HIV entry: Env-mediated Cell Fusion Measured in Cell-Based System Using Plate Reader - 7013-01_Inhibitor_Dose_CherryPick_Activity
Although some inhibitors of HIV-1 entry exist, these are not suitable for use in a prophylactic setting because of limitations in potency, breadth and/or adaptability to a microbicide formulation. HIV-1 Envs expressed on the surface of cells can mediate the fusion of cells with target cells expressing the CD4 and CCR5/CXCR4 receptors, such as CEM lymphoblasts. We have established an assay that more ..
BioActive Compounds: 1019
Depositor Specified Assays
Keywords: cell fusion, Env protein, HIV, luciferase
Although some inhibitors of HIV-1 entry exist, these are not suitable for use in a prophylactic setting because of limitations in potency, breadth and/or adaptability to a microbicide formulation. HIV-1 Envs expressed on the surface of cells can mediate the fusion of cells with target cells expressing the CD4 and CCR5/CXCR4 receptors, such as CEM lymphoblasts. We have established an assay that monitors the cell-cell fusion activity of HIV-1 Envs with a firefly luciferase readout. In our primary high-throughput screen (HTS), a modified HeLa cell line that inducibly expresses the Envs of a primary HIV-1 strain (HIV-1JRFL) will be co-cultivated with receptor-bearing target cells (CEM21) in the presence of the compounds to be screened. CEM21 expresses a Tet-activator responsive Luciferase reporter. This is a Tetracycline regulated system and when the Tet-activator from the Env-expressing cell line is transferred into the target cell upon successful fusion, the Luciferase reporter will be activated. Detection of Luciferase will occur using the SteadyGlo reagent (Promega, Madison, WI). Maraviroc, a known CCR5 antagonist, that blocks Env-mediated cell fusion will be used as a positive control.
Expected Outcome: Compounds that inhibit cell fusion will cause a decrease in Luciferase signal.
HIV Fusion Assay Protocol (Streamlined version for HTS)
1.Grow JRFL effector cells in DMEM passage medium with Doxycycline. Split 1:3 or 1:4 for passing. For HTS: 4 days prior to plating, thaw 3 vials (1.0 x 10;7 cells/ vial) into 4 triple flasks with 120 mL DMEM passage medium/flask. After 3 days, transfer to 3 Hyperflasks (HF) with ~550 mL RPMI induction media (~33 million cells per HF)
2.Day 0: Pass JRFL cells to DoxFree. Wash cells 2x with PBS & add 15 ml Accutase per triple to detach cells. Stop Accutase by adding RPMI induction media. Spin down cells & seed 9-10 x10;6 cells in 120 ml of RPMI induction medium in triple flask.
3.Day 1: Plating of JRFL cells. Wash cells with PBS & add 15 ml Accutase for triple or 50 ml for HF to detach cells (@ 37 degrees C). Stop by adding RPMI induction medium. Spin down the cells at 1200 rpm for 5 min & re-suspend in phenol red-free RPMI induction medium (with Puromycin).
4.Count cells, adjust to 1.25 x10;5 cells/mL & seed 2,500 cells per well in 20 microl of RPMI (no Phenol Red) induction medium in Aurora white, opaque 384 well plates.
5.Day 2: Pinning & adding CEM21 cells Spin down & count CEM21 cells. Adjust to 5.0x10;5 cells/ml in RPMI fusion medium. Dispense 5,000 cells at 10 microl/well in RPMI induction media. Pin transfer compounds to the plate. Positive control is Maraviroc to a final concentration of ~300 nM. Incubate the plate 37 degrees C O/N.
6.Day 3: SteadyGlo & Read. Add 10 microl of room temperature Steady-Glo substrate. Wait 10 minutes & read luminescence with ultrasensitive settings on Perkin-Elmer EnVision with 0.5 second read time per well.
DM EM passage medium (1 L):
DMEM 882 mL
Heat Inactivated-FBS 100 mL
Pen-Strep-L-Glu 10 mL
*Doxycycline (1 mg/mL)2 mL
Puromycin (10 mg/mL)100 microL
Geniticin (50 mg/mL)4 mL
RPMI induction medium (DoxFree) (1 L):
RPMI (-Phenol red)890 mL
10 % Tet approved HI-FBS 100 mL
Pen-Strep-L-Glu 10 mL
Puromycin (10 mg/mL)100 microL
.*Doxycycline is light sensitive so minimize light exposure
.All cells have to be in exponential growth phase. JRFL & Hela-F-Luc cells are usually split 1:3 every 2-3 days, CEM#21 are split 1:10 every 3 days. Cells should not be allowed to overgrow in the flasks.
.CEM21 cells take 1-2 weeks after thawing to grow at an optimal rate
.Cells must be spun down after dissociation to remove StemPro Accutase from the medium.
.Cells can be allowed to fuse 16 to 24 hours.
.Effector (JRFL) cells should be propagated for no more than 30 passages or 3 months whichever is sooner.
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)