1. CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
3.2 Methods
3.2.3 Virus titration and phenotypic drug susceptibility testing
Phenotypic drug susceptibility assays were conducted to determine the drug susceptibility profiles of 18 selected viruses. Two drugs were used; LPV and DRV (both obtained from the AIDS Research and Reference Reagent Programme, Division of AIDS, NIAID, NIH).
Viruses employed in this assay were selected based on data from replication capacity assays and sequence analysis (described further in the results section).
For the current study, a TZMBL cell based two-cycle phenotypic drug susceptibility assay was employed (31). A two-cycle assay was selected, since the inhibitory effect of PIs cannot be established in a single cycle. Viral titres were also conducted using a two-cycle assay (31).
3.2.3.1 Preparation of TZMBL cells
TZMBL cells are a luciferase reporter based HeLa cell line. These cells are adherent, they express CD4 and CCR5 and have been engineered to include a luciferase gene which is under the control of the HIV-1 promoter. As such they are replication competent and allow for detection of infected cells by luminescence. TZMBL cells, were obtained from the AIDS Research and Reference Reagent Programme, Division of AIDS, NIAID, NIH.
An aliquot of TZMBL cells (1 ml) was removed from a liquid nitrogen freezer and immediately placed in a water bath at 37ºC. The cells were transferred to a 15 ml falcon tube containing 10 ml of pre-warmed Dulbecco’s Modified Eagle’s Medium (DMEM;
Sigma), supplemented with 10% FBS (Gibco), 50 U/ml penicillin streptomycin and 10 mM
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HEPES. The 15 ml falcon tube was centrifuged at 1,200 rpm for 10 minutes. Supernatant was removed, pellets were re-suspended in fresh DMEM (5 ml) and transferred to a T25 flask containing an additional 5 ml of DMEM. The T25 flask was incubated at 37ºC and 5%
CO2 for 48 hours. After 48 hours, cells were visualised using a Zoe® fluorescent imager (Bio-Rad). If cells were not 100% confluent, media was removed and replaced with fresh pre-warmed DMEM. If cells were 100% confluent, they were counted and used in experiments or seeded accordingly.
Counting of cells required that they be dislodged from the monolayer. This was achieved by removing DMEM from the flask, rinsing the monolayer with PBS and adding 2.5 ml of 0.25% trypsin-EDTA (Sigma) to the cell monolayer. The flask was incubated at room temperature for 2 minutes, followed by removal of trypsin-EDTA and subsequent incubation at 37ºC and 5% CO2 for 4 minutes. Thereafter, 10 ml of pre-warmed DMEM was added and the wall of the T25 flask, containing the cell monolayer, was repeatedly rinsed in order to dislodge cells. The contents of the flask was thoroughly mixed. Cells were then counted as described for GXR cells in section 3.2.1.2.
The required number of cells were removed and used in experiments, whilst the remaining cells were maintained at 250,000 cells/ml in DMEM in a T25 culture flask, incubated at 37ºC and 5% CO2. Cells were monitored by microscopy, fed and split every 48 hours.
Cells were maintained for a maximum of one month, after which time a new aliquot of cells was prepared for use in experiments.
3.2.3.2 Two-cycle virus titration
As per Puertas et al., (2012), a two-cycle infection assay was conducted in the absence of drug, in order to determine the amount of virus required to obtain the mean 50% tissue culture infective dose (TCID50) of each virus. The main aim of this approach was to standardise the virus input thereby attaining a similar rate of infection for each virus stock in order to exclude failure of the experiment due to fitness costs inherently associated with PR RAMs (31).
Virus titrations were set up in a 96 well tissue culture plate (Corning Costar). A total of 100 µl of DMEM was added to all wells of the plate. Thereafter, 25 µl of virus (generated in 3.2.1) was added to the first 3 wells (i.e. in triplicate). A 5-fold serial dilution was then
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performed. All wells of column 12 were virus free; this served as the cell control. A total of 10,000 TZMBL cells in 100 µl of DMEM and 0.05 g/µl of diethylaminoethyl-dextran hydrochloride (DEAE dextran; Sigma) was added to all wells. The plate was then incubated at 37ºC and 5% CO2 for 48 hours. Following incubation, the contents of each well was mixed and 100 µl of supernatant was removed from each well and added to the corresponding wells of a new 96 well culture plate, containing 10,000 TZMBL cells in 100 µl of DMEM and 0.05 g/µl of DEAE-dextran. The new culture plate was incubated at 37ºC and 5% CO2 for a further 48 hours. After the second incubation (i.e. second-cycle of infection), 100 µl of culture medium was removed from each well and replaced with 100 µl of Bright Glo luciferase reagent (Promega). The plate was incubated at room temperature for 2 minutes, the contents of wells were thoroughly mixed and 100 µl of culture from each well was transferred to corresponding wells of a 96 well black solid bottom microplate (Promega). Luminescence (i.e. indicator of infectivity) was measured using a Glomax®- Multi Microplate Multimode reader (Promega).
