Diana V. Pastrana, Christopher B. Buck, Douglas R. Lowy and John T. Schiller
Laboratory of Cellular Oncology
Center for Cancer Research
National Cancer Institute
Building 37 Room 4106
9000 Rockville Pike
Bethesda, MD 20892-4263
Key Words: Papillomavirus, papillomaviral, vector, pseudovirus, pseudovirion, neutralize, neutralization, capsid, virion, pseudogenome, transduction, antibody, serum, Optiprep, iodixanol, ultracentrifugation, gene transfer.
Abstract
It has recently become possible to generate high titer papillomavirus-based gene transfer vectors. The vectors, also known as papillomavirus pseudoviruses (PsV), have been useful for studying papillomavirus assembly, cellular entry and neutralization and may have future utility as laboratory gene transfer tools or vaccine vehicles. In this protocol, PsV encapsidating a secreted alkaline phosphatase (SEAP) 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 SEAP activity in the culture supernatant using a highly sensitive chemiluminescent reporter system. Antibody-mediated PsV neutralization is detected by a reduction in SEAP activity. The neutralization assay has similar analytic sensitivity and higher specificity compared to a standard virus-like particle (VLP) ELISA.
1. Introduction
Several methods for in vitro production of papillomavirus virions or pseudovirions (PsV) have been reported. They include production in keratinocyte raft culture, in cultured monolayers of mammalian cells after infection with recombinant Vaccinia or Semliki Forest Virus vectors expressing L1 and L2, or in the test tube after reassembly of capsomeres in the presence of plasmid DNA 1234. However, none of these strategies efficiently produces high infectious titers. Because high-titer PsV carrying an easily scored marker gene were unavailable, papillomavirus neutralization assays have been laborious, both in terms of production of the infectious capsids and in the conduct of the neutralization assays. At our websitewe provide a procedurefor generating purified PsV with titers in excess of one billion transducing units per ml. We have used PsV produced by this method to develop a simple high throughput assay for detecting papillomavirus-neutralizing antibodies.
PsV encapsidating a secreted alkaline phosphatase (SEAP) reporter plasmid were used to develop the in vitro neutralization assay presented below. PsV transduction of 293TT cells is monitored by SEAP activity in the culture supernatant using a highly sensitive chemiluminescent reporter system. Antibody-mediated PsV neutralization is detected by a reduction in SEAP activity. This is the first papillomavirus neutralization assay to be adapted to a high-throughput 96 well plate format. A single 75 cm2flask can produce sufficient SEAP PsV for conducting thousands of neutralization assays. The neutralization assay appears to be as sensitive as, but more specific than, a standard VLP-based ELISA, and requires similar operator effort as an ELISA. The assay should have utility in both vaccine and sero-epidemiology studies.
2. Materials
3. Methods
3.1 Culture of 293TT Cells
3.1.1 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.
3.1.2 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 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.
3.2 Neutralization Assay
The methods described below outline (1) titration of the SEAP-PsV stock, (2) luminometry to detect SEAP production, and (3) determination of the neutralization titer of test sera. The PsV used for this assay encapsidates a reporter plasmid, pYSEAP, encoding secreted placental alkaline phosphatase (SEAP). When PsV infect 293TT cells, the pYSEAP reporter plasmid, which carries an SV40 ori, is replicated to high copy number by SV40 T antigen. This leads to high-level production of alkaline phosphatase that is secreted into the culture medium, and so can be easily assayed. Antibody–mediated neutralization of the PsV results in a corresponding reduction in SEAP expression.
3.2.1 Titration of SEAP-PsV Stocks
Before assaying for neutralizing activity of test sera, it is important to titrate the PsV stock to determine the inoculum that will be used in each assay. The goal of the titration is to determine the minimum amount of PsV required to give a robust signal in the SEAP assay (Subheading 3.2.2) that is well above background, but within the linear range of the assay. Typically, this falls in a range between 30 and 100 Relative Light units (RLUs) in the absence of neutralizing antibodies, with a background of no more than 1 RLU when the PsV is maximally neutralized with the positive control antibody or heparin. The method to titrate the stock follows.
10.Dilute the positive neutralization antibody (or heparin) such that it is 5-fold more concentrated than its known 95% neutralizing dilution. For example:
V5 (anti-HPV16 monoclonal) at 1:250,000
5B6 (anti-BPV monoclonal) at 1:25,000
Rabbit anti-VLP polyclonal sera at 1:10,000 to 1:1,000,000
Heparin H-4784 at 1 mg/ml
Dilute the antibody another 5-fold by adding 20 µl of diluted antibody (above) to triplicate wells containing 80 µl of diluted PsV.
11.Once the PsV and positive neutralization control(s) are combined, place on ice for 1 hour
13.Return to the incubator for 72 hours.
14.The media should not be replaced during these 72 hours (seeNote 3).
3.2.2 Chemiluminescent Detection of Secreted Alkaline Phosphatase
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. Although SEAP activity can be detected colorimetrically(seeNote 4), chemiluminescent methods, such as the one described here, are generally preferable since they offer a much higher signal to noise ratio.
