Merkel Cell Polyomavirus Neutralization Assay

Diana V. Pastrana and Christopher B. Buck

Laboratory of Cellular Oncology

Key Words: Polyomavirus, vector, neutralize, neutralization, capsid, virion, transduction, antibody, serum, Optiprep, iodixanol, ultracentrifugation, gene transfer.

Reference:  Pastrana et al (2009) PLoS Pathogens 5:e1000578, PMID 19750217

Abstract

It has recently become possible to generate high titer Merkel cell polyomavirus (MCV)-based gene transfer vectors.  The vectors are useful for studying assembly, cellular entry and neutralization and may have future utility as gene therapy or vaccine vehicles.  In this protocol, MCV-based vectors encapsidating a secreted Gaussia luciferase (Gluc) reporter plasmid are used for a high throughput in vitro neutralization assay in a 96 well plate format.  Infection of 293TT cells is monitored by Gluc activity in the culture supernatant using a highly sensitive chemiluminescent reporter system. Antibody-mediated neutralization is detected by a reduction in Gluc activity.  The neutralization assay has superior sensitivity and accuracy compared to a standard virus-like particle (VLP) ELISA.

1. Materials

1. 293TT cells¹.  Although, Invitrogen has developed a conceptually similar cell line, 293FT(cat# R700-07), 293FT generally do not perform as well as 293TT in neutralization assays.  
2. DMEM-10: DMEM, 10% 56ºC inactivated fetal calf serum, 1% nonessential amino acids, 1% Glutamax-I (Invitrogen)
3. 50 mg/ml Hygromycin B stock (Roche)
4. 0.05% Trypsin / EDTA (Invitrogen)
5. MCV-based reporter vectorcarrying reporter plasmid phGluc
6. Siliconized pipet tips (VWR) (Optional)
7. Neutralization/growth media: DMEM with HEPES and without phenol red (seeNote 1) or sodium pyruvate (Invitrogen #21063-029), 10% 56ºC heat inactivated fetal calf serum, 1% MEM non essential amino acids (Invitrogen # 11140-050), 1% Glutamax-I (Invitrogen # 35050-061), 1% Antibiotyc-antimycotic (Invitrogen # 15240-062)  
8. Dilution plates:  untreated sterile U-bottom 96 well (Corning Costar #3788)
9. 96-well flat-bottom tissue culture treated plates (Corning Costar # 3596)
10. Assay plates: Optiplate-96, white for luminescence, isotopic & fluorescence (Perkin Elmer #6005299)
11. Multichannel pipettor 
12. Sterile reservoir for use with multi-channel pipettor
13. Polystyrene 15 or 50 ml conical tubes
14. Positive controls for neutralization assay: i.e. pooled human serum or sera from VLP-immunized rodents. 
15. Microplate Luminometer (e.g., BMB Labtech: POLARstar OPTIMA)

1. Gaussia Luciferase Assay kit (New England Biolabs # E3300L)

2.  Methods

2.1 Culture of 293TT Cells

2.1.1 Thawing 293TT Cells

            293TT cells are cultured in DMEM-10.  To thaw 293TT cells, place the thawed cells directly into a 150 cm²flask with 25 ml of DMEM with a total of 20% FCS. It is not necessary (or desirable) to spin the cells out of the freezing medium.  Like other types of 293 cell lines, 293TT do not adhere tightly.  It may take as many as three days for the cells to attach after thawing.  If cells do not attach after two days, it may help to spin them out of the medium, wash once with calcium-free PBS, then pellet and resuspend for five minutes at 37ºC in 1 ml of trypsin/EDTA.  Resuspend the trypsin-treated cells in 25 ml of DMEM-10 and plate in a 75 cm²flask.

