Study design and Population
A case controlled study was conducted in which archived blood samples from the “Investigation of host genetic factors for HIV control in Ugandan Elite and Viremic controllers (ELITE) study” were used(6). Among its target population, the ELITE study recruited 42 HIV-infected participants’ ≥18 years(6). The participants were ART naïve by 31st December 2015 and had been under care at Makerere University Joint AIDS Program (MJAP), Mulago ISS clinic for at least 5 years with documented serial CD4+T cell counts ≥ 500cells/ml and viral load < 2,000 copies/ml. Their average duration in care at the clinic was 7.36 years (95% CI 6.58-8.13)(6). Of these participants, 36 were ECs, 2 were VCs and 4 were progressors. In addition, ELITE study recruited 15 HIV-infected individuals ≥18 years with documented CD4+T count below 500 cells. Peripheral blood mononuclear cells (PBMCs) were harvested and frozen in liquid nitrogen in the Immunology laboratory of Makerere University College of Health Sciences located on the 1st floor of the Clinical Research building. Figure 2 summarizes the flow of in vitro assays performed on PBMCs from 31 LTNPs and 15 progressors.
Ethical considerations
We conducted our study in compliance with recognized international standards, including the International Conference on Harmonization (ICH) and the guidelines of the Declaration of Helsinki. We obtained written informed consent from all study participants. Ethical approval was granted by the Makerere University School of Biomedical Sciences, Higher Degrees Research and Ethics Committee (SBS-HDREC) and the Uganda National Council for Science and Technology in Kampala, Uganda.
Laboratory Methods
PBMC thawing: Aliquots of PBMCs from LTNPs and progressors were removed from liquid nitrogen followed by thawing by a brief incubation of the vials in a 37oC water bath until there was a small ice pellet left. Cells were washed immediately with thawing medium composed of 90% RPMI 1640 medium (Thermofischer Scientific, Grand Island USA, catalogue no. 11875093) and 10% Fetal Bovine Serum (Thermofischer Scientific, South America, catalogue no. 10270106) in a 15ml centrifuge tube.
Enrichment/purification of CD4+ T cells: CD4+T cell enrichment was performed using EasySep™ Human CD4+T Cell Isolation Kit (Stem Cell Technologies, Catalog no. 17952). Briefly, a cell suspension was prepared at a concentration of 4 x 108 cells/mL in 1.6ml PBS/10% FBS using 5ml capacity round bottomed polystyrene falcon tubes. EasySep™ Dextran Rapid Spheres were added to the sample at a concentration of 50 μl /mL. PBS/10%FBS was added to top up the sample up to 2.5mL. The mixture was then mixed by gently pipetting up and down 2-3 times and placed the tube (without lid) into the EasySep magnet and incubated for 3 minutes at room temperature. The magnet was then picked up, and in one continuous motion inverted with the tube, pouring the enriched cell suspension into a new falcon tube. To assess CD4+T cell purity, samples were stained with 5µg of mice anti-human CD4-allophycocyanin (APC) (RPAT4 clone), (BioLegend, San Diego, CA, USA, catalogue no. 300552) and flow cytometry was then performed with a FACS Calibur H (Becton Dickinson, Franklin Lakes, NJ) ( Figure 5A illustrates the gating strategy used).
HIV-1 pseudo viruses used for the infectivity assays: Three strains of HIV-1 pseudovirus were used in this experiment i.e (1) strain I: vesicular stomatitis viral glycoprotein (VSV-G) enveloped HIV-1, an HIV-1 pseudotyped virus encoded with a gene reporter-green fluorescent protein (GFP). We used VSV-G enveloped HIV-1 as a positive control to demonstrate that purified and activated CD4+T cells from cases and controls were in good condition to take up HIV-1. (2) strain II: ADA enveloped HIV-1 pseudotyped virus particles, a wild type R5-tropic HIV-1 subtype B isolate which utilizes CCR5 as a coreceptor and strain III: NL4.3, an X4-tropic HIV. The three HIV strains were obtained from Dr Richard Sutton laboratory at Yale University, USA.(9)
Titration of HIV-1 pseudovirus: The infectious dose of HIV-1 particles had been standardized by titration protocols in the Sutton Laboratory at Yale University(9, 10). We therefore adopted and used the optimal dose of 1ml of the virus as previously described in GHOST Hi5 and CD4+T cells in the Sutton Laboratory. A dose of 1ml gave a maximum infectivity without compromising the viability of the CD4+T cells(9, 10).
In vitro CD4+T Cell activation: EasySep enriched CD4+T cells (200,000 cells per well) were activated in tissue culture as follows. 10µg of anti-CD3 (OKT3 clone, eBioscience™, San Diego, CA, Catalog no.14-0037-82) in 1 ml of cold PBS was coated on a 96 well plate. After overnight incubation at 4oC, the plate was washed three times with cold PBS. A solution of 200IU/ml of IL-2 and 10µg/ml of mouse anti-human-CD28 (Clone L293, BD biosciences, catalogue no. 348040) in 1 ml of RPMI/10%FBS was added to the coated plate. 200,000 cells were added to the well and incubated for 3 days.
The activation state of CD4+T cell was monitored daily by microscopy for phenotypic signs (clumping and increase in size). Three days post activation, CD4+T cell size was measured based on Forward scatter (FSC) using flow cytometry. The percentage CD4+T cells with FSC>200 were computed. In addition, we measured the expression of CD69 and CD25 on CD4+T cells three days post-infection using flow cytometry. Briefly, samples were stained with 5µg/ml of mouse anti-human CD69- fluorescein isothiocyanate (FITC) (BD biosciences, catalogue no 555530) and 5µg of anti-human CD25- pyridine chlorophyll protein (PerCP) (BD bioscience catalogue no. 560503). Flow cytometry analysis was then performed with a FACS Calibur H (Becton Dickinson, Franklin Lakes, NJ). Figure 5B illustrates the gating strategy used. To confirm CD4+T cell activation, we based on the level of expression of CD25 and CD69 on CD4+T cells from cases and controls. The choice of CD25 and CD69 as our markers of T cell activation was based on prior experiments of HIV-1 infectivity assays reported by Walker and colleagues(10)
Infection with HIV pseudoviruses: Activated CD4+T cells were then cultured in M24 plates separately with 1ml of each of the three strains of pseudotyped HIV particles for 3 days. A third culture was setup in which no virus was added to the activated cells (Negative control). After 3 days of CD4+T cell-pseudotyped virus co-culture, we measured percentage GFP/YFP positive cells by flow cytometry using FACS Calibur H (Becton Dickinson, Franklin Lakes, NJ). In brief, we collected CD4+ T cell from the culture plate by pipetting up/down. We transferred cells into flow tubes with 50µl of fixation buffer (2% paraformaldehyde, PFA in PBS) and analyzed percentage GFP/YFP positive cells with flow cytometer. Figure 6 illustrates the gating strategy used.
Biostatistical analysis
Data was analyzed using flow Jo version 9.3.2 (Tree Star Inc., Ashland, OR) and GraphPad prism version 8.0. Data was graphically presented using column scatter plots for cases and control groups displaying group means and standard deviations or medians and ranges. We compared means/medians of the cases and control group. Statistical comparison was done using Mann Whitney U test for two groups and Wilcoxon rank sum test for more than two groups. Statistical significance was considered if p-value ≤ 0.05.