for GBV-C/HGV RNA and Anti-E2 antibodies by reverse transcription polymerase chain reaction (RT-PCR) and an enzyme linked immunosorbent assay (ELISA), respectively. Overall 43.9% (43/98) of patients with chronic liver disease; 60 % (30/50) of patients with alcoholic liver disease; 47.1% (33/70) of haemodialysis patients; 60% (21/35) of alcoholics without liver disease and 31.9% (74/232) of blood donors (Africans] 44/76; 5.9%); Asians (5/52; 9.6%); Whites (15/49; 30.6%) and "Coloureds" [mixed origin] (9/54; 16.6%)]) were exposed to GBV-C/HGV infection as determined by the detection of Anti-E2 &/or RNA in serum. There was a significant difference in the prevalence of GBV-C/HGV infection (RNA &/or anti E2) between African blood donors and the other racial groups (p < 0.001), between blood donors and haemodialysis patients (p = 0.02) and or patients with chronic liver disease (p =0.04). There was no significant difference in the prevalence of GBV-C/HGV between African blood donors (45/76, 59.2%) and alcoholics with and without liver disease (30/50, 60% and 21/35, 60%, respectively). Anti-E2 antibodies and GBV-C/HGV RNA were almost mutually exclusive. GBV-C/HGV infected dialysis patients tended to have had more transfusions (p = 0.03) and had a longer duration of dialysis than non infected patients, indicating that the majority of patients on maintenance haemodialysis acquire their GBV-C/HGV infection through the transfusions they receive. There was no evidence for in utero and/or intrapartum transmission of GBV-C/HGY. However, there is some mother-to-infant transmission of GBV-C/HGV, though it is very probable that in KZN GBV-C/HGV is transmitted by as yet undefined non-parenteral routes. Sequence and phylogenetic analysis of the 5' non-coding region (5' NCR) and E2 gene segments of the GBV-C/HGV genome identified an additional "genotype" (Group 5) of GBV-C/HGV that is distinct from all other known GBV-C/HGV sequences (Groups 1-4). Although there is a high prevalence of Group 5 GBV-C/HGV isolates in KZN, there was no significant difference in liver biochemistry between GBV-C/HGV infected and noninfected patients with liver disease or between blood donors in each of the four racial groups. There was no significant differences in CD4 (461.12 ± 163.28 vs 478.42 ± 181.22) and CD8 (680.83 ± 320.36 vs 862.52 ± 354.48) absolute cell counts between HIV positive patients co-infected with GBV-C/HGV and those not infected with GBV-C/HGV, respectively. However, significantly higher relative CD3 [80.0 ± 4.17% vs 70.99 ± 19.79%] (p = 0.015), gamma delta T cells (yLT) [3.22± 1.30% vs 2.15 ± 29.12%] (p = 0.052) and lower CD 30 [35.45 ± 17.86% vs 50.59 ± 9.20%] (p = 0.041) status were observed in GBV-C/HGV positive compared to GBV-C/HGV negative HIV infected patients, respectively. Although there is a high prevalence of novel Group isolates of GBV-C/HGV in KZN, the lack of elevated liver enzymes and clinical hepatitis in blood donors and haemodialysis patients suggests that GBV-C/HGV is not associated with liver disease. HBV and not GBV-C/HGV modifies the course of alcoholic liver disease. The relatively higher number of CD3 cells and increased yLT expression, together with a decrease in CD 30 cells tends to suggest an association with protection and or delayed progression of HIV disease in GBV-C/HGV infected patients. Whilst GBV-C/HGV is not associated with liver disease, it may be an important commensal in HIV infected patients.
Genotypic and phenotypic characterization of HIV-1 from cerebrospinal fluid and blood compartments in patients with cryptococcal meningitis.
(2017) Sojane, Katlego.; Ndung'u, Peter Thumbi.
The combination of HIV and cryptococcal meningitis (CM) is a major cause of morbidity and mortality in sub-Saharan Africa. The phylogenetic relatedness of HIV-1 subtype C (HIV-1C) variants in peripheral blood and the central nervous system (CNS) compartments of individuals with CM is unknown. Additionally, the major and alternative coreceptor usage of HIV-1C in those compartments are not clear and these have implications for the pathogenesis of the virus, and the use of coreceptor blocking therapies. For genotypic studies, 16 antiretroviral therapy naïve individuals with CM were randomly selected. We conducted single-genome, or bulk PCR amplification and sequencing of full-length HIV-1 env genes from plasma and/or cerebrospinal fluid (CSF) of the participants. Additionally, we estimated the prevalence of CXCR4-using variants in our cohort using coreceptor usage prediction algorithms (CPAs). Next, we evaluated the usage of CCR3, CCR5 and CXCR4 expressed on NP2/U87-CD4 cells by HIV-1C Envs derived from the plasma and/or CSF of 14 of the 16 participants. CCR3 alone, or in combination with the other receptors. Overall, our results have improved the understanding of HIV-1C pathogenesis in the peripheral blood and CNS compartments of individuals with end-stage infection and CM, and provides clinically relevant information for therapies including coreceptor antagonists in this setting.