2.1.2 Passaging 293TT Cells

            Split 293TT cells 1:5 to 1:20 when they reach 80-90% confluence.  Allowing 293TT cells to become super-confluent can irreversibly reduce their performance.  Detach cells by gently rinsing the flask once with several milliliters of trypsin, followed by a 5-10 minute incubation in 2 ml of fresh trypsin in a humidified 37ºC incubator.  It is important to trypsinize the cells thoroughly since insufficient trypsinization can lead to shredding of cell clumps during trituration (resuspension). Inactivate trypsin by adding 10 ml of DMEM-10.  Resuspend the cells and transfer a portion of the cell suspension directly into a fresh flask.  It is not necessary (or desirable) to spin the cells out of the residual trypsin, since it is inactivated by the fetal calf serum in DMEM-10.

After the cells have fully recovered from thawing, DMEM-10 can be supplemented with 400 µg/ml hygromycin B to promote maintenance of T antigen expression.  Although 293TT cells can typically be passaged for several months without alteration of PsV production or titration characteristics, early passages should be frozen in aliquots for long-term storage.

To freeze 293TT cells, reserve several ml of supernatant (conditioned medium) from a sub-confluent flask of cells. Trypsinize cells as described above, then resuspend in the reserved conditioned medium.  Mix the cell suspension 1:1 with freeze medium (fetal calf serum + 20% DMSO).  Freeze in 1ml aliquots of a few million cells per aliquot.  Place aliquots in a “Mr. Frosty” isopropanol bath pre-cooled to 4ºC. Place Mr. Frosty at -80º C overnight, then transfer aliquots into liquid nitrogen for long-term storage.

2.2 Neutralization Assay

The methods described below outline (1) titration of the MCV-Gluc vector stock, (2) luminometry to detect Gluc production, and (3) determination of the neutralization titer of test sera.  The MCV used for this assay encapsidates a reporter plasmid, phGluc, encoding secreted Gaussia Luciferase(Gluc).  When MCV vectors infect 293TT cells, the phGluc reporter plasmid, which carries an SV40 ori, is replicated to high copy number by SV40 T antigen.  This leads to high-level production of Gluc that is secreted into the culture medium, and so can be easily assayed. Antibody–mediated neutralization of the vector results in a corresponding reduction in Gluc expression. 

2.2.1 Titration of Gluc-vector Stocks

Before assaying for neutralizing activity of test sera, it is important to titrate the vector stock to determine the inoculum that will be used in each assay.  The goal of the titration is to determine the minimum amount of vector required to give a robust signal in the Luciferase assay (Subheading 2.2.2) that is well above background, but within the linear range of the assay.  Typically, this falls in a range between 50,000 and 600,000 Relative Light units (RLUs) in the absence of neutralizing antibodies, with a background of no more than 900 RLU when the vector is maximally neutralized with the positive control antibody (or when no virus is applied to the cells). The method to titrate the stock follows. 

1. Calculate the number of plates needed to titer the vector.
2. Trypsinize 293TT cells and suspend in neutralization/growth media.
3. Dilute the cells to 300,000/ml in the neutralization/growth media, place in a sterile reservoir.
4. Using a multichannel pipettor deliver 100 µl of cell suspension to each of the internal wells of a 96-well tissue culture treated plate.  To avoid evaporation, do not use external wells and instead fill surrounding wells with 120-150 µl of medium with phenol red (Fig. 1).
5. Replace cover and return the cells to the incubator for 2-5 hours (see Note 2) while setting up MCV vector / antibody mixtures.
6. Make serial dilutions of the MCV-Gluc vector stock.  Each dilution is tested in triplicate and enough should be diluted for at least 6 wells; three with and three without positive control neutralizing serum.  Depending on the vector stocks and polyomavirus type, appropriate dilution ranges are typically between 1:200 and 1:5,000.
7. Place 96 µl of the diluted vector into the wells of a U-bottom dilution plate.  
8. Add 24 µl of neutralization/growth media to “no antibody” control wells.
9. Dilute rabbit anti-MCV polyclonal serum (positive control) 1:500.  Add 24µl of diluted serum to diluted vector wells. 

10.Once the vector and positive neutralization control(s) are combined, gently mix the plate and place on ice for 1 hour

1. Add 100 µl of vector-antibody mixture to the preplated cells.

12.Return the plate to the incubator for 72 hours.

13.The media should not be replaced during these 72 hours (seeNote 3). 

