Studies of drug resistance in minor HIV quasispecies

Sammanfattning: The main objective was to study drug resistance mutations (DRM) in the HIV-1 reverse transcriptase (RT) gene of minor HIV-1 quasispecies, not detectable with standard techniques. Sensitive allele-specific PCR (AS- PCR) and next-generation sequencing (NGS) were developed to study resistance to drugs of relevance in low- and middle-income countries (LMIC); the nucleoside analogue RT inhibitor (NRTI) lamivudine (3TC) and the non-nucleoside RT inhibitors (NNRTIs) efavirenz (EFV) and nevirapine (NVP). In Paper I and II, AS-PCR was used to detect M184I/V mutations which confer high-level resistance to 3TC. We addressed the selection of drug- resistant HIV quasispecies occurs during the initial phase of viral decay after treatment initiation and their emergence in two viral reservoirs, blood plasma and cerebrospinal fluid (CSF). Selection of M184I/V was found to be rare during the first phase of viral decay in patients with primary HIV- 1infection (PHI) or advanced chronic infection initiated on a three- or four- drug antiretroviral treatment (ART), containing 3TC. In contrast, drug- resistant quasispecies were more commonly detected in patients given dual ART, implicating that highly potent ART is necessary to avoid drug resistance during the early phase of viral decay. In patients who had ART- failure during 3TC containing therapy differences in drug resistance patterns, in both minor and major viral populations, were observed in the blood and CSF. However, the differences observed were most likely a result of differences in the selective pressure of ART rather than unique evolutionary pathways. In Paper III, in order to study transmitted drug resistance (TDR) AS-PCR was used to detect K103N and Y181C mutations, which confer high-level resistance NVP and EFV, in treatment-naïve patients from Ethiopia, from East Africans and Caucasians living in Stockholm. The AS-PCR was highly sensitive and detected K103N and Y181C in minor quasispecies inboth subtype B (HIV-1B) and subtype C (HIV-1C) infected patients. These NNRTI mutations were found in the minor HIV-1 populations in all three patient groups. In Paper IV, we developed a feasible, cost-efficient and easy-to-use high throughput NGS protocol for detection RTI mutations, including M184V, K103N and Y181C, to be applied in large scale surveillance of DRM in LMIC. The NGS assay was applicable to both HIV-1 C and HIV-1 B. It showed good concordance with standard population sequencing in detecting major DRMs and was also able to detect additional low abundance DRMs. In summary, standard population sequencing assays underestimate the prevalence of important DRM in ART naïve and ART experienced patients. AS-PCR and easy to use high throughput assays can be useful in large scale surveillance in LMIC and to address the clinical significance of drug resistance in minor HIV-1 quasispecies.