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A rapid, high-throughput vaccinia virus neutralization assay for testing smallpox vaccine efficacy based on detection of green fluorescent protein
Journal of Virological Methods 150 (2008) 14–20
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
A rapid, high-throughput vaccinia virus neutralization assay for testing smallpox vaccine efficacy based on detection of green fluorescent protein Matthew C. Johnson, Inger K. Damon, Kevin L. Karem ∗ Poxvirus Program, Division of Viral and Rickettsial Diseases, US Centers for Disease Control and Prevention, 1600 Clifton Road, Mail Stop G-43, Atlanta, GA, United States
a r t i c l e
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Article history: Received 9 November 2007 Received in revised form 14 February 2008 Accepted 21 February 2008 Available online 2 April 2008 Keywords: Vaccinia Neutralization Smallpox GFP Hoechst
a b s t r a c t Virus neutralization remains a vital tool in assessment of vaccine efficacy for smallpox in the absence of animal smallpox models. In this regard, development of a rapid, sensitive, and high-throughput vaccinia neutralization assay has been sought for evaluating alternative smallpox vaccines, use in bridging studies, as well as understanding the effects of anti-viral immunotherapeutic regimes. The most frequently used method of measuring vaccinia virus neutralization by plaque reduction is time, labor, and material intensive, and therefore limiting in its utility for large scale, high-throughput analysis. Recent advances provide alternative methods that are less labor intensive and higher throughput but with limitations in reagents needed and ease of use. An innovative neutralization assay is described based on a modified Western Reserve vaccinia vector expressing green fluorescent protein (WR-GFP) and an adherent cell monolayer in multi-well plate format. The assay is quick, accurate, provides a large dynamic range and is well suited for large-scale vaccination studies using standard adherent cell lines. Published by Elsevier B.V.
1. Introduction Successful eradication of smallpox in the last century occurred due to the efficacy of vaccinia as a vaccine, but ironically in the absence of good indicators of correlates of protective immunity. A “take” at the vaccination site remains the most reliable indicator of successful vaccination. Virus neutralization provides another measure of inferred protective immunity. Traditional orthopoxvirus limiting-dilution neutralization methods are laborious, particularly for large numbers of test samples and take up to 48–72 h for plaque formation (Katz, 1987). Analysis of results takes additional time and data output may be subjective since plaques are counted manually. Manual counting based on visual readout represents a source of non-standardized error (user based) and can weaken the interpretation in number of events counted per sample (usually <50) and can also induce subjective differences if plaque size variation is observed. Nevertheless, these assays are vital for both clinical diagnosis of viral infection and research into the humoral immune response to infection. These limitations in traditional plaque reduction assays provide an impetus for development of high-throughput, automated neutralization assays that do not rely on plaque formation, and subsequent manual counting, as a measurement of anti-virus activity. In recent years there have been several assays developed that are high-throughput, semi-automated, and that do not rely on
∗ Corresponding author. Tel.: +1 404 639 1598; fax: +1 404 639 1060. E-mail address: [email protected] (K.L. Karem). 0166-0934/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jviromet.2008.02.009
plaque formation and manual counts. Some of these detect aggregate cell infection as indicated by enzyme immunoassay (Eyal et al., 2005) or the expression of recombinant reporter genes such as galactosidase (BGZ), green fluorescent protein (GFP) and luciferase (Chakrabarti et al., 1985; Dominguez et al., 1998; Manischewitz et al., 2003; Rodriguez et al., 1988), while others utilize recombinant GFP expression coupled with flow cytometry to produce an individual cell-based readout as an additional advance in sensitivity (Cosma et al., 2003; Earl et al., 2003). Perceived difficulties of these assays may include the use of cell suspension cultures for GFP assays, which may be laborious to maintain, and a lower dynamic range observed with enzymatic (BGZ) assays. Described here is an alternative GFP fluorescent assay in a high-throughput format that uses adherent cells (monolayer) and provides exceptional dynamic range without the use of flow cytometry and single cell suspensions.