HIV cell-to-cell spread leads to a differential transcriptional response and slows evolution of drug resistance relative to cell-free infection.
(2020) Hunter, Jessica Rose.; Sigal, Alexander.
HIV transmits between hosts but also transmits between cells in the same host. How this latter, cellular transmission occurs has been the subject of extensive study. Yet why HIV transmits between cells using two different infection modes: cell-to-cell spread and cell-free infection, is not clearly understood. This is because cell-to-cell spread is a more efficient mode of infection, where the virus is able to be successfully transmitted between cells despite natural inhibitors such as antibodies. Here, using in vitro experimentation, I have determined some of the implications of cell-to-cell spread of HIV for cell death, evolution, and inflammation. I have discovered potential costs to this infection mode, such as increased cell death, slowed evolution of resistance, and an increased interferon response which may interfere with viral replication. Hence, costs associated with cell-to-cell spread may prevent it being the dominant infection mode in cellular transmission.
The HIV-1 gag and protease: exploring the coevolving nature and structural implications of complex drug resistance mutational patterns in subtype C.
(2019) Marie, Veronna.; Gordon, Michelle Lucille.
Due to the high prevalence of HIV-1 subtype C infection coupled with increasing antiretroviral (ARV) drug treatment failure, the elucidation of complex resistance mutational patterns arsing through protein coevolution is required. Despite the inclusion of LPV and DRV in second- and third-line, many patients still fail treatment. In this study, protease (PR) inhibitor resistance mutations were identified by comparing treatment versus naïve sequences datasets in Gag and PR. Thereafter, to investigate Gag-PR coevolution and pathways to LPV resistance, phylogenetic analyses and Bayesian networks were constructed. Following this, structural analyses combining homology modelling, molecular docking and molecular dynamic simulations were carried out on specific patterns of protease resistance mutations (PRMs). To complement these analyses, the structural impact of a mutated Gag cleavage site on PR resistance dynamics was also evaluated. Accordingly, this study identified 12 major PRMs and several resistance combinations. Of these, the M46I+I54V+V82A pattern frequently occurred. The second most frequently recurring pattern included L76V as a fourth mutation to the above triplet. Coevolution analyses revealed correlations between positions 10, 46, 54 and 82 in PR. Of these, minor PRM L10F occurred in 6.4% of the dataset and was involved in pathways to LPV resistance. Additionally, Gag cleavage site (CS) mutation A431V was also correlated with L10F and the major PRMs. Distinct changes in PR’s active site, flap and elbow regions due to the PRMs (L10F, M46I, I54V, L76V, V82A) were found to alter LPV and DRV drug binding. When the PRMs were combined with the mutant Gag CS binding was greatly exacerbated. While the A431V Gag CS mutation coordinated several amino acid residues in PR, the L76V mutation was found to have a significant role in substrate recognition rather than directly inhibiting the drugs. These data show that the co-selection of mutations in Gag-PR greatly contributes to resistance outcomes and that our understanding on drug resistance is largely lacking, particularly where structure is concerned.
HIV-1 integrase inhibitor mutations: analysis of structural and biochemical effects.
(2021) Mbhele, Nokuzola Brightness.; Gordon, Michelle Lucille.; Khan, Rene Bernadette.
Introduction. Combination antiretroviral therapy (cART), composed of drugs from different drug classes, is an effective HIV-1 treatment strategy. As part of cART, integrase strand transfer inhibitors (INSTIs) have become essential drugs and are now recommended for use in first-line, second-line, and subsequent HIV-1 treatment regimens. Though highly potent, the use of first-generation INSTIs Raltegravir and Elvitegravir still resulted in the development of integrase drug resistance mutations. Second-generation INSTIs Dolutegravir, Bictegravir, and Cabotegravir were developed to combat the emerging resistant virus strains to first-generation INSTIs and are considered some of the best antiretroviral drugs in HIV-1 treatment. Despite the fundamental changes and improved performance in second-generation INSTIs, they are not immune to drug resistance. This highlights the need to understand the molecular mechanisms of resistance to INSTIs. This thesis, through a combination of structural and biochemical methods, seeks to understand resistance development in South African HIV-1 subtype C (HIV-1C) viruses and the effect of resistance mutations on enzyme-substrate binding, DNA binding, and 3’ processing. Methods. A total of 48 HIV-1C sequences were analyzed in this study, of which 7 had a virologic failure (i.e. plasma viral loads >1000 copies/mL) and 41 were INSTI naïve isolates (32 treatment-naïve South African HIV-1C integrase sequences downloaded from GenBank and 9 INSTI-naïve isolates amplified in our laboratory). Virologic failures were receiving at least 6 months of INSTI-based cART and presented at the King Edward VIII hospital, a 3rd line regimen referral hospital in Durban, South Africa. Viral RNA was extracted, and the integrase region was amplified and sequenced using Sanger sequencing. To investigate the effect of mutations on the integrase structure, wild-type and representative mutant isolates were modeled on the SWISS model online server and visualized in Chimera v1.13.1. Raltegravir, Elvitegravir, and Dolutegravir were docked into each of the structures using the AutoDock-Vina Plugin available on Chimera, and molecular dynamics simulations were conducted using the AMBER 18 package. Integrase biochemical assays were carried out using a wild-type protein and the 3 mutant recombinant proteins that were expressed and purified. Integrase - LTR binding and 3’ processing assays were then performed. Results. Only one of the 7 (14,28%) INSTI-treated isolates had major mutations (i.e., G140A and Q148R). In addition, this isolate harboured the E157Q minor mutation and previously identified polymorphisms. Interestingly, S119T & V151I, located near the integrase active site, were only found in INSTI failures. Structural analysis results showed a reduced binding affinity for the mutants, which was supported by their weaker hydrogen-bond interaction compared to the wild-type. Our findings showed that the G140A+Q148R double mutant had the strongest effect on the HIV-1C protein structure and binding of EVG and RAL with binding free energies of -12.49 and -11.45 kcal/mol for EVG and RAL, respectively, which are approximately three times lower than the wild-type binding energy. Biochemical assays performed with purified integrase showed a decrease in integrase-LTR binding for all mutants. The 3’ processing activity was slightly decreased in the mutants compared to the wild-type protein; however, no appreciable differences were observed across the mutant isolates. Conclusions Changes near the highly conserved active site residues in HIV-1C integrase core domain and mutations in the 140’s loop have a negative effect on in vitro integrase activity, suggesting that these changes impact viral integration. While they are still few reports of INSTI resistance-associated mutations (RAMs) in South Africa , identification of the G140A+Q148R double mutant for the first time in South African HIV-1 clinical samples, and the identification of S119T and V151I in INSTI-treated patients warrants further investigation. This data broadens the understanding of HIV-1C resistance against INSTIs and adds to the available knowledge of drug resistance mutations that guide therapeutic decisions.
The impact of antiretroviral therapy and Immunological factors on preterm and small for gestational age deliveries in HIV infected pregnant women.
(2020) Mdletshe, Nontlantla Cecilia.; Ndung'u, Peter Thumbi.; Tshabalala, Christina Thobakgale.
Introduction Antiretroviral therapy (ART) during pregnancy may be associated with an increased risk of adverse pregnancy outcomes, including preterm delivery (PTD) and small-for-gestational-age (SGA) but the underlying biological mechanisms remain unclear. Immune activation as well as the use of ART have been associated with adverse outcomes during pregnancy. We explored the association between adaptive and innate immune cell activation markers ex vivo in HIV-infected women initiating ART during or before pregnancy with PTD or SGA. Materials and methods Study participants were women living with HIV drawn from the PIMS cohort, based in Cape Town South Africa and initiated ART during pregnancy or conceived while on ART. Participants were enrolled at median 15 week’s gestation; and were analyzed for immune markers, matched on ART initiation timing (15 women initiated pre- and 15 during pregnancy). There were 30 PTD (delivery <37 weeks), 30 SGA (weight for age ≤10th centile) cases and 30 controls (term, weight for gestational age >25th centile) as outcomes. Immunological parameters were compared T cell activation, antigen presenting cell subsets, activation and function, regulatory T cell phenotypes and functions and plasma cytokine profiles. Results We found that CD8+ T cell, monocyte and dendritic cell activation were lower in PTD women initiating ART in pregnancy when compared to SGA cases and AGA controls over time. Classical (CD14+CD16-) and intermediate (CD14+CD16+) monocyte frequencies were higher in PTD than in SGA cases and AGA women initiating ART in pregnancy compared to those stable on ART. There was lower inflammatory monocyte (CD14dimCD16+) frequencies over time. Monocytes and mDCs but not pDCs showed higher levels of activation in patients initiating ART compared to those stable on ART. A lower activation of APCs (monocytes, mDCs and pDCs) was associated with the PTD outcome. When APCs were stimulated with TLR ligands, a lower IFN-α production by monocytes following TLR4 was associated with PTD. A similar trend was also observed for TLR9 and TLR7/8 stimulation at some time points. Some plasma cytokine levels were higher in participants initiating treatment in pregnancy compared to those stable on ART but there was no link of cytokine levels with birth outcomes. Regulatory T cell frequencies did not differ between ART initiators and those stable on ART, did not change over the course of pregnancy and were not associated with pregnancy outcomes. Conclusion Overall, we noted that lower levels of monocyte activation and reduced functionality (IFN-α production) of monocytes in response to TLR stimulation were associated with PTD. A similar trend of reduced production of MIP-1β and TNF-α by monocytes was noted for PTD cases. This suggests that reduced responsiveness to antigen stimulation may be an underlying factor for PTD, especially for women initiating ART in pregnancy. The markers of immune activation described here may be potential biomarkers to identify women at risk for PTD. Our results also suggest that PTD and SGA have distinct underlying immunological determinants that warrant further investigation.