Data expressed as relative light units (RLUs) was analysed according to the method by Reed and Muench (32). Positive infection was quantified using a cut-off of 2.5 times that of the background RLU.
3.2.3.3 Two-cycle phenotypic drug susceptibility assay
The two-cycle phenotypic assay was performed as previously described (31). Drug concentrations for LPV and DRV used in this study ranged from 1 µM to 0.00032 µM.
These drug concentrations were within the range of that used in a previous susceptibility assay (33). Each assay comprised of a cell control (cells only, no virus or drug), a virus control (virus and cells only, no drug) and virus experiments (virus, cells and drug).
Briefly, in the first round of infection 10,000 TZMBL cells (in 100 µl of DMEM, 0.05 g/µl of DEAE dextran) and 3-fold serial dilutions of the respective PI was infected with the relevant amount of virus that yielded 50 TCID50s (as calculated in 3.2.3.2), in a 96 well culture plate. The plate was incubated for 48 hours at 37ºC and 5% CO2. Following incubation, the contents of each well was mixed and 100 µl of culture was transferred to corresponding wells of a 96 well plate containing 10,000 TZMBL cells (in 100 µl of DMEM and 0.05 g/µl of DEAE dextran). The plate was once again incubated at 37ºC and 5% CO2
for an additional 48 hours, this constituted the second cycle of infection. After 48 hours,
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150 µl of supernatant was removed and replaced with 100 µl of Bright Glo reagent. The plate was incubated for 2 minutes at room temperature. Thereafter, the contents of each well was mixed and transferred to corresponding wells of a black solid bottom microplate.
HIV-1 infection was measured by luciferase expression using the Glomax luminometer
The extent to which the drug inhibited viral replication (i.e. percent inhibition) was calculated by determining the difference in RLUs between the test wells and the cell control wells (i.e. negative/cells only) and dividing this value by the difference between the virus control wells (i.e. virus without exposure to drugs) and the cell control well and multiplying the result by 100 for each virus. The calculation is given below:
%inhibition = (test wells – cell control wells) ÷ (virus control wells – cell control wells)
The concentration of drug required to inhibit viral replication by 50% (i.e. IC50) was calculated by fitting the percent inhibition data to a sigmoidal dose-response curve (with a variable slope) in GraphPad Prism.
Fold change in drug susceptibility (i.e. variations in amount of drug required to achieve the IC50 between the virus of interest and a reference virus) was calculated by dividing the IC50
of each virus by the IC50 of the NL43-WT reference strain, which was known to be susceptible to LPV and DRV.
For this study, two classifications of drug susceptibility was used to categorize viruses these included: susceptible (S) and reduced susceptibility (RS). For viruses to be considered as susceptible, their FC was required to be below the lower FC cut-off established as part of this study. To be categorized as having reduced susceptibility the FC values were required to be greater than the lower FC cut-off level.
The lower FC cut-off level for LPV and DRV was calculated using the 99th percentile of the average IC50 for the NL43-WT reference virus, which was known to be susceptible to LPV and DRV (50). This data was obtained from multiple (i.e. 20) independently repeated phenotypic susceptibility assays for both LPV and DRV. Table 3.1 shows the calculated lower FC cut-off values for LPV and DRV used in this study. The lower FC cut-off for LPV was 2.42 whilst DRV was 1.49. A previous study reported the lower biological cut-off for
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LPV to be 1.6 and DRV to be 2, using the Antivirogram-Virco kit (33) whilst the clinical cut- off for both LPV and DRV has been reported to be 10 (54). The cut-off values used in the current study are thus similar to those which have been previously reported.
The drug susceptibility of each virus was measured in triplicate in each assay, with each virus being phenotyped in at least duplicate, in independently repeated experiments.
Table 3-1Overview of data used in the calculation of the lower FC cut-off levels for lopinavir and darunavir
Lopinavir Darunavir
Average 0.013 0.040
Standard deviation 0.004 0.004
95% confidence interval 0.011 0.038
Number (n=) 20 20
Minimum 0.010 0.033
Median 0.012 0.040
Maximum 0.024 0.048
(Q1) 0.010 0.040
(Q3) 0.013 0.042
1st Percentile 0.010 0.033
99th Percentile 0.024 0.048
Lower FC cut-off 2.42 1.49
Table 3-2 FC cut-off levels used to categorize viruses as being susceptible or conferring reduced susceptibility to LPV and DRV.
Susceptible (S) Reduced susceptibility (RS)
Lopinavir <2.42 >2.42
Darunavir <1.49 >1.49