10.Read on MLX Microplate Luminometer (Dynex Technologies) set at Glow-Endpoint 0.20 sec/well RAW Data Handling Average readings at 20 min after adding substrate.
The relative light units (RLUs) obtained from triplicate samples should not vary by more than 10 or 15%. If they vary more than that check the notes section to try to troubleshoot the problem.
3.2.3 Neutralization Assay
Once the PsV has been titrated, test sera (seeNotes 5 and 6) 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 PsV or serum, (2) at least four wells of PsV-infected cells to which no antibody was added, (3) cells treated with PsV pre-incubated with a known serum with at least 4 dilutions that span the 50% neutralizing titer that has been recorded in other experiments; and (4) cells treated with PsV pre-incubated with at least 1 dilution of a known non-neutralizing serum. See Fig. 2 for a typical arrangement of samples.
10.The medium should not be changed.
11.The supernatant is then assayed for presence of SEAP (Subheading 3.2.2). The titer is defined as the reciprocal of the highest dilution of serum that reduces the SEAP activity by at least 50% in comparison to the reactivity in the wells that received PsV but no antibody.
If the same PsV stock is used to repeat the neutralization, then the 50% neutralizing titer would should be the same, or vary by 3 or 4 fold. If the results vary by more than 4 fold, the assay should be repeated a third time. Report the geometric mean titer of all assays performed.
4. Notes
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A |
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B |
| HPV 16 PsV@ 1:300 no Ab or heparin | HPV 16 PsV@ 1:600 no Ab or heparin | HPV 16 PsV@ 1:800 no Ab or heparin |
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C |
| HPV 16 PsV@ 1:300 V5 MoAb @ 1:250,000 | HPV 16 PsV@ 1:600 V5 MoAb @ 1:250,000 | HPV 16 PsV@ 1:800 V5 MoAb @ 1:250,000 |
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D |
| HPV 16 PsV@ 1:1000 no Ab or heparin | HPV 16 PsV@ 1:2000 no Ab or heparin | HPV 16 PsV@ 1:4000 no Ab or heparin |
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E |
| HPV 16 PsV@ 1:1000 V5 MoAb @ 1:250,000 | HPV 16 PsV@ 1:2000 V5 MoAb @ 1:250,000 | HPV 16 PsV@ 1:2000 V5 MoAb @ 1:250,000 |
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F |
| HPV 16 PsV@ 1:5000 no Ab or heparin |
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G |
| HPV 16 PsV@ 1:5000 V5 MoAb @ 1:250,000 |
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Fig. 1 Schematic drawing for a 96-well plate for titering PsV. Shaded cells should be filled with 120µl of medium with phenol red to avoid evaporation from inner wells.
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| Test serum 1 @ 1:40 | Test serum 2 @ 1:40 | Test serum 3 | Test serum 4 | Test serum 5 | Test serum 6 | Test serum 7 | Test serum 8 | HPV16 + V5 @ 1:2.5x105 | HPV16 with no Antibody |
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| Test serum 1 @ 1:160 | Test serum 2 @ 1:160 | HPV16 + V5 @ 1:2.5x106 |
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| Test serum 1 @ 1:640 | Test serum 2 @ 1:640 | HPV16 + V5 @ 1:2.5x107 |
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| Test serum 1 @ 1:2560 | Test serum 2 @ 1:2560 | HPV16 + V5 @ 1:2.5x108 |
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F |
| Test serum 1 @ 1:10240 | Test serum 2 @ 1:10240 | HPV16 + 5B6 @ 1:2.5x105 | No PsV no Antibody |
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G |
| Test serum 1 @ 1:40960 | Test serum 2 @ 1:40960 | HPV16 + 5B6 @ 1:2.5x105 |
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Fig. 2: Schematic drawing for a typical set up for determining neutralizing titer of unknown sera with HPV16 PsV. Shaded wells should be filled with 120µl of medium with phenol red to avoid evaporation from inner wells.
References
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3. Unckell, F., Streeck, R. E. & Sapp, M. (1997). Generation and neutralization of pseudovirions of human papillomavirus type 33. J Virol71, 2934-9.
4. Touze, A. & Coursaget, P. (1998). In vitro gene transfer using human papillomavirus-like particles. Nucleic Acids Res26, 1317-23.
5. Buck, C. B., Pastrana, D. V., Lowy, D. R. & Schiller, J. T. (2004). Efficient intracellular assembly of papillomaviral vectors. J Virol78, 751-7.
6. Selinka, H. C., Giroglou, T., Nowak, T., Christensen, N. D. & Sapp, M. (2003). Further evidence that papillomavirus capsids exist in two distinct conformations. J Virol77, 12961-7.
7. Klasse, P. J. & Sattentau, Q. J. (2002). Occupancy and mechanism in antibody-mediated neutralization of animal viruses. J Gen Virol83, 2091-108.