Browsing by Author "Gordon, Michelle Lucille."

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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.
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) Maharaj, Shevani.; Gordon, Michelle Lucille.
The World Health Organization (WHO) has recommended Tenofovir disoproxil fumarate (TDF) as one of the preferred first-line antiretrovirals (ARVs). TDF and Abacavir (ABC) were introduced into the South African National Antiretroviral Treatment Guidelines in 2010. However, exposure to TDF and ABC can result in the development of the K65R and L74V resistance mutations, respectively. The K65R mutation occurs preferably in subtype C viruses, due to the unique polymorphisms found at codons 64 and 65 (which are not present in subtype B). This is a cause for concern in South Africa, where subtype C is the most common HIV-1 subtype. In addition, these mutations may be present in the minor viral population (i.e <20% of the viral population) and it has been shown that the presence of a resistance mutation in a frequency as low as <0.5% may be associated with an increase in the risk of virological failure. This study investigated the prevalence of K65R and L74V in the minor viral population, using Allele-specific PCR (ASPCR), in a cohort of subtype C infected patients that failed their first-line treatment regimen that did not include TDF or ABC. RNA was extracted from stored plasma samples from a subset of the South African Resistance Cohort Study (SARCS) and the pol region was reverse transcribed and amplified using a one-step RT-PCR kit (Invitrogen; California, USA). For both the K65R and L74V mutations, ASPCR was performed using specific and non-specific primers. A specific and non-specific standard curve was optimised for each mutation (using a mutant plasmid control) and these standard curves were used to perform an absolute quantification. Subsequently, the percentage of each mutation (in each sample) was calculated by dividing the quantity of mutant sequences in the sample by the quantity of total viral sequences in the sample and multiplying this ratio by 100. The Limit of Detection (LOD) of the K65R ASPCR was 0.72%. Of the 84 patients that were assayed, the K65R mutation was detected in 7 (8.33%) of the patients. Five of the 7 samples were detected above 1% (i.e 3 were approximately 2%, 1 was 9.48% and 1 was 100%) and 2 were detected below 1% (i.e 1 was 0.88% and the other was 0.93%). The limit of detection for the L74V ASPCR was 0.013%.We found the L74V mutation to be prevalent in 9 (10.7%) of 84 patients. In 4 of the 9 patients, the L74V mutation was found in ≥1% of the viral population (viz. 2.82%, 10.10%, 12.02% and 18.22%) and in the other 5 patients, the L74V mutation was detected in <1% of the viral population (2 were between 0.5% and1%, while 3 were detected between 0.013% and0.5%). In this study, ASPCR detected additional K65R and L74V mutations in the minor viral population of TDF and ABC-inexperienced patients that were missed by standard genotyping. These minorityK65R mutations could contribute to treatment failure in these patients when switched to TDF or ABC-containing ARV regimens. ASPCR is a useful tool for screening for minority mutations before starting or switching regimens.
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.
Characterization of anti-HIV-1 neutralizing and binding antibodies in chronic HIV-1 subtype C infection.
(Elsevier., 2012) Archary, Derseree.; Rong, Rong.; Gordon, Michelle Lucille.; Boliar, Saikat.; Madiga, Maphuti C.; Gray, Elin Solomonovna.; Dugast, Anne-Sophie.; Hermanus, Tandile.; Goulder, Philip Jeremy Renshaw.; Coovadia, Hoosen Mahomed.; Werner, Lise.; Morris, Lynn.; Alter, Galit.; Derdeyn, Cynthia A.; Ndung'u, Peter Thumbi.
Neutralizing (nAbs) and high affinity binding antibodies may be critical for an efficacious HIV-1 vaccine. We characterized virus-specific nAbs and binding antibody responses over 21 months in eight HIV-1 subtype C chronically infected individuals with heterogeneous rates of disease progression. Autologous nAb titers of study exit plasma against study entry viruses were significantly higher than contemporaneous responses at study entry (p=0.002) and exit (p=0.01). NAb breadth and potencies against subtype C viruses were significantly higher than for subtype A (p=0.03 and p=0.01) or B viruses (p=0.03; p=0.05) respectively. Gp41-IgG binding affinity was higher than gp120-IgG (p=0.0002). IgG–FcγR1 affinity was significantly higher than FcγRIIIa (p<0.005) at study entry and FcγRIIb (p<0.05) or FcγRIIIa (p<0.005) at study exit. Evolving IgG binding suggests alteration of immune function mediated by binding antibodies. Evolution of nAbs was a potential marker of HIV-1 disease progression.
