Genotypic and phenotypic resistance profiles of HIV-1 infected patients failing salvage antiretroviral therapy
Date
2020
Authors
Davis, Ashlyn Samantha Christal
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Abstract
Antiretroviral drug resistance is a major concern for HIV-1 patients failing third line regimens
or salvage therapy. Currently, South African patients who fail third line antiretroviral therapy
(ART) have no further antiretroviral drug options or resistance test recommendations, therefore
remain on heavily compromised regimens and will likely develop AIDS and die. A phenotypic
susceptibility assay that could determine which antiretroviral drugs may still be effective
against HIV-1 from these patients would be a valuable tool in their successful long-term
treatment success. Thus, the overall aim of this study was to establish a locally relevant in vitro
phenotypic susceptibility assay to allow for the phenotypic profiling of HIV-1 from South
African HIV-1 positive patients failing a third-line regimen. Viral RNA was extracted from six
South African HIV-1 positive patients failing a third-line regimen, and the approximately 4.5
kb gag-pol regions were successfully RT-PCR amplified and sequenced using a newly
established protocol. Nucleotide sequences were submitted to the Stanford University HIV drug
resistance database to describe any known antiretroviral drug resistance mutations present, and
manually aligned to look at mutations in the Gag cleavage sites and PTAP insertions. Overall,
all six patients were infected with HIV-1 subtype C, with a complex mixture of known drug
resistance mutations to the various ARV drug classes, in line with their current (and previous)
drug regimen. Looking at the Gag cleavage sites, CA/P2 showed no changes, whereas various
mutations were detected in MA/CA, P2/NC, NC/P1 and P1/P6. Intact PTAP motifs were
observed in all six patient samples, and virus from two patients had an additional full or partial
PTAP motif. An in vitro phenotypic inhibition assay was successfully set up using the wild type HIV-1 subtype B pNL4.3.Luc.R-E- and its performance and robustness was tested against
a panel of ARV drugs. IC50 values against pNL4.3.Luc.R-E- were obtained for one protease
inhibitor, six reverse transcriptase inhibitors and two integrase inhibitors. The next step was to
create patient derived gag-pol pseudoviruses/expression vectors for substitution of pNL4-
3.Luc.R-.E- in these assays. Within the time constraints of the study, multiple attempts using
Gibson assembly and traditional restriction/ligation cloning of gag-pol into the pNL4-3.Luc.-
R.-E backbone (and a psPAX2 backbone) were unsuccessful. Overall, genotypic sequence
analysis of the Gag-pol confirmed that the known HIV-1 drug resistance profiles from each of
the six patients used in this study were unique, justifying future phenotypic testing, and
compensatory mutations in Gag may contribute to overall resistance. Future optimization of the
protocols described here to successfully create a locally relevant HIV-1 subtype C in vitro
phenotypic susceptibility assay for use by patients failing third line ART is critical.
Description
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2020