Multidisciplinary viral analyses in people living with HIV-1C and receiving second-line combination antiretroviral therapy (cART) in South Africa

Obasa, Adetayo Emmanuel Adegbenga (2019-12)

Thesis (DSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: The use of combination Antiretroviral Therapy (cART) has grown since its first introduction into the South African public sector. cART has significantly reduced the mortality rate caused by human immunodeficiency virus (HIV) in both high- and low-to-middle-income countries. The development of drug resistance has challenged the outcome of cART. This has led to the introduction of Integrase (IN) strand transfer inhibitors (InSTIs) as part of the first-line cART regimen. Due to their superior efficacy and high genetic barrier, this class of drugs was previously reserved as salvage therapy. The World Health Organization (WHO) supports InSTIs as first-line regimen non-nucleoside reverse transcriptase inhibitors (NNRTIs) particularly in regions where pre-treatment drug resistance to NNRTIs reaches 10%. Therefore, this study aimed to (i) to investigate the prevalence of InSTI mutations in treatment-naïve and treatment-experienced PLHIV using genotypic assays, which included Sanger sequencing, next-generation sequencing (NGS) and molecular modelling; (ii) analysed Long Terminal Repeats (LTR) to identify transcription factor binding sites. Chapter 2: Ninety-one (n = 91) treatment-naïve patients were obtained before the start of antiretroviral treatment in South Africa. Furthermore, we included 314 South African patient sequences obtained from the Los Alamos National Library database (www.lanl.gov). The IN gene ~ 900 base pairs [bps] was amplified and sequenced using conventional DNA Sanger sequencing. Homology structure was generated using the cryoEM structure of HIV-1B IN intasome (PDB file 5U1C) using ‘Prime’ of Schrodinger Suit. Chapter 3: Ninety-six (n = 96) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained for further analyses. We performed conventional DNA Sanger sequencing to analyse the complete pol gene (~3011bps) and sequences were analysed using the Stanford HIV drug resistance database to assess genotypic resistance associated mutations (RAMs). Chapter 4: Fifty-six (n = 56) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained. We performed a high-throughput (HT) sequence analyses on the complete pol gene using Illumina HiSeq2500, followed by bioinformatics analysis to quantify the RAMs according to the Stanford HIV drug resistance database. Chapter 5 and 6: We performed in-silico analyses on diverse HIV-1 subtypes based on 8114 sequences. These included treatment naïve and downloaded sequences from the HIV Los Alamos National Library Database (www.lanl.gov). Homology derived molecular models of HIV-1 IN tetramers from different subtypes were generated using cryoEM structure of the HIV-1B IN intasome. Chapter 7: Fifty-six (n = 56) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained. We performed Sanger sequencing to analyse the LTR gene (~ 474 bps) followed by bioinformatics analyses to identify transcription factor binding sites.The data indicates that in South Africa, the prevalence of RAMs against InSTIs is low and InSTIs can be used as a potential viable salvage therapy option and/or first-line regimen. Molecular modelling was done for IN structural analyses, which revealed how naturally occurring polymorphisms might affect structural stabilities, viral DNA binding and drug-binding propensity. This study represents a true baseline InSTI resistance rate as the treatment-naïve patients were obtained before the cART introduction. We propose GRT for people living with HIV (PLHIV) before treatment initiation and we recommend continued InSTIs drug resistance monitoring when introduced on a larger scale in South African.

AFRIKAANSE OPSOMMING: Kombinasie- antiretrovirale terapie (cART) het heelwat toegeneem sedert dit die eerste keer in die Suid-Afrikaanse openbare sektor bekend gestel is. cART het ’n aansienlike verlaging in die sterftesyfer in sowel hoë- as lae-/middelinkomstelande teweeggebring. Die ontwikkeling van middelweerstandigheid en oorgeërfde middelweerstandige mutasies hou egter ’n uitdaging vir die uitkoms van cART in. Daarom is integrasestringoordraginhibitors (InSTI’s) by die eerstelinie-ART-regime ingesluit. Hierdie klas middels is voorheen uitsluitlik as reddingsterapie gebruik weens hulle uitmuntende doeltreffendheid en hoë genetiese skans. Die Wêreldgesondheidsorganisasie ondersteun InSTI’s as eerstelinieregime, veral in streke waar voorbehandelingsweerstand teen nie-nukleosied-omgekeerdetranskriptase-inhibitors 10% bereik. Vir hierdie studie is ’n deursneeondersoek uitgevoer wat bestaan het uit die genotipiese en molekulêre modellering van InSTI’s vir die volgende:Behandelingsnaïewe pasiënte voor bekendstelling van die Suid-Afrikaanse nasionale menslike-immuniteitsgebreksvirus-(MIV-)behandelingsprogram en die beskikbaarheid van InSTI’s in Suid-Afrika. Behandelingservare pasiënte wat tweedelinie-ART in die Suid-Afrikaanse nasionale behandelingsprogram ontvang. Hoëdeursetreeksvorming (HTS) by behandelingservare pasiënte wat tweedelinie-ART in die Suid-Afrikaanse nasionale behandelingsprogram ontvang. Die strukturele implikasie van genotipiese variasies in MIV-1-integrase (IN) by sowel behandelingservare as behandelingsnaïewe pasiënte. Vir behandelingsnaïewe pasiënte is genetiese ontledings van 91 plasmamonsters sowel as 314 pasiënte se databasisafkomstige reekse gedoen. Genotipiese weerstandigheidstoetsing (GRT) het op die Q148-roete afgekom, wat tweedegenerasie-dolutegravir uit die databasisafkomstige reekse kan beïnvloed. Nietemin is geen weerstandigheidsverwante mutasies (RAM’e) opgemerk in monsters wat voor die bekendstelling van die nasionale MIV-behandelingsprogram bekom is nie. Vir behandelingservare pasiënte is die genotipiese Sanger-reeksvormingsessai gebruik om belangrike RAM’e soos T66I, Y143R en T97A te identifiseer. Hierdie RAM’e bied weerstand een raltegravir (RAL) en elvitegravir (EVG). HTS is gebruik om geringe (<20% van die populasie) en erge variante binne die MIV quasispecies (>20% van die populasie) by behandelingservare pasiënte te kwantifiseer. HTS het InSTI-RAM’e soos Y143R, S147G en E138R aan die lig gebring, wat weerstand bied teen RAL en EVG. Molekulêre modellering is vir IN- strukturele ontledings uitgevoer, wat getoon het hoe natuurlike polimorfismes strukturele stabiliteit, virale DNS-binding en middelbindingsgeneigdheid kan beïnvloed.Die studiedata dui op ’n lae voorkoms van RAM’e teen InSTI’s in Suid-Afrika. InSTI’s kan as ’n potensieel lewensvatbare reddingsterapie en/of eerstelinieregime gebruik word. Die studie bied ’n ware basislynaanduiding van InSTI-weerstandigheid omdat die behandelingsnaïewe pasiënte voor die bekendstelling van cART gewerf is. Nogtans beveel die navorsers GRT aan vir MIV-geïnfekteerde persone voor die aanvang van behandeling, en word die gebruik van HTS-GRT as ’n standaard-GRT voorgestel.

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