2. Materials and methods 2.1. Virus Western Reserve vaccinia vector expressing green fluorescent protein (WR-GFP) (VV.NP-S-EGFP) was kindly provided by Patricia Earl (Laboratory of Viral Diseases, NIAID, NIH). This recombinant vaccinia virus is referred to as WR-GFP to reflect that it was derived from the Western Reserve strain, and contains a chimeric gene that encodes the influenza virus nucleoprotein, ovalbumin SIINFEKL peptide, and enhanced GFP regulated by the P7.5 early–late
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´ et al., 1999; Mackett et al., 1984; Norbury et al., promoter (Anton 2002). Nuclear localization of GFP expression and accumulation has been observed. The virus was propagated in BSC-40 cells and was purified before storage at −80 ◦ C until needed. 2.2. VIG and human sera Vaccinia immunoglobulin (VIG) (Cangene VIGIV, Product 172, Lot#1730403, 2006, 53 mg/mL) was obtained for use in vaccine efficacy studies using traditional plaque reduction assays (Wittek, 2005). Human serum samples were obtained as part of CDC vaccine study and public health response efforts. Assay control sera were derived for ELISA-based serodiagnostics and represent na¨ıve donors and vaccine recipients. 2.3. ArrayScan HCS Reader and target acquisition software from Cellomics Inc. The ArrayScan system is an automated fluorescent microscope coupled with image-processing and analytical software that can autonomously record the size, location, and fluorescent intensity (in several channels) of a large number of objects on a 96-well plate (Cellomics Inc., Pittsburgh, PA). For this assay, cells are identified by Hoechst 33342 nuclear stain and the average GFP intensity per cell is observed in order to determine the level of vaccinia virus gene expression (through both mean average GFP intensity, and the percentage of GFP expressing cells). The instrument has the capability to record images in six separate fluorescent channels: Hoechst, FITC (GFP), TRITC, Cy5, YFP, and TexasRed. The protocol described below relies on a two-channel scan with nuclear (Hoechst) staining in channel 1 and GFP in channel 2. The Cellomics target acquisition software identifies objects in the visual field via channel 1 and records features such as location, size, and shape in order to identify cell-like objects. Multichannel (Hoechst and GFP) features such as average fluorescent intensity (by object area) and object responder status (as determined by comparing object average intensity to a cutoff value determined by controls) are recorded, as well. Well level features, which include mean average GFP intensity and the percentage of GFP responders, are then calculated from the object level values.
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inhibitor, was added to stop viral replication, and to inhibit late gene expression. This would permit only one round of viral gene expression, in an effort to achieve greater reproducibility. With Ara-C, it was found that the average GFP intensity per well and the overall number of GFP expressing cells reached a maximum value around 20 h post-infection (HPI), and stayed constant until roughly 68 HPI, when cell death began to occur, due to media depletion. Based on these initial studies, a general use protocol was developed as follows. (i) Vero E6 cells were diluted with 10% DMEM from a stock cell suspension to 2.5E5 cells/mL, and 200 L of the resulting suspension was added to each well on a Costar 3606 96well flat bottom assay plate. The plates were rocked gently for 15 min before being placed in a 37 ◦ C CO2 incubator for 24 h. (ii) Serial dilutions of heat-inactivated serum were diluted in an 8 × 12 rack of Titertek microtubes with 2% DMEM such that each tube then contained 180 L of diluted serum. Each microtube rack also contained 12 tubes containing 180 L of media only, for infection without serum, and 12 tubes containing 360 L media, for uninfected wells. (iii) Previously titered stock WR-GFP was thawed on ice for 1 h before sonication at 40% power for three bursts of 60 s, with 20 s on ice between bursts. The virus was then diluted in 2% DMEM and 180 L viral suspension was added to the necessary tubes and mixed by pipette. The microtube rack was rocked briefly and placed in a 37 ◦ C CO2 incubator for 2 h, rocking every 30 min. (iv) The media on the 96-well plates was carefully aspirated and replaced with the contents of the microtubes, 100 L per well, three wells per tube. The plates were rocked briefly and placed in a 37 ◦ C CO2 incubator for 2 h, and rocked every 30 min. (v) The inoculant was then aspirated, and each well received 100 L 2%DMEM with 46 g/mL Ara-C before being placed back in a 37 ◦ C CO2 incubator for 21–26 h. (vi) The cells were fixed by adding 25 L 10% formalin to each well, rocking briefly, and incubating for 20 min. The plates were aspirated as before and 100 L of Hoechst 33342 (10 g/mL in DDI H2 O) was added to each well before rocking briefly and incubating an additional 20 min, aspirating a final time, and adding 100 L PBS to each well before being sealed. The plates were then either scanned immediately, or wrapped in aluminum foil and stored at 4 ◦ C. 2.5. Traditional plaque reduction neutralization titer assay (PRNT)