Impact of immune-driven sequence variation in HIV-1 subtype C Gagprotease on viral fitness and disease progression.
(2011) Wright, Jaclyn.; Ndung'u, Peter Thumbi.
Understanding of the viral and host factors that determine time for progression to acquired immunodeficiency syndrome (AIDS) in individuals infected with human immunodeficiency virus type 1 (HIV-1) could aid in the design of an effective HIV-1 vaccine. Human leukocyte antigen (HLA) class I profile is strongly and consistently associated with differential rates of HIV-1 disease progression, however the mechanisms explaining this are not well understood. It has been hypothesised that “protective” HLA alleles select escape mutations in functionally important epitopes in the conserved group specific antigen (Gag) protein resulting in HIV-1 attenuation, which may result in slower disease progression. Many of the studies investigating the fitness cost of Gag escape mutations have concentrated on a few pre-selected mutations and have not assessed fitness consequences in the natural sequence background. Furthermore, the majority of studies have focussed on HIV-1 subtype B, while HIV-1 subtype C is the most prevalent subtype worldwide. Therefore, in the present study, a large population-based approach and clinically-derived Gag-protease sequences were used to comprehensively investigate the relationship between immunedriven sequence variation in Gag, viral replication capacity and markers of disease progression in HIV-1 subtype C chronic infection. The influence of Gag function on HIV-1 disease progression was further investigated in early HIV-1 subtype C infection. It was also hypothesised that Gag may contribute significantly to overall HIV-1 fitness and towards fitness differences between HIV-1 subtypes. Materials and Methods Recombinant viruses encoding Gag-protease, derived from antiretroviral naïve HIV-1 subtype C chronically (n=406) and recently (n=60) infected patients as well as a small subset of HIV-1 subtype B chronically infected patients (n=25), were generated by electroporation of an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid. The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C (n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication capacity was defined as the slope of increase in percentage infected cells from days 3-6 following infection, normalised to the growth of a wild-type NL4-3 control. Replication capacities were related to patient HLA alleles and markers of disease progression (viral load, CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral set point and rate of CD4+ T cell decline in recently infected patients). Replication capacities were compared between isolates and recombinant viruses encoding Gag-protease from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease amplicons was performed and mutations were identified that were significantly associated with altered viral replication capacity. The fitness effect of some of these mutations was directly tested by site-directed mutagenesis followed by assay of the mutant viruses. Results In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81 (p<0.0001), were associated with lower replication capacities. HLA-associated mutations at low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed mutagenesis experiments (p<0.001), and the co-varying mutations tested did not significantly compensate for this fitness cost. Replication capacity also correlated positively of an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid. The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C (n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication capacity was defined as the slope of increase in percentage infected cells from days 3-6 following infection, normalised to the growth of a wild-type NL4-3 control. Replication capacities were related to patient HLA alleles and markers of disease progression (viral load, CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral set point and rate of CD4+ T cell decline in recently infected patients). Replication capacities were compared between isolates and recombinant viruses encoding Gag-protease from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease amplicons was performed and mutations were identified that were significantly associated with altered viral replication capacity. The fitness effect of some of these mutations was directly tested by site-directed mutagenesis followed by assay of the mutant viruses. Results In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81 (p<0.0001), were associated with lower replication capacities. HLA-associated mutations at low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed mutagenesis experiments (p<0.001), and the co-varying mutations tested did not significantly compensate for this fitness cost. Replication capacity also correlated positivelyof an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid. The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C (n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication capacity was defined as the slope of increase in percentage infected cells from days 3-6 following infection, normalised to the growth of a wild-type NL4-3 control. Replication capacities were related to patient HLA alleles and markers of disease progression (viral load, CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral set point and rate of CD4+ T cell decline in recently infected patients). Replication capacities were compared between isolates and recombinant viruses encoding Gag-protease from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease amplicons was performed and mutations were identified that were significantly associated with altered viral replication capacity. The fitness effect of some of these mutations was directly tested by site-directed mutagenesis followed by assay of the mutant viruses. Results In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81 (p<0.0001), were associated with lower replication capacities. HLA-associated