Characterization of nucleoside reverse transcriptase inhibitor-associated mutations in the RNAse H region of HIV-1 subtype c infected individuals.
(Viruses., 2017) Ngcapu, Sinaye.; Theys, Kristof.; Libin, Pieter.; Marconi, Vincent C.; Sunpath, Henry.; Ndung'u, Peter Thumbi.; Gordon, Michelle Lucille.
Abstract available in pdf.
Characterizing protease inhibitor failure in HIV-1 subtype C, using ultra deep pyro-sequencing and homology modelling.
(2015) Singh, Avashna.; Gordon, Michelle Lucille.
The extensive roll-out of combination antiretroviral therapy (cART) has significantly improved the life expectancy for HIV-1 infected individuals in South Africa. Despite the inclusion of potent Protease Inhibitors (PIs) in second-line cART, many patients still fail treatment. The extent to which PI resistance contributes to treatment failure is not completely clear. In this study we report the prevalence of PI mutations amongst individuals failing a second-line Lopinavir (LPV/r) inclusive regimen. We also investigated if low frequency minority variants at LPV/r failure influence Darunavir (DRV/r) failure in a subset of patients using Ultra Deep Pyro-sequencing. Structural changes at DRV/r failure were investigated using Homology modeling. Models were constructed using the SWISS-MODEL webserver and visualized in Chimera v1.8.1. Darunavir was docked into each of the structures using the CLC Drug Discovery workbench ™ and Molecular Dynamics simulations was performed using the AMBER12 package. Our study reports a 24% prevalence of PI resistance mutations, slightly higher than other studies. A distinct pattern of PI resistance mutations was found: M46I+I54V+L76V+V82A, present in 13/37 (35%) of those with PI mutations. Darunavir resistance mutations detected following DRV/r failure included V11I, V32I, L33F and I54L. There were no minority variants detected at LPV/r failure that could have influenced DRV/r failure. Distinct conformational changes were evident in both the LPV/r-resistant and DRV/r-resistant model. Molecular docking showed that the inhibitory potency of DRV was lowered in the mutated DRV/r-resistant model and to a lesser extent in the LPV/r-resistant model. These results show that resistance mutations greatly contribute to DRV drug susceptibility. This work will contribute to the clinical management of patients failing treatment and will also assist in the design of new and improved ARVs.
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.
The differential influence of HIV-1 subtype C,nucleoside analog resistance mutations: K65R, A62V, S68N and Y115F susceptibility to tenofovir.
(2019) Didamson, Onyisi Christiana.; Gordon, Michelle Lucille.
The use of Tenofovir Disoproxil Fumerate (TDF) for the treatment of HIV-1 infection has been recommended for the first-line as well as a second-line antiretroviral regimen in South Africa, due to its high antiretroviral activity and low toxicity level. However, the efficacy of the drug could be threatened by the emergence of drug resistance mutations. The development of TDF resistance poses a public health threat. TDF resistance can be acquired through a selection of the K65R mutation or the K70E mutation (though less frequently) under TDF selection pressure. Besides, K65R and K70E mutations, recent studies have identified other mutations associated with TDF resistance such as A62V, K65N, S68G/N/D, K70E/Q/T, L74I, V75L, and Y115F. These mutations were particularly observed to be in association with the K65R mutation and were reported to be more common in HIV-1 subtype C viruses. Also, these mutations could cause high-level resistance to TDF, especially when in combination with K65R. However, in-vitro studies are required to demonstrate their influence on viral fitness and TDF susceptibility. In this study, we investigated the impact of K65R, A62V, S68D, Y115F, and K65R+S68N on replication capacity and TDF susceptibility. The reverse transcriptase (RT) region was amplified from a drug-naive HIV-1 subtype C isolate obtained from a patient