Characterizing protease inhibitor failure in HIV-1 subtype C, using ultra deep pyro-sequencing and homology modelling.

Master of Medical Sciences in Virology. University of KwaZulu-Natal, Medical School 2015.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

Tuberculous meningitis is associated with higher cerebrospinal HIV-1 viral loads compared to other HIV-1-associated meningitides.

To gain a better understanding of the immunopathogenesis of tuberculous meningitis (TBM) and identify potential diagnostic biomarkers that may discriminate TBM from other HIV-1-associated meningitides, we assessed HIV-1 viral load levels, drug resistance patterns in antiretroviral therapy (ART)-experienced patients with persistent viremia and soluble immunological analytes in peripheral blood and cerebrospinal fluid (CSF) of HIV-1 infected patients with TBM versus other meningitides. One hundred and three matched blood and CSF samples collected from HIV-1 infected patients with TBM or other meningitides presenting at a hospital in Durban, South Africa, from January 2009 to December 2011 were studied. HIV-1 RNA and 28 soluble immunological potential biomarkers were quantified in blood plasma and CSF. Viremic samples were assessed for HIV-1 drug resistance mutations. There were 16 TBM, 46 probable TBM, 35 non-TBM patients, and six unclassifiable patients. TBM and non-TBM patients did not differ in median plasma viral load but TBM patients had significantly higher median CSF viral load than non-TBM participants (p = 0.0005). No major drug resistance mutations were detected in viremic samples. Interleukin (IL)-1β, IL-17, platelet derived growth factor (PDGF)-BB, granulocyte colony stimulating factor (G-CSF) and cathelicidin were significantly elevated in the CNS of TBM participants compared to other patients although these associations were lost after correction for false discovery. Our data suggest that TB co-infection of the CNS is associated with enhanced localized HIV-1 viral replication but none of the evaluated soluble immunological potential biomarkers could reliably distinguish TBM from other HIV-associated meningitides

Comparison of demographic and clinical characteristics of TBM and non-TBM study participants.

<p>Comparison of demographic and clinical characteristics of TBM and non-TBM study participants.</p

Clustering patterns of potential biomarkers according to participant clinical status (TBM, non-TBM, ART-naïve and ART-experienced).

<p>Hierarchical clustering heat map of potential biomarker profiles for (A) plasma analytes and (B) CSF analytes of patients with and without TBM (on ART and not on ART). Blue represents low concentration of biomarker below the median and red depicts high concentration of biomarker above the median value. (C) PCA plots of biomarkers in the plasma and (D) CSF of TBM and non-TBM patients. The % of variance explained by each PC is displayed on the respective axis.</p

HIV-1 viral loads in plasma and cerebrospinal fluid (CSF).

<p>(A) Plasma viral loads are significantly higher than CSF viral loads in ART-naïve participants. (B) For ART-experienced participants with meningitis, there was no significant difference in HIV-1 viral load levels when comparing plasma versus CSF. (C) HIV-1 viral loads in the CSF versus plasma of ART-naïve patients with tuberculous meningitis (TBM), showing no significant differences between the two compartments. (D) ART-naïve non-TBM patients had significantly higher plasma compared to CSF viral loads. (E) Plasma viral loads of ART-naïve patients with TBM versus other meningitides are not significantly different. (F) Significantly higher HIV-1 viral loads in CSF of ART-naïve TBM patients compared to those with other meningitides. P-values are shown for each comparison and are bolded if the differences are statistically significant.</p

Demographic, clinical characteristics and summary of drug resistance testing profiles of patients with persistent viremia treated with first-line antiretroviral therapy.

<p>Demographic, clinical characteristics and summary of drug resistance testing profiles of patients with persistent viremia treated with first-line antiretroviral therapy.</p

Comparison of plasma and CSF potential biomarker levels for all ART-naïve assayed participants.

<p>Comparison of plasma and CSF potential biomarker levels for all ART-naïve assayed participants.</p

Flow diagram of the study participants and their grouping.

<p>Participants with matched plasma and CSF samples were included in the current study. Quantification of viral loads and potential biomarkers were performed on all participants, then viral loads and potential biomarkers comparison analysis were restricted to definite TBM versus non-TBM groups.</p