1. CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
3.2 Methods
3.2.1 Generation of Gag-Protease recombinant virus stocks
Recombinant viruses for 80 participant derived samples were generated by co-transfection of a CEM derived T-cell line (i.e. GXR cells) with an NL43-deleted-gag-protease (NL43∆gag-protease) backbone and patient derived Gag-Protease amplicons (generated in section 2.2.3) using a method previously described and validated (19-22).
3.2.1.1 Generation of pNL43∆Gag-pro backbone
A pNL43∆gag-protease plasmid, supplied in E. coli STBL3 cells by Dr Jaclyn Mann (University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South Africa), was used as a backbone for generation of recombinant viruses. The plasmid contained a BstE II restriction enzyme recognition site in the Gag-Protease coding region, enabling linearization of the plasmid as required for recombination with patient-derived Gag- Protease amplicons (19-21).
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Large quantities of pNL43∆gag-pro plasmid stocks were generated by adding 30 µl of the pNL43∆gag-pro plasmid (in STBL3 cells) to a mixture of Luria-Bertani (LB) (Sigma Aldrich, St Louis, USA) broth and ampicillin (100 ml of LB broth and 100 µl ampicillin). This was incubated in a shaking incubator (Infors HT, Bottmingen, Switzerland) at 37ºC and 230 rpm for 16 hours. Thereafter, the plasmid was extracted and purified using the Qiagen Plasmid Maxi kit (Qiagen) as per manufacturer’s instructions, and quantified using a nanodrop spectrophotometer (Thermo Scientific, Delaware, USA). Quantified plasmid was aliquoted and stored at -80ºC.
3.2.1.2 Preparation of CEM-GXR25 cells
A CEM-GXR25 green fluorescent protein (GFP) reporter T-cell line (i.e. GXR cells) (23) was supplied by Dr Mark Brockman (Simon Fraser University, Vancouver, Canada). GXR cells are replication competent as they express the CD4 receptor and both the CXCR4 and CCR5 co-receptors. Additionally, they encode a Tat-inducible HIV-1 LTR- GFP expression cassette, which is responsible for the expression of GFP during infection thereby facilitating detection of infected cells using flow cytometry (23).
A frozen aliquot (i.e. 1 ml) of approximately 1 million GXR cells (stored in dimethylsulfoxide [DMSO], Sigma) was transferred from a liquid nitrogen freezer (Custom Biogenics Systems, Romeo, USA) directly into a preheated 37ºC water bath. The tube of cells was gently agitated in the water bath until the contents were completely thawed. Thereafter the tube of cells was transferred into a T25 flask (Corning-Costar, New York, USA) containing 4 ml of pre-warmed R10 culture medium, and incubated at 37ºC and 5% CO2 for 24 hours in a humidified Heraeus incubator (Thermo Scientific). R10 media comprised of RPMI- 1640 (Sigma), supplemented with 50 U/ml penicillin streptomycin (Gibco, New York, USA), 10 mM N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid (HEPES; Gibco), 2 mM L- glutamine (Sigma) and 10% foetal bovine serum (FBS; Gibco).
After 24 hours, the contents of the T25 flask was transferred into a 15 ml falcon tube and centrifuged at 1,500 rpm for 10 minutes (Heraeus multifuge 3SR+, Thermo Scientific) in order to remove DMSO. Cells were then re-suspended in 1 ml of pre-warmed R10 and transferred into a T25 flask containing 9 ml of pre-warmed R10. The flask was then incubated at 37ºC and 5% CO2 for a further 24 hours.
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Following incubation, cells were counted by adding 10 µl of thoroughly mixed cell culture to 10 µl of trypan blue (Bio-Rad, Hercules, USA). A total of 10 µl of this mixture was inserted into a TC20 cell counting slide (Bio-Rad) which was subsequently loaded into a TC20 automated cell counter (Bio-Rad). The output of the TC20 cell counter was the cell concentration (i.e. cells/ml). The volume of cell culture used to obtain a required number of cells was calculated as follows:
Volume of cell culture (ml) = number of cells required ÷ cell concentration (cells/ml)
Cells were maintained at a concentration of 250 000 cells/ml in a final volume of 30 ml in a T75 flask (Corning). Cell growth was monitored every second day and if not used for experiments, 80% of the cell culture was removed and replaced with fresh pre-warmed R10. Cells were maintained for a maximum of two months, after which time a new aliquot of GXR cells were thawed and prepared for use.
Aliquots of GXR cells were also stored in liquid nitrogen for use in future experiments, within two weeks of thawing. Briefly, 5 million GXR cells re-suspended in 900 µl of R10 was added to a cryovial (Greiner Bio-One, Germany). Immediately thereafter, 100 µl of DMSO was added drop by drop with simultaneous gentle agitation. The DMSO functioned to prevent the formation of water crystals during cryopreservation (24). The cryovials were then stored in a strata-cooler (Agilent Technologies, Waldbronn, Germany) at -80ºC overnight. The Strata cooler functioned to gradually reduce the temperature of the cells to - 80ºC (at a controlled rate of 0.4-0.6ºC per minute) prior to transfer into a liquid nitrogen freezer (25).
3.2.1.3 Co-transfection
Recombinant viruses were prepared as previously described (26). Briefly, 2 hours prior to co-transfection, 10 µg of pNL43∆gag-pro plasmid (prepared in 3.2.1.1) was digested with 2 µl of a 10 U/µl stock of BstE II enzyme (Promega, Madison, USA) for 2 hours in a water bath set to 60ºC. This functioned to linearize the plasmid. During this time, GXR cells were prepared (described in section 3.2.1.2). A total of 5 million cells were required for each co- transfection. One million of which was re-suspended in 1 ml of R10 and 4 µl of polybrene (10 µg/µl)18 (Sigma) and subsequently added to an additional 8 ml of R10 in a T25 flask.