enrolled in the Tropism study (BREC: BF088/07) and cloned into a TOPO vector using a TOPO TA cloning kit. The HIV-1 RT mutations (K65R, A62V, S68D, Y115F, K65R+A62V, K65R+S68D, K65R+S68G, K65R+S68N, and K65R+Y115F) were introduced into the TOPO+RTsubC recombinant using the Quikchange lightning Multi site-directed mutagenesis kit. Next, recombinant viruses were created by co-transfection of the mutant RT amplicons and a pNL4-3-deleted-reverse transcriptase (RT) (pNL43ΔRT) backbone into GXR cells by electroporation. The replication capacity of the mutant viruses was assessed using a replication method that utilized a green fluorescent protein (GFP) reporter cell line and flow cytometry. We evaluated the replication capacity using the exponential growth curve function in Excel to determine the percentage GFP-expressing cells between days 2 and 6. The impact of the mutant viruses on susceptibility to TDF was performed in a luciferase-based assay. The 50% inhibitory concentration (IC50) was calculated using Graph Pad Prism. Drug susceptibility was expressed as the fold change in IC50 of mutant virus compared with the wild type virus. Of the 5 TDF- selected mutants analysed: A62V, K65R, and Y115F mutants display a reduction in replicative fitness whereas, S68D and K65R+S68N showed high viral fitness. Interestingly, the TDF- selected resistance mutations we analysed, showed high susceptibility (A62V, S68D, and Y115F) and reduced susceptibility (K65R and K65R+S68N) to TDF. Our findings support the hypothesis that TDF- selected mutations only confer reduced susceptibility to TDF. Hence, further study is needed on various combinations of TDF-selected resistance mutations to further solidify this claim.
Distribution of PML-RARA isoforms in Acute promyelocytic leukemia patients from a tertiary hospital in KZN, South Africa using qPCR.
(2017) Safiya, Ebrahim.; Gordon, Michelle Lucille.; Parsanath, Sharlene.
The study of genetic epidemiology of cancers in Africa is unique as compared to first world countries, as it entails the combination of gene-environment interaction, poor socio-economic conditions and the high prevalence of infectious (e.g. tuberculosis and HIV) and non-infectious diseases. Acute Promyelocytic Leukemia (APL), a subtype of Acute Myeloid leukemia (AML) if not diagnosed within 24 hours because of its hemorrhagic tendencies, becomes a medical emergency. It has become one of the most treatment-responsive cancers due to its excellent response to all trans-retinoic acid (ATRA). Advances in molecular diagnostics have resulted in a reversetranscriptase polymerase chain reaction (RT-PCR) test to detect the PML-RARA (retinoic acid receptor alpha) transcript found in APL’s. Globally, many centers have investigated the different breakpoint cluster regions (bcr) to classify the patients into different prognostic groups for specific molecular targeted treatment. However, there are no reports from Africa on the frequency of the different isoforms in APL patients. In this study we aim to identify and determine the frequency of bcr isoforms in APL patients from a tertiary hospital in Kwa Zulu Natal (KZN) by quantitative RT-PCR (qPCR). The correlation of the hematological parameters with the different isoforms was analyzed by descriptive non-parametric statistical analysis. The qPCR confirmed bcr1 to be the predominant isoform (63,6%) followed by bcr3 (31,8%) and bcr2 (4,5%). There was a median age group of less than 45 in our patient cohort. Patients with the bcr3 isoform had a poor prognosis according to their clinical risk stratification but this did not necessarily result in poor overall survival when monitored for minimal residual disease (MRD). The HIV-infected APL patients with different isoforms responded to “standard of care” treatment in the same way as noninfected HIV patients.
The echocardiographic manifestations of an urban, working class community with a high cardiovascular risk profile.
(2013) Prakaschandra, Dorcas Rosaley.; Naidoo, Datshana Prakesh.; Gordon, Michelle Lucille.