mutations at low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed mutagenesis experiments (p<0.001), and the co-varying mutations tested did not significantly compensate for this fitness cost. Replication capacity also correlated positively with baseline viral load (p<0.0001) and negatively with baseline CD4+ T cell count (p=0.0004), but not with subsequent rate of CD4+ T cell decline (p=0.73). In HIV-1 subtype C recent infection, replication capacities of the early viruses did not correlate with subsequent viral set points (p=0.37) but were significantly lower in individuals with below median viral set points (p=0.03), and there was a trend of correlation between lower replication capacities and slower rates of CD4+ T cell decline (p=0.09). Overall, the proportion of host HLA-specific Gag polymorphisms in or adjacent to epitopes was negatively associated with replication capacities (p=0.04) but host HLA-B-specific polymorphisms were associated with higher viral set points (p=0.01), suggesting a balance between effective Gag CD8+ T cell responses and viral replication capacity in influencing viral set point. A moderate statistically significant correlation was found between the replication capacities of whole isolates and their corresponding Gag-protease recombinant viruses (p=0.04) and the replication capacities of the subtype C recombinant viruses were significantly lower than that of the subtype B recombinant viruses (p<0.0001). The subtype-specific difference in the consensus amino acids at Gag codons 483 and 484 was found in site-directed mutagenesis experiments to largely contribute to the fitness difference between subtypes, possibly by influencing budding efficiency. Discussion The data support that protective HLA alleles, in particular HLA-B*81, attenuate HIV-1 through HLA-restricted CD8+ T cell-mediated selection pressure on Gag. Results suggest that viral replication capacity determined by sequence variability in Gag-protease has an impact on HIV-1 disease progression, but also indicate that a balance between HLA-driven fitness costs and maintenance of effective CD8+ T cell responses is important in determining clinical outcome. Gag-protease was observed to significantly contribute to overall HIV-1 replication capacity and variability in this region between HIV-1 subtypes B and C is suggested to partly explain the difference in viral fitness between these subtypes. Specific mutations in Gag-protease associated with viral attenuation were identified and it was also observed that mutations in conserved Gag regions carried the greatest cost to HIV-1 replication capacity. Overall, the data support the concept of, and may assist in the rational design of, an HIV-1 vaccine in which immune responses are directed towards several conserved epitopes, particularly in Gag, with the aim to constrain immune escape (thereby maintaining effective CD8+ T cell responses) and attenuate HIV-1 (in the event of partial escape), resulting in slower disease course and reduced HIV-1 transmission at the population level.
Impact of injectable hormonal contraceptives on innate immune environment in the genital tract in women at high risk for HIV-1 infection.
(2015) Ngcapu, Sinaye.; Abdool Karim, Quarraisha.; Passmore, Jo-Ann Shelley.
Abstract available in PDF file.
In vitro modelling of the impact of anti-inflammatory drugs on cellular cytotoxicity, activation and inflammation.
(2020) Cromarty, Ross Thomas.; Archary, Derseree.
The relationship between inflammation and HIV has been a major focus of HIV research. In people living with HIV (PLWH), HIV-associated immune activation drives HIV disease progression. While genital inflammation has been significantly associated with increased risk for HIV acquisition and transmission, immune correlates for reduced HIV risk remain less well defined. In HIV-exposed seronegative individuals, the immune quiescent phenotype, characterised by regulated immune activation and inflammation, has been implicated in reducing HIV acquisition risk. Targeted management of inflammation, therefore, is a plausible strategy to mitigate the risk of HIV infection, and to slow HIV disease progression. Therefore, we sought to investigate how anti-inflammatory drugs affect TLR-mediated inflammation and impact HIV infection of CD4+ T cells. This study utilized an in vitro peripheral blood mononuclear cell (PBMC) model. PBMCs were either treated with the anti12 inflammatory drugs ibuprofen (IBF) or betamethasone (BMS) or were left untreated. Thereafter they were either left unstimulated or were stimulated with phytohaemagglutinin (PHA) or Toll-like receptor (TLR) agonists Pam3CSK4 (TLR1/2), LPS (TLR4) or R848 (TLR7/8) before exposure to HIV NL4-3 AD8. To assess inflammation, multiplexed ELISA was used to measure 28 proinflammatory, chemotactic, growth-related, adaptive response-related or regulatory cytokines. Flow cytometry was used to measure activation (CD38, HLA-DR and CCR5) and HIV infection (p24 production) of CD4+ T cells. Despite minimal immune activation, TLR stimulation elicited significant cytokine responses (p<0.05). TLR4 stimulation significantly reduced HIV infection of CD4+ T cells (p<0.01). With the addition of IBF, minimal immunosuppressive effects were observed. In contrast, BMS significantly dampened inflammation (p<0.05) and immune activation (p<0.05) regardless of the stimulation condition. Regardless of global immunosuppression, only with TLR4 stimulation did BMS significantly reduce HIV infection of CD4+ T cells (p=0.02). The finding that TLR4 stimulation reduces rather than increases susceptibility of CD4+ T cells to HIV infection, while BMS only affected HIV infection in the TLR4 condition, strongly suggests that additional factors, and not only inflammation play a powerful, although complex, role in determining HIV infection risk. Together, these data emphasize the importance of understanding signalling pathways induced during inflammation to identify novel targets to mitigate HIV infection.
