Masters Degrees (Virology)

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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.
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.
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.
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.
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.
The role of the protease cleavage sites in viral fitness and drug resistance in HIV-1 subtype C.
(2010) Giandhari, Jennifer.; Gordon, Michelle Lucille.
There is an increasing number of patients failing second line highly active antiretroviral therapy (AZT, DDI and LPV/r) in South Africa, where HIV-1 subtype C predominates. Mutations at gag cleavage sites (CS) have been found to correlate with resistance mutations in protease (PR). Therefore, it is important to collect data on subtype C protease and gag sequences from patients as these mutations may affect the efficacy of protease inhibitor (PI) containing drug regimens. In this study, 30 subtype-C infected second-line failures were genotyped using the ViroSeqTM resistance genotyping kit and the gag region from these isolates were then characterised. These sequences were then compared to 30 HIV-1 subtype C infected first-line failures (PI-naïve) and subtype B, C and group M naïve sequences that were downloaded from the Los Alamos Sequence Database. Amino acid diversity at the CS was measured using Mega version 4.0. To investigate the effect of CS mutations on replication capacity, a mutation was introduced by site-directed mutagenesis (Stratagene’s QuikChange Site-Directed Mutagenesis kit). Of the 30 second-line failures that we genotyped, only 16 had resistance mutations in PR and 23 in gag. The most frequent major PI mutations were: I54V/L, M46I, V82A, and I84V and in gag CS were V390L/I and A431V. Interestingly the A431V mutation significantly correlated with protease mutations M46I/L, I54V and V82A. The virus carrying the A431V mutation in vitro was found to have a lower replication capacity compared to the wild type. These findings emphasize the need for further investigation of gag mutations and their contribution to the evolution of HIV resistance to PIs.
Sequence analysis of an HIV-1 subtype C acutely infected cohort from Durban, South Africa.
(2018) Carries, Stanley.; Gordon, Michelle Lucille.
The Human Immunodeficiency Virus is a global public health concern. The Joint United Nations Programme on HIV/AIDS estimated that 36.9 million people were infected with HIV globally at the end of 2017. Almost 20% of these resided in South Africa, making this the highest global HIV burden held by any one country. It is thus important that HIV infection be detected early as this may have important implications in the control of the pandemic. The early recognition of acute HIV infection could present early treatment options that could alter the natural history of the disease, or even eliminate infection. Detecting acute infection early could also provide a unique opportunity to understand HIV transmission and pathogenesis, including early host-virus interactions. In the present study, blood samples were collected from 18-23 year old HIV-1 subtype C acutely infected women from Umlazi Township in KwaZulu-Natal, South Africa, that had participated in a study called Females Rising through Education, Support and Health (FRESH). Eleven blood samples from this cohort, collected within 24 hours of onset of plasma viremia, were used for this study. The aim of the present research was to identify sites within pol that were experiencing positive selective pressure and the likely implications of these mutations on viral functional domains and host cytotoxic T-lymphocyte (CTL) epitopes. The study also sort to observe the loss of drug resistant mutations (DRM) in the viral sequences of participants who had multiple timepoints and to correlate mutation loss to structural changes. Datamonkey and Phylogenetic Analysis by Maximum Likelihood (PAML) were used to detect positively selected sites. Putative functional domains were detected using Prosite and CTL epitopes were identified using the Los Alamos Molecular Immunology Database. Ancestral reconstruction was performed using PAML and Bayesian Evolutionary Analysis by Sampling Trees (BEAST) was used to calculate the time to the most recent common ancestor. Altogether 16 unique positively selected sites were identified in this cohort. Putative functional domains were highly conserved in protease, while positive mutations in reverse transcriptase resulted in either a loss of functional domains in conserved regions or in the gain of functional sites in non-conserved regions. Owing to the important role that protease plays in viral maturation and infectivity, mutations within these conserved regions could possibly lead to defective viral particles with reduced viral infectivity. The K103N in reverse transcriptase, observed in one participant, was the only DRM inherited from its common ancestor. The major limitation of this study was the small sample size.

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Browsing Masters Degrees (Virology) by Author "Gordon, Michelle Lucille."