The metabolic syndrome (MS), consequent upon the pandemic of obesity and diabetes, is associated with an increased risk for cardiovascular (CV) disease. Development of sub-clinical cardiac structural and functional changes associated with CV disease risk factors may be detected on echocardiography. The extent to which these structural changes and CV risk factors are dependent on genetic factors is not clearly established. This project was designed to investigate the relationship between CV disease risk factors, cardiac structural and functional changes and underlying genetic abnormalities. Specifically, the risk factor profile and the presence of the MS were determined. This was then correlated with the echocardiographic findings and gene polymorphisms. Method: A randomly selected cohort of 1428 subjects from the Phoenix community was studied. Demographic data was collected using the WHO STEPS instrument. Blood samples for biochemistry and genetic analysis, together with anthropometric measurements, were collected. Blood pressure and echocardiography was performed on all subjects. The metabolic syndrome was classified according to the National Cholesterol Education Panel (NECP) Adult Treatment Panel III (ATP III) and International Diabetes Federation (IDF) criteria. The Lipoprotein Lipase and Human Paraoxonase-1 genes were genotyped on a Light Cycler 480 Real-Time PCR instrument, using allele-specific probes and sequencing. Results: There was a high prevalence of CV risk factors in this sample; particularly increased waist circumference (79%), obesity (64%) insulin resistance (58%) and hypertension (50%) across the age groups. This translated into a high prevalence of MS (38% using NCEP ATPIII and 46% using IDF criteria). There were significant echocardiographic differences between subjects with and without MS for chamber dimensions (p<0.001), left ventricular wall thickness (p<0.001) and mass (p<0.001), diastolic indices (E-wave {p<0.001}, trans-mitral ratio {p=0.017}) and sub-epicardial adipose tissue (SEAT) thickness (p<0.001). Stepwise multivariate analysis identified age (95% CI 0.975; 0.998), gender (95%CI 0.48; 0.9) and hypertension (95% CI 0.53; 0.99) as independent risk factors for diastolic abnormalities. Logistic regression identified age as the most significant contributor to diastolic abnormalities (OR=1.02; 95%CI 1.009; 1.03; Wald=13.4), followed by the waist circumference (OR=1.025; 95%CI 1.014; 1.037) and BMI (OR=1.075; 95% CI 1.035; 1.117). Genetic analysis showed significant associations between the heterozygous variant of Q192R genotype (PON-1 gene) and elevated HDL levels and also between this variant and obese women (p= <0.05). Conclusion: The high prevalence of CV risk factors and MS in this community has reached epidemic proportions. Although the MS was associated with significant remodelling of cardiac structure, alteration of diastolic indices and increased sub-epicardial adipose tissue thickness, BMI and waist circumference were stronger promoters of altered cardiac physiology. This augurs poorly for this population group unless intervention is introduced to address the markedly high prevalence of these culprit drivers.
Genetic contribution to the risk for metabolic syndrome : an investigation of candidate gene polymorphisms related to lipid and carbohydrate metabolism.
(2015) Maistry, Tanya.; Gordon, Michelle Lucille.; Naidoo, Datshana Prakesh.
Abstract available in PDF file.
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.
Identification of mutational pathways to tenofovir resistance in subtype C isolates using a Bayesian Network.
(2016) Maphumulo, Ntombikhona F.; Gordon, Michelle Lucille.
No abstract.
Impact of p2/NC cleavage site polymorphisms on HIV-1 subtype C viral fitness.
(2012) Wilson, Serron.; Gordon, Michelle Lucille.
Subtype C accounts for the majority of HIV infections and in South Africa, is the dominant subtype. The Gag cleavage sites of subtype C viruses show a high degree of natural variation compared to subtype B and group M sequences, with the p2/NC site having the highest degree of variation among all cleavage sites and between all subtypes. This study therefore aimed to determine the functional effect of this variation on viral fitness. A library of drug naïve subtype C sequences were screened using computational analysis to predict binding affinity between HIV protease and the Gag substrate at the p2/NC site. Ligands with high predicted affinity had hydrophobic cleavage sites with substantial diversity at positions P5-P3. Lower ranking ligands were mostly similar to the consensus subtype C. Three ligands were selected for fitness assays from each the high ranking and low ranking groups. Chimeric viruses expressing selected cleavage sites were generated by site directed mutagenesis. Replication capacity assays of these viruses showed moderate differences in fitness but failed to demonstrate a correlation with computational estimates of binding affinity. Enzymes assays were performed to further investigate substrate preferences and the binding mechanism of protease. To this end, recombinantly expressed HIV-1 protease was tested against a range of substrates the matching the p2/NC cleavage sites used in the replication capacity assay. Results of the enzyme assay did not correlate with either the computation studies or the replication capacity assay results, suggesting a sequence independent binding and recognition mechanism of HIV-1 protease. Taken together the results suggest that processing of Gag is determined by tertiary folding of the polyprotein and not amino acid sequence at the cleavage site.