Investigation of the relationships between host genetics and COVID-19 disease progression among different ethnic groups in South Africa.
(2024) Naidoo, Lisa.; Ramsuran , Veron.
Abstract available in PDF.
Maedi-Visna virus : the development of serum and whole blood immunodiagnostic assays.
(1997) Boshoff, Christoffel Hendrik.; York, Denis Francis.; Conradie, Jan D.
This thesis describes the development of serum and whole blood immunodiagnostic assays for Maedi-Visna virus (MVV). All previously described recombinant MVV ELISA assays utilised either the core p25 or transmembrane (TM) proteins alone, or combined, but as individual proteins. The p25 and TM genes of MVV were cloned individually into the pGEX-2T expression vector. Both proteins were expressed as a combined fusion protein in frame with glutathione S-transferase (GST). The purified recombinant antigens (GST-TM and GST-TM-p25) were used to develop a MVV ELISA. Sera from 46 positive and 46 negative sheep were tested using the GST-TM and GST-TM-p25 ELISAs and a commercial p25 EIA kit. A two-graph receiver operating characteristic (TG-ROC) analysis program was used to interpret the data. The GST-TM-p25 ELISA was more sensitive than the commercial assay which is based on the p25 antigen alone and more specific than the GST-TM ELISA. The GST-TM-p25 ELISA showed a sensitivity and specificity of 100%. The human AIDS lentivirus transmembrane (TM) glycoprotein portion of the envelope viral protein has been identified as the antigen most consistently recognised by antibodies. There is suggestive evidence that the same applies to MVV as the GST-TM fusion protein, expressed in E. coli, has comparable sensitivity to the GST-TM-p25 fusion protein, but lacks specificity. However, due to the hydrophobic nature of the MVV TM protein, purification of the expressed fusion protein required lengthy purification protocols. This was despite the fact that only a truncated version of the TM protein was expressed. This prompted investigating an alternative expression system that could possibly circumvent the above mentioned problems. The yeast Pichia pastoris is known to be suitable for the high-level expression of heterologous proteins which are secreted into the culture supernatant. These features made P. pastoris an attractive host for the expression of the hydrophobic TM protein of MVV. However, limited success was achieved as only low expression levels were obtained and detection and quantification was only accomplished by means of ELISA. Evaluation of the diagnostic performance of the P. pastoris expressed MVV TM-polypeptide was performed using a panel of 36 confirmed negative and positive sera, and evaluated using a TG-ROC analysis programme, which yielded an equal Se and Sp of 83%. The use of a novel rapid immunoassay system, which allows the detection of circulating antibodies in whole blood, has been investigated for use as a MVV diagnostic assay. The central feature of this immunoassay lies in a monoclonal antibody against a glycophorin epitope present on all sheep erythrocytes. A Fab'-peptide conjugate was constructed by coupling a synthetic peptide, corresponding to a sequence from MVV TM protein, to the hinge region of the Fab' fragment of the antisheep erythrocyte antibody. Within the limited number of 10 seronegative and 10 seropositive samples the autologous red blood cell agglutination assay had a sensitivity of 90% and a specificity of 80%. Despite the limitations and difficulties encountered, the use of such rapid whole blood immunodiagnostic assays for MVV holds promise.
Molecular characterisation of endogenous loci related to jaagsiekte sheep retrovirus.
(2007) Hallwirth, Claus Volker.; York, Denis Francis.; Fan, Hung Y.