2.2.2 Chemiluminescent Detection of Gaussia Luciferase

For this section of the protocol use a multi-channel pipettor when transferring liquids from one plate to the other. Make up kit reagents as indicated below and transfer to a reservoir so you can also use a multi-channel pipettor for those steps. 

If an injection-capable luminometer is not available, it may be possible to use a stabilized Gluc substraterecently developed by New England Biolabs.

1. After the 72 hour incubation, vigorously shake, swirl and pat culture plate to obtain a homogeneous distribution of Gluc in the supernatant. 
2. Transfer 25 µl of supernatant to the corresponding wells of a white optiplate-96 assay plate 
3. Use Gaussia Luciferase reagents according to manufacturer’s instructions. Briefly: 
4. Add 1 part of Luciferase substrate to 100 parts of Luciferase Assay Buffer. Make enough substrate to inject 50µl per well
5. Read on BMG Labtech POLARstar OPTIMA luminometer with 3 seconds of shaking after substrate injection reading at 1 second intervals for 12 seconds total. Integrate the signal observed from seconds 7 through 12 after substrate injection.

The relative light units (RLUs) obtained from triplicate samples should not vary by more than 15 or 20%.  If they vary more than that check the notes section to try to troubleshoot the problem.  

2.2.3 Neutralization Assay

Once the vector has been titrated, test sera (seeNote 5) can be assayed to determine endpoint neutralization titers. To monitor inter-assay variability, the following controls should be included for each plate: (1) at least two wells of cells in neutralization/growth media without vector or serum, (2) at least eight wells of vector-transduced cells to which no antibody was added.  For initial runs of the assay, it is useful to include a dilution series for a known neutralizing serum (rabbit-anti-MCV VLP or pooled human serum).   See Fig. 2 for a typical arrangement of samples. 

1. Trypsinize 293TT cells and suspend at 300,000/ml in the neutralization/growth media. Transfer suspension to a sterile basin.
2. Using a multichannel pipettor deliver 100 µl of cell suspension to each of the internal wells of a 96-well tissue culture treated plate.  To avoid “edge effects” due to evaporation of outermost wells, use only the inner wells of the plate (see Figure 2).  Fill outer wells with 100 µl of culture medium or PBS (Fig. 1). Replace cover and return the cells to the incubator for 2-5 hours (see Note 2) while setting up MCV vector / antibody mixtures.
3. Optional:  heat inactivate serum by incubating at 56ºC for 20 minutes (see Note 6).  Perform serial dilutions of the unknown sera in sterile polystyrene U-bottom plates.  Typically, unknown sera are diluted 1:20 (for example, 4 µl of serum into 76 µl of culture medium) then subject to 4-fold serial dilution (for example, 20 µl of diluted serum onto 60 µl of culture medium).  
4. Pre-wet a sterile multichannel pipet reservoir with neutralization/growth media to prevent sticking of vector to the reservoir. 
5. Dilute vector stock in neutralization/growth medium to the concentration determined inSubheading 2.2.1in a 15 or 50 ml polystyrene centrifuge tube.  For most MCV-Gluc vector stocks, a dilution of 1:3000 is appropriate. 
6. Vortex briefly and decant diluted vector stock into the pre-wetted reservoir. 
7. Transfer 96 µl of the diluted vector to each well of a 96-well dilution plate.  Optional: use of siliconized pipette tips may help avoid loss of vector titer due to non-specific adsorption.  
8. Add 24 µl of the test or control sera to the 96µl of diluted vector stock and incubate on ice for 1 hour. 
9. Add 100µl of the virus-antibody mixture to the corresponding wells of the plate using a multi-channel pipettor.

10.Return the plate to the incubator for 72 hours. 

11.The medium should not be changed.  

12.The supernatant is then assayed for presence of GLuc (Subheading 2.2.2).  

2.2.4 Data analysis

To calculate the effective 50% neutralizing concentration (EC₅₀) Prism software (Graphpad) is used to fit a sigmoidal dose-response curve to the luminometric data points. Although Prism can automatically calculate the top and bottom of the inhibition curve, we’ve found that it’s more reliable to standardize the curve to the average of the “no serum” and “no virus” control well values.