Minority HIV-1 drug resistance mutations in patients failing highly active antiretroviral therapy (HAART).
(2014) Khumalo, Phumzile.; Gordon, Michelle Lucille.
Abstract not available.
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.
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.
(2012) Ngcapu, Sinaye.; Gordon, Michelle Lucille.
Background: The South African national treatment program includes NRTIs in both first and second line highly active antiretroviral therapy regimens. Recently, mutations in the RNase H domain have been associated with resistance to NRTIs. Here we investigated the prevalence and association of RNase H mutations with NRTI resistance in isolates of HIV-1 subtype C infected individuals. Methods: RNase H sequences from 134 NRTI treated (104 adults and 30 children) and 134 drug-naïve sequences (30 KZN isolates and 104 downloaded from the Los Alamos Database) were analyzed. Spearman’s rank correlation and a Bayesian network were used to explore the relationship between mutations occurring within the RNase H domain and NRTI treatment. Results: 130 of 134 samples clustered phylogenetically with HIV-1 subtype C, with one subtype A, two subtype B and two subtype D. All 30 sequences from HAART-naїve patients were classified as subtype C. Five mutations in the RNase H region had significantly higher frequency when comparing ART-naïve and NRTI-experienced patients. These were: (E438GKR, L517ISV, K527GENQR, E529DK and Q547HKR) (Table 1). Three mutations (E432D, A446SVY and Q507HK) showed decreased proportions in treatment-experienced isolates when compared to ART- naїve isolates. E438GKR was seen in 6.72% of treated versus only 0% of naїve isolates (p= 0.0034), L517IV was found in 17.16% of treated isolates versus 7.46% of naїve isolates (p= 0.0245). Similarly, K527GENQRS was found in 41.04% of treated isolates versus 26.12% of naїve isolates (p= 0.0138), and E529DK was more prevalent in treated (17.91%) when compared to 2.99% of naїve subtype isolates (p <0.001). Finally, Q547HKR was seen in 5.22% of treated versus 0% of naïve subtype C patients (p= 0.0144). Interestingly, samples of twenty treatment experienced individuals that did not show of the classical NRTI mutations in the RT domain harbored E438GKR, L517ISV, K527GENQR, E529DK and Q547HKR. Conclusion: Results obtained from this study suggested that drug resistance could be caused by mutations in the RNase H domain either alone (T470S), or in combination with mutations in the pol region (D67N and L491P). Phenotypic studies are required to understand the prevalence and impact of RNase H mutations, particularly E438GKR, T470S, L517ISV, K527GENQR, E529DK and Q547HKR on NRTI resistance in HIV-1 subtype C as suggested by our data. Further studies using site-directed mutagenesis may also reveal the impact of these mutations on viral fitness.
Pharmacokinetic influences of selected phytochemical compounds from herbal medicine used by HIV- positive patients on drugs-metabolising proteins of HIV-1 protease inhibitor drugs.
(2020) Idowu, Kehinde Ademola.; Gordon, Michelle Lucille.; Nlooto, Manimbulu.