2.4. HCS–GFP neutralization assay Initial efforts using HCS–GFP attempted to identify virus presence in cells within a few cycles to increase the sensitivity of an assay that measures abundance of virus in cells after some limited number of rounds of viral replication, before cell lysis. This would enhance signal generation, hopefully prior to reaching saturation kinetics (due to unlimited bursts of viral replication), which would confound the observation to be measured: is the virus neutralized by the sera—readout being ability to infect a cell. This initial assay strategy was capable of observing qualitatively serum neutralization of vaccinia virus, however the well to well levels of measurable GFP were found to vary considerably in the virus only controls, presumably due to variations in the number of lysed cells per well, making it impossible to produce an accurate and sensitive measure of neutralization. It was found that the accuracy of the assay could be greatly improved by fixing cells 6 h post-infection and thus ensuring that viral replication and subsequent cell lysis did not progress, but this increase in accuracy brought about an extreme decrease in sensitivity due to the low levels of GFP production at this early time point (only one cycle of viral replication). Subsequent infections and fixing of cells were tested from 6 to 45 h to determine GFP intensity levels at varying time points. In addition to testing various time points for GFP expression, cytosine arabinoside (Ara-C), an antimetabolic DNA replication
The following protocol was adapted from a standard lab PRNT protocol (Newman et al., 2003). (i) Vero E6 cells were diluted from a stock suspension in 10% DMEM to 2.2E5 cells/mL, and 2 mL of the resulting suspension was added to each well on a Costar 3506 6well plate. The plates were rocked gently for 15 min before being placed in a 37 ◦ C CO2 incubator for 48 h. (ii) Six twofold serial dilutions of heat-inactivated serum were prepared in 5 mL Falcon tubes with 2% DMEM such that each tube then contained 1.2 mL. There was also a tube contained 1.8 mL media only, for infection without serum present, and one tube containing 3.6 mL media only, for uninfected wells. (iii) Previously titered stock WR-GFP was thawed on ice for 1 h before sonication at 40% power for three bursts of 60 s, with 20 s on ice between bursts. The virus was then diluted in 2% DMEM to 100 PFUs per mL and 1.2 mL was added to every tube except for those for uninfected wells. The tubes were then vortexed briefly and placed in a 37 ◦ C CO2 incubator for 2 h, pausing every 30 min to vortex briefly. (iv) The media in the plates was carefully aspirated and replaced with the contents of the tubes, 1 mL per well, two wells per tube, such that each non-serum containing well received 50 PFUs. The plates were rocked briefly and placed in a 37 ◦ C CO2 incubator for 2 h, pausing every 30 min to rock briefly. (v) The inoculant was then aspirated, and each well received 2 mL 2% DMEM. The plates were then incubated in a 37 ◦ C CO2 incubator for 48 h. (vi) The wells were fixed and stained by adding 2 mL crystal
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violet to each well. After 20 min, the plates were rinsed and allowed to air-dry before manually counting the plaques. 2.6. Data analysis Three output-measured values were obtained. The HCS–GFP neutralization assay recorded the mean average GFP intensity (MAI) per well, and the percentage of GFP responders (%R) per well, while the PRNT assay measured the number of plaques per well. MAI and %R were both observed due to similarities to the assays developed by Manischewitz et al. (MAI) and Earl et al. (%R). To compare results between assays, these values were gated using their respective minimum control (mock-infected wells) and maximum control (infected, serum-negative wells) values. This was done by calculating a “relative” value, defined as the fraction between the controls: relative value = (measured value − minimum control)/(maximum control − minimum control). The relative value represents a given variable on a range from zero (same as minimum) to one (same as maximum); linearity in this case is assumed due to the low MOI. For the MAI, for instance, this fraction is represented as: RMAI = (sample MAI − