Investigation into the effect of HIV viral proteins on endothelial function in the HIV-infected population
Background: Infection with HIV is associated with an increased risk of cardiovascular disease (CVD). This may be due to the negative effect of viral proteins (Nef and Tat) on endothelial function. This has been proven in in vitro experiments but not in clinical studies. Currently, the measurement of Nef and Tat in human serum is hampered by the lack of availability of validated assays. Furthermore, the viral nef and tat genes contain numerous sequence variants, which may be associated with different levels of endothelial dysfunction. Therefore, the aims of this study were to investigate whether endothelial dysfunction is present in African subjects with HIV infection, identify its possible determinants in this population and assess if it is related to variants in the viral nef and tat genes. Lastly, to develop an efficient method for the quantification of HIV viral proteins in human serum. Materials and Methods: Eighty HIV-infected treatment-naïve Black South Africans were recruited and compared against 60 healthy age-matched HIV-uninfected individuals. The HIV infected participants were followed up for 18 months (n=13) following initiation of anti retroviral therapy (ART). Blood pressure, height, weight, body mass index (BMI), waist and hip circumference, triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol, glucose, viral load and CD4 counts were measured. Serum biomarkers of endothelial function i.e. intercellular adhesion molecule-1 (ICAM-1), endothelial leukocyte adhesion molecule-1 (E-selectin), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1), as well as markers of inflammation i.e tumor necrosis factor-α (TNF-α), interleukin 6 (IL6) and interleukin 8 (IL8) were quantified using the Luminex platform, while von Willebrand Factor (vWF) and HIV-1 Nef were quantified by ELISA. HIV-1 nef and tat genes were sequenced from 30 and 34 patients respectively, and sequence analysis performed to identify potential mutations associated with high levels of biomarkers of endothelial function and inflammation. A second cohort consisting of 140 HIV-infected individuals on ART was recruited for whom carotid intima medial thickness (CIMT) measurements were available. This cohort was divided into 2 groups: CIMT < 0.50 and CIMT > 0.58 mm. The HIV-1 nef gene was successfully sequenced from 60 patients, and variant frequencies compared between the two groups. An in silico tryptic digest was performed with HIV-1 nef sequences to identify conserved, endogenous target Nef peptides for quantification by tandem mass spectrometry (LC-MS/MS) using the Sciex QTrap 5500 with MicroLC. Results: The HIV-infected cohort had higher levels of ICAM-1, VCAM-1 and vWF compared to the HIV-uninfected cohort of which ART initiation decreased plasma levels of ICAM-1 and VCAM-1 despite increases in serum levels of TNF-α, IL-6 and IL-8 post-treatment. Serum levels of VCAM-1 and vWF correlated positively with low CD4 counts and high viral load, respectively. HIV-1 polymorphisms that were significantly associated with markers of endothelial dysfunction and inflammation were seen with the following HIV-1 Nef peptide sequence variants: V16I, H40Y, T50H,A, S169N and H188Q,S with ICAM-1; Y202F with VCAM-1; K182M with MCP-1; and D205N with TNF-α. In the HIV-infected cohort on ART, the following HIV-1 Nef peptide sequence variants were found to be significantly associated with higher CIMT: K39R, H40Y, D177E, F143Y and V180T. The Nef assay method developed on the LCMS identified the target Nef peptide standards and endogenous Nef in human serum. Conclusions: Collectively, our data shows evidence of endothelial dysfunction in the South African HIV-infected population, despite ART administration, and several variants in HIV Nef associated with elevated serum levels of markers of endothelial dysfunction and CIMT, confirming a possible role for this viral protein in impacting endothelial function. Thus, targeting HIV Nef is a potential therapeutic strategy to circumvent the development of CVD in HIV-infected patients. A LC-MS/MS method for the quantification of Nef in human serum was developed but requires further refinement.
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Medicine (Chemical Pathology) to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2020