Introduction: Sub-Saharan Africa has the highest incidence of HIV/AIDS and AIDS-related deaths in the world. Although there is currently no cure for the disease, significant progress has been made in developing antiretroviral drugs (ARVs) that can inhibit disease progression. However, despite the availability of these ARVs, HIV-positive patients use traditional herbal medicines (THMs) either alone or simultaneously with conventional ARVs. This simultaneous usage may cause serious adverse effects due to herb-drug interactions, although there are also possible positive effects such as the enhanced bioavailability of the ARVs or possible antiviral activity. Aim: These potential interactions prompted this study which examined the pharmacokinetic properties and influences of selected phytochemical compounds (PCs) commonly found in THMs on drug-metabolising proteins involved in the metabolism of protease inhibitor drugs (PIs) as well as their potential as inhibitors of HIV-1 protease. Method: The potential inhibitory activities of fifteen PCs (Epigallocatechin gallate (EGCG), Fisetin (FST), Ellagic acid (EGA), Cholesta-4,6-dien-3-ol (CHD), Lanosteol (LNT), Benzyl Isothiocyanate (BIT), Gallic acid, (GA), Isosteviol (IST), Stigmasterol (STG), Phthalic acid (PTA), Naringenin (NGN), Kaempferol-7-glucoside (K7G), Luteolin (LUT), Geranin (GER), Apigenin (APG)) against the South African sub-type C HIV-1 protease enzyme and PIs’ drug-metabolizing proteins were investigated, using molecular dynamic (in-silico) techniques. Furthermore, an in vitro evaluation of the cytotoxicity assays, cell viability profiles and modulatory influences of the most promising antiviral PCs on the mRNA and protein expressions of the drug-metabolising proteins in two human cell lines (liver (HepG2) and kidney (HEK293)) was carried out. Result: Four of the fifteen PCs (EGCG, K7G, LUT and EGA) were predicted to be potential inhibitors of HIV-1 protease, as well as inhibitors of cytochrome P450 3A4 (CYP3A4) and Pglycoprotein P-gp/ABCB1. Results from the in vitro study showed that these four PCs were not toxic to HepG2 cells at their IC50 (50% cell viability) and IC20 (80% cell viability). ATP (adenosine triphosphate) levels increased at IC20, with no significant change at IC50. In addition, no significant change in LDH (lactate dehydrogenase) was seen (with the exception of LUT).In the HepG2 cells, ABCB1 protein expression (western blot) decreased overall. While all PCs decreased CYP3A4 protein expression at IC20, (with the exception of LUT) xxv protein expression increased at IC50. mRNA levels were decreased for EGCG and K7G at IC20. InHEK293 cells, all PCs were non-toxic. ATP concentrations were similar to the control except for EGCG which decreased at IC20, and K7G which increased at IC50. LDH concentration decreased when exposed to the PCs at IC20, but a significant (p < 0.05) increase was recorded in LUT IC50. ABCB1 protein expression increased at both IC20 and IC50 concentrations, although LUT and EGA mRNA expression decreased at IC50. The decreased protein activities of CYP3A4 in K7G IC50 and LUT IC20 correlates with increased intracellular ATP. Conclusion: The study therefore suggests that EGCG, K7G, LUT and EGA could decrease the biotransformation of drugs, and eventually increase drug plasma concentrations in the systemic circulation. These natural compounds that can serve as inhibitors of drugmetabolizing proteins and the HIV-1 protease enzymecould be useful in the treatment of HIV-1.
Phenotypic effects and predictions of HIV-1 subtype C reverse transcriptase C-terminal domain mutations on reverse transcriptase inhibitors.
(2018) Mkhwanazi, Nompumelelo Prudence.; Gordon, Michelle Lucille.
Antiretroviral drug therapy has been shown to reduce the death of HIV-1 infected individuals. However, the emergence of HIV-1 drug resistance has hindered the success of HIV-1 treatment. Genotyping tests mainly concentrate on the polymerase domain of HIV-1 RT leaving the rest understudied. Recently, data from HIV-1 C suggested that drug resistance could be caused by mutations in the connection and RNase H domains either alone or in combination with mutations in the polymerase region. Here, the phenotypic effects of the specific RNase H domain mutations in HIV-1 subtype C on RTIs were investigated. The predictions of HIV-1 subtype C reverse transcriptase C-terminal domain mutations on reverse transcriptase inhibitors were also investigated. Material and methods Viral RNA was extracted from 500μl of plasma using the Viral RNA extraction kit (Qiagen,Germany) according to the manafacturer’s instructions, and stored at -80oC until utilisation. The RNA was amplified using the Superscript III One-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen, Life Technologies Corporation, Carlsbad, CA, California). The HIV-1 RT amplicon was cloned into a TOPO vector using the TOPO TA cloning kit (Invitrogen, Life Technologies). Viral mutants were constructed using site-directed mutagenesis, introducing D67N in the polymerase domain and E529D, L517I, T470S, and T470P mutations in the RNase H domain. Viral replication capacity and drug susceptibilities were determined using the single