18 Polybrene functions to make the cell wall porous, in order to facilitate virus entry into the cell.
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These flasks were incubated at 37ºC and 5% CO2. The remaining 4 million cells were re- suspended in 300 µl of R10 and added to a 4 mm electroporation cuvette (Bio-Rad).
A total of 80-90 µl of patient-derived Gag-Protease amplicons (i.e. 5-20 ng/µl) (generated in section 2.2.3), with either end of Gag and Protease exactly complementary to that of NL43, was thawed and added to the respective cuvette.
Thereafter, approximately 15–20 µl of digested pNL43∆gag-pro plasmid (i.e. 10 µg of plasmid) was added to the cuvette. The contents of the cuvette was mixed using a 200 µl pipette. This was followed by electroporation in a Gene Pulser II electroporator (Bio-Rad), with conditions set to 250 V and 950 µF. Electroporation served to temporarily disrupt the cell wall enabling virus and plasmid to enter cells (27). Cuvettes were left at room temperature for 5 minutes, to allow for cell recovery. Thereafter the contents of cuvettes were transferred into the T25 flask containing the 1 million GXR cells in 9 ml R10 and 4 µl polybrene.
Flasks were incubated at 37ºC and 5% CO2 for five days. On the fifth day an additional 5 ml of R10 was added to each flask followed by further incubation for five more days. On the tenth day, and every two days thereafter, 2 ml of culture was removed and replaced with 2 ml fresh pre-warmed R10. The 2 ml of culture removed was used to monitor viral growth.
No more than 10 recombinant viruses were generated at a time. Additionally, each experiment included a negative control (GXR cells only) and a positive control (NL43-WT virus). The NL43-WT virus was generated as part of this study by transfection of GXR cells with 10 µg of NL43-WT plasmid.
3.2.1.4 Monitoring viral growth by flow cytometry
Viral growth was monitored from the tenth day since the linearized plasmid and Gag- Protease amplicons required time to recombine and replicate (26).
From the 2 ml of culture removed from each flask (see section 3.2.1.3), a total of 1 ml was added to matrix cluster tubes (Corning Costar) and centrifuged at 1,500 rpm for 10 minutes, in order to pellet the cells. The majority of supernatant was discarded, leaving
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behind very little residual supernatant to re-suspended the pelleted cells. Thereafter, 200 µl of 2% paraformaldehyde (PFA) was added to each tube and vortexed to mix thoroughly.
Paraformaldehyde was prepared by adding 10 g of paraformaldehyde powder (Sigma) to 400 ml of Dulbecco’s phosphate buffered saline (PBS; Gibco) and incubating the mixture at 55ºC for 45 minutes in a water bath. Thereafter the pH was adjusted to 7–7.4 and 100 ml more of Dulbecco’s PBS was added. The 2% PFA solution was aliquoted and stored at -20ºC.
Following the 10 minute incubation of cells with PFA, matrix cluster tubes were inserted into FACs tubes (BD Biosciences, San Jose, USA), vortexed and flowed using the BD FACS Calibur (BD Biosciences). A negative control (i.e. GXR cells only) was always flowed first in order to gate live cells and distinguish between infected and uninfected cells (Figure 3.2). A total of 15,000 cells were gated. All data from the flow cytometer was further analysed using FlowJo (28). Figure 3.2 shows the gating of live cells; expression of
>0.05% GFP indicated cell infection.
Figure 3-2 Example of the gating strategy used to distinguish between live and dead GXR cells and infected versus uninfected GXR cells. (a) Gating of a negative control identified that 87.3% of cells were alive. (b) The threshold to distinguish between infected and uninfected cells was set at 0.05% (c) Flow cytometry of a sample showed that a total of 3.15% of cells in one of the test samples was infected with HIV.
3.2.1.5 Harvesting recombinant viruses
Viruses were harvested once 25–30% of GXR cells were infected, as previously described (26). For harvesting, the contents of the T25 flask, containing the viral culture, was emptied into a falcon tube and centrifuged at 1,700 rpm and 4ºC for 5 minutes. The
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supernatant was then aliquoted into cryovials (1 ml aliquots) and stored at -80ºC for use in titre, replication capacity and phenotyping assays.
3.2.1.6 Validation of recombinant viruses
A total of eight randomly selected recombinant viruses were subject to RNA extraction, amplification and sequencing of the Gag-Protease region (Chapter 2, section 2.2.2, 2.2.3 and 2.2.4). Virus sequences were analysed in conjunction with matched HIV-1 RNA plasma sequences in order to determine if sequences of recombinant viruses were representative of the matched plasma sequences. This was achieved by using the highlighter tool in Los Alamos to quantify similarity between pairs of sequences (i.e.
recombinant virus sequence and plasma sequence) (29). Sequences were also assessed for contamination using a neighbour joining tree drawn in Paup version 4.0 which was edited in Figtree version 1.4.2 (30).
3.2.1.7 Validating consistency of the replication capacity assay
The consistency of the replication capacity assay was also validated. Stored RT-PCR products from 10 samples which were analysed for replication capacity in a previous study conducted in 2008, at the HIV Pathogenesis Programme laboratory, (26) were used to generate recombinant viruses for the current study. These recombinant viruses were subject to replication capacity assays, conducted in duplicate. Replication capacity data from 2008 was then correlated to the replication capacity data for the same samples generated in 2014.