The study of retroviruses has been of pivotal significance to the field of biomedical science, where it has provided fundamental insights into the processes underlying both viral and non-viral carcinogenesis. Ovine pulmonary adenocarcinoma (OP A), a contagious lung cancer of sheep and goats, has emerged over the past three decades as an invaluable model of human epithelial cancers. It is one of the very few animal models of retrovirus induced neoplasia of epithelial tissues, whereas most other such animal models of human cancers pertain to the haematopoietic system. OP A represents a unique, naturally occurring, inducible, outbred animal model of peripheral lung carcinomas, and is caused by a betaretrovirus - jaagsiekte sheep retrovirus (JSRV) - that is receiving increasing attention in the fields of retrovirology and lung cancer research. JSRV exists in two highly homologous, yet molecularly distinct forms. The first is an exogenous form of the virus that is transmitted horizontally from one animal to another. This form is infectious and the direct cause of OP A. The other is an endogenous form, 15 to 20 proviral copies of which reside benignly in the genome of sheep and are transmitted vertically from one generation to the next. At the time this study commenced, no knowledge existed regarding the underlying pathogenic mechanism by which JSRV causes OPA. Even though the nucleotide sequence of exogenous JSRV had been elucidated seven years earlier, only limited sequence information was available on endogenous JSRVs. With a view towards identifying genetic regions or elements within exogenous JSRV that could potentially be implicated in its pathogenic function, this study began with the cloning of the first three full-length endogenous JSRV loci ever isolated from sheep. The DNA sequences of these full-length endogenous JSRV loci were determined and comprehensively analysed. Comparison with exogenous JSRV isolates revealed that the two forms of the virus are highly homologous, yet can be consistently distinguished in three short regions within the coding genes. Two of these reside in the gag gene, and one at the end of the env gene. These regions were named the variable regions (VRs) of sheep betaretroviruses. The JSRV VR3 in env was linked by our collaborators to the virus's ability to transform cells in tissue culture. The effects and biological significance of VRI and VR2 in gag are subtler and more difficult to determine. After identifying these regions, it became the objective of this study to develop relevant molecular tools that could be used to discern the significance of these variable regions in vivo, and to characterise these tools in vitro to assess their suitability for in vivo studies. The development of these tools entailed the design of a novel strategy that was implemented to precisely substitute the endogenous VRI and VR2 (individually and in combination) into an infectious molecular clone of exogenous JSRV. These chimeric constructs were shown to support retroviral particle release into the supernatant of transiently transfected cells in tissue culture. These particles were confirmed by independent experiments to have arisen specifically from transfection with the chimeric clones. Finally, the particles were shown to be capable of infecting cultured cells and of productively integrating their genomes into those of their host cells, rendering these particles fully competent retroviruses that can be used in the context of in vivo studies to determine the biological significance of VRI and VR2. This study has made a significant contribution to the further development of the OP A / JSRV model system of human epithelial lung cancers. It has also led to the design of a molecular substitution strategy that can be adapted to introduce any genetic region into a cloned DNA construct, regardless of the degree - or lack of interrelation - of the two DNA sequences, thereby creating a highly versatile molecular biological tool.
Molecular characterization of HIV-1 Subtype C strains from KwaZulu-Natal, South Africa, with a special emphasis on viral fitness and drug resistance.
(2004) Gordon, Michelle Lucille.; Cassol, Sharon.
As South Africa begins its National HIV-1 treatment program, it is urgent that we collect data that will help define the phylogenetic relationships, transmissibility and drug responsiveness of C viruses. In this thesis, data is presented on the genetic diversity of locally circulating drug naive subtype C strains, as an indication of their natural susceptibility to antiretroviral drugs, prior to the national roll-out of antiretroviral therapy. At the time this thesis was initiated, antiretroviral therapy was only available in South Africa in a few clinical trials and in the private sector, and it was therefore difficult to obtain large numbers of samples from treatment-experienced patients. Nevertheless, valuable information on the prevalence and patterns of resistance mutations in subtype C infected patients was obtained from small studies on patients receiving HAART, concomitant HAART and TB treatment, HAART and treatment for Kaposi Sarcoma, and single dose nevirapine for the prevention of mother-to-child transmission of HIV-1 infection. The results show that the general antiretroviral drug naive population do not harbour any major resistance-associated mutations to the currently available protease and reverse transcriptase inhibitors, with no differences in genetic variation between the different ethnic groups infected with subtype C. Phenotyping of some of these isolates showed that they were susceptible to the available protease and reverse transcriptase inhibitors, and hyper-susceptible to the protease inhibitor, Lopinavir. Phylogenetic analysis of recent and retrospective subtype C isolates showed that there are multiple lineages of subtype C viruses circulating in South Africa, indicative of multiple introductions of subtype C across its many borders. Polymorphisms in the protease, reverse transcriptase and C2-V5 region of envelope in these drug naive samples lead to significant variation in the number, type and location of potential phosphorylation sites. There was also variation in the cleavage sites controlling the initiation and rate of Gag and Gag-Pol processing (p2/NC) and the activation of protease (TFP/p6gag) suggesting that there may be important differences in the way that B and C viruses regulate polyprocessing and virion assembly. Similar to studies on subtype B, 10 to 18% of the patients on HAART developed drug resistance. However, those on concomitant HAART and TB treatment developed resistance as early as one month after starting treatment. Generally, the resistance mutations that were seen were consistent with those seen in treatment experienced subtype B isolates. Of note was the high level of resistance to the entire class of NNRTIs. This could be reflective of the predominant use of NNRTI-based regimens, as well as the low genetic barrier in this class of drugs. The NNRTI mutations included the V106M mutation that is considered a signature mutation of EFV experienced subtype C isolates. Resistance was high (40%) in mothers and infants 6 weeks after each received a single dose of NVP. K103N was most common mutation in the mothers, while Y181C was most common in the infants. Of note were the changes in functional properties caused by these mutations, by the introduction or alteration of putative myristoylation and phosphorylation sites in the RT. Taken together, these data suggests that the pattern of resistance in African patients will be similar to that observed for the treatment of subtype B infection. However, patients should be closely monitored for viral rebound very early on in treatment. Also, given the high rate of resistance in mothers and infants after single dose NVP, the search for safer regimens to prevent MTCT should be intensified. Although the mechanisms are unknown, our results indicate that several of the phosphorylation-related substitutions in the pol and env genes of KZN and other C viruses are highly conserved and positively selected. It will be important to determine whether these sites play an important role in the replicative capacity and proteolytic processing of C viruses, and in viral entry. These data provide important benefits for public health policy and planning and for future patient treatment management.