cycle luciferase assay in TZM-bl cells. To identify the HIV-1 connection domain (CN) mutations associated with drug resistance, HIV-1 subtype C sequences were downloaded from the Los Alamos and Stanford HIV- drug resistance xvi databases from drug naïve and treated-experienced patients. The presence of connection domain (CN) mutations were identified using REGA HIV-1 subtyping tools (Universiteit Leuven, Belgium). Bayesian Network (BN) analysis (B-course) was used to determine the association of connection domain (CN) mutations (condon 320-440) with other TAMS. The effect of RNase H domain mutations on the structure of reverse transcriptase was determined using Swiss Model and viewed in Chimera. Results The replication capacities of T470S, T470P, L517I, E529D RNase H domain mutations were lower than the HIV-1 subtype C wild type in the absence of drugs. The D67N mutation alone had a lower replication capacity compared with the wild-type. Combination of L517I and D67N showed a further decrease in replication capacity compared to the wild type in the absence of drugs. E529D mutation replication capacity was assessed in TZM-bl cell line in both HIV-1 subtype B and C. Although not statistically significant, both competent subtype B and C E529D mutant had a decreased growth and infectivity rate compared to their respective wildtypes. The RNase H domain mutation T470S showed a moderate level of resistance to NVP (10.2X), ETR (8.75X), d4T (5X) and no resistance to AZT and EFV. Interestingly, T470P showed a moderate level of resistance to NVP (6X) and no resistance to ETR and d4T, as well as EFV. It did however show a 5-fold change to AZT when compared to the wild-type virus. As expected, the thymidine analog mutation, D67N, showed a high level of resistance to AZT (103.3X), moderate level of resistance to d4T (6.2X) and low level of resistance to ETR (3.2X) and no resistance to NVP and EFV. The RNase H domain L517I mutation showed moderate level of resistance to AZT (5.2X), d4T (6.0X) and NVP (10.79X), and low level of resistance to ETR (3.50X). L517I mutation caused hypersusceptibility to EFV. The combination of RNase H and TAM (L517I+D67N) showed high xvii level of resistance to AZT (157.3X), moderate level of resistance to NVP (11.3X), low level of resistance to d4T (3.9X) and no resistance to ETR and EFV. Subtype C E529D mutation conferred up to 2-fold stavudine (d4T), 3-fold zidovudine (AZT) and nevirapine (NVP) resistance, respectively. These findings demonstrate that RNase H mutation E529D can confer mild resistance to nucleotide (AZT and d4T) and non-nucleotide (NVP) reverse transcriptase inhibitors. New connection domain mutations identified were: D324G/N/P, T338S, I341F/L/V, M357R, E370D, M377T/L, A376S, I434M/L, A437V/I. E370D and A437V/I were directly associated with treatment in the BN, while N348I was only indirectly associated with treatment. Discussion and Conclusions Overall, the RNase H domain mutations impaired replication capacity in the absence of drugs, suggesting that they are acquired at a fitness cost (as withmost drug resistance mutations). While T470S decreased drug susceptibility to ETR, it was shown to be hypersusceptible to EFV. Interestingly, T470S is very common in subtype C RTI treated patients and could indicate that a switch to the newer NNRTI might not be as beneficial as expected. The phenotypic data also suggests that the resistance pathways for T470S and T470P could be different; however further studies are required to investigate their mechanism of resistance. The L517I mutation alone only minimally decreased drug susceptibility to both NRTI and NNRTIs. However it futher decreased drug susceptibility to the NRTIs when in combination with D67N, compared to D67N alone. D67N is known to affect the NRTIs, and the combined effect of D67N and L517I on NVP, a NNRTI, was surprising. The decreased replication capacity in the L517I indicated that it has additive effect in fitness loss of the viral. Further site-directed mutagenesis studies are needed to understand the effect of these RNase H mutations alone and in xviii combination with other polymerase domain mutations or with connection domain mutations on nucleoside reverse transcriptase inhibitors. Structural analysis showed that the T470S/P mutations cause an inward movement of the RNase H active site amino acids residues, which may have an affect on RNase H activity. There was no interaction between L517I and E529D with the RNase H active site amino acid residues observed. The observed interaction was between the amino acids that form part of the RNase H primer grip and these RNase H mutations. New HIV-1 subtype C connection domain mutations were identified and phenotypic studies are required to investigate their role in HIV-1 drug resistance. In conclusion, this is the first study to show that T470S/P, L517I and E529D in HIV-1 subtype C affect NNRTI drug susceptibility. This provides further support for the monitoring of C-terminal domain mutations in relation to NNRTI, as well as NRTI drug resistance. In addition, these mutations need to be taken into account when designing newer NNRTI with the ability to retain activity against these mutants.