To start, transfer the RLU values into an Excel spreadsheet.  Calculate average no serum and no virus values.

1.  Make a column listing the serum dilution series (i.e., 100, 400, 1600, etc).  For reporting data, we make the conservative assumption that the biologically relevant antibody-virion equilibrium is reached during the pre-mix (step 8).  Thus a typical “1:100” top dose in a series is obtained when serum is first diluted 1:20 (Section 2.2.3 step 3), then diluted 1:5 with diluted vector stock (step 8). Under this convention, we do not take into account the additional 1:2 dilution into the cell culture well (step 9).

2.  Arrange the RLU values for each serum sample into columns next to the column listing dilution values.  Open Prism. Chose “XY” tab.  Paste the assay data as a block (including the dilution values) into box X1 of the Prism worksheet.  

3.  Click “=Analyze” button. 

- Under “Data manipulations” find “Transforms.” Click OK.  

-Click the “Transform X values using” box near the top of the pop up window.  Pull down “X=Log(X).”  Click OK. For some reason Prism prefers to deal with the X axis after conversion to log values.

4.  Click “=Analyze” button again.  Under “Curves & regression” find “Nonlinear regression (curve fit)”. Click OK button.  

-Highlight “Sigmoidal dose-response (variable slope).”  

-Click “Constraints” button.  Next to BOTTOM pull down “Constant equal to” and type the “no virus” average into the Value window.  Next to TOP, pull down “Constant equal to” and type in “no serum” control value. Click OK. 

5.  EC₅₀values will be listed in the “Table of results” sheet, curves will be plotted in the Graphs folder under “Transform of Data 1.”  

In repeat assays, the EC₅₀of a given serum should vary by no more than about three fold.  

3.  Notes

1. The presence of phenol red tends to give a higher background in the chemiluminescent detection assay; therefore medium without phenol red is preferred for the assays.

1. The timing of plating of cells in the neutralization assay is important. Preplating overnight, or just before adding vector results in suboptimal transduction.
2. Since 293TT cells detach very easily, replacement of the media once the procedure has been started is not recommended.
3. With many vector/ antibody combinations, the neutralization assay probably operates under conditions of antibody excess (for an excellent review of this important concept, see Klasse and Sattentau²). For this reason, modest changes in the amount of input vector typically do not alter the 50% neutralization cutoff (Pastrana et al. Plos Pathgens submitted). 

1. Sera frequently have non-specific interfering activity at dilutions of 1:50 or lower, so 1:100 is a typical starting dilution.  Highly neutralizing sera may need to be diluted by as much as ten million fold to encompass the 50% neutralization value. 
2. Complement can dramatically enhance antiviral neutralizing titer via mechanisms that aren’t fully understood.  Complement is unstable and will decay over the course of several days storage of a serum at 4ºC.  Freshly thawed sera may display a higher EC50 value immediately after thawing compared to re-testing after storage at 4ºC.  A 20 minute incubation of serum at 56ºC will destroy complement, thus immediately stabilizing the observed EC50.  Antibodies are highly stable and the heating therefore does not alter EC50 when compared to sera stored for at least a week at 4ºC.

Fig. 1 Schematic drawing for a 96-well plate for titering Merkel Cell Polyomavirus vector.  Shaded cells should be filled with 120µl of medium with phenol red to avoid evaporation from inner wells. 

Fig. 2: Schematic drawing for a typical set up for determining neutralizing titer of unknown sera with Merkel Cell Polyomavirus vector.  Shaded wells should be filled with 120µl of medium with phenol red to avoid evaporation from inner wells.  

References

1.         Buck, C. B., Pastrana, D. V., Lowy, D. R. & Schiller, J. T. (2004). Efficient intracellular assembly of papillomaviral vectors. J Virol78, 751-7.

2.         Klasse, P. J. & Sattentau, Q. J. (2002). Occupancy and mechanism in antibody-mediated neutralization of animal viruses. J Gen Virol83, 2091-108.