Mucosal-associated invariant T (MAIT) cell heterogeneity in peripheral blood and bronchoalveolar compartment: implications for TB and HIV immunity.
(2020) Khuzwayo, Sharon Jabu.; Ndung'u, Peter Thumbi.; Wong, Emily.
Tuberculosis (TB) is the leading cause of infectious mortality globally and the leading cause of death in people living with human immunodeficiency virus (HIV). Understanding the mechanisms leading to impaired anti-TB immunity at mucosal sites and how this is altered during HIV infection, may assist in the development of more effective TB vaccines or immune-based therapies. Mucosalassociated invariant T (MAIT) cells are depleted and dysfunctional in the peripheral blood during HIV infection, but little is known about HIV’s impact on their quantity and quality at the lung’s mucosal surface, the site of TB infection. We aimed to characterise phenotypic, functional and transcriptomic features of MAIT cells in the peripheral blood and lung mucosa of people with latent Mycobacterium tuberculosis (Mtb) infection and HIV co-infection. Matched peripheral blood and bronchoalveolar lavage fluid were collected from consenting participants with confirmed latent TB infection, either with or without HIV. Characterisation of MHC class I-related protein 1 (MR1) 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) tetramer-positive T cells from the lung was performed by cloning, immunophenotyping, functional assays and T cell receptor sequencing. The phenotype, function and transcriptome of MAIT cells from both compartments were characterised by surface marker staining, intracellular cytokine staining, as well as single-cell and bulk RNA-sequencing of MR1 5-OP-RU tetramer-positive T cells from HIVnegative and HIV-positive individuals. Peripheral blood MAIT cells were characterised by the CD161++CD26++ phenotype and produced more interferon (IFN)-γ (P = 0.016) than bronchoalveolar MAIT cells in HIV-negative individuals. Bronchoalveolar MR1 5-OP-RU tetramer-positive cells, included subpopulations with the atypical CD161-CD26++ and CD161-CD26- phenotypes. T cell cloning demonstrated that cells from the typical and atypical CD161/CD26 phenotypic subpopulations all had MR1-restricted function and MAIT cell consistent T cell receptors. In HIV infection, the frequency of both peripheral blood and bronchoalveolar MAIT cells was reduced (P = 0.035 and P = 0.047 respectively), with peripheral blood MAIT cells producing less interleukin (IL)-17 (P = 0.025) and expressing higher levels of the inhibitory co-stimulatory molecule T cell immunoglobulin and mucin domain-containing protein (TIM)-3. Interestingly, the phenotype and function of bronchoalveolar MAIT cells remained relatively unchanged by HIV. Single-cell transcriptional analysis confirmed MAIT cell heterogeneity in the bronchoalveolar compartment which contained two distinct transcriptional subsets, one associated with typical MAIT cell features and effector functions and the other associated with alternative MAIT cell functions including tissue-repair. We report previously undocumented phenotypic and transcriptional heterogeneity in bronchoalveolar MAIT cells, which were also less pro-inflammatory than those in peripheral blood. HIV infection led to depletion of MAIT cells in both compartments, but phenotypic and functional alterations were more pronounced in the peripheral blood compartment. The preservation of function and heterogeneity in bronchoalveolar MAIT cells may represent a potential avenue for therapeutic targeting to restore normal MAIT cell function in people living with HIV. This data suggests that understanding immune responses requires compartment-specific analyses, which may lead to the development of more effective vaccines and immunotherapies targeted at inducing immune responses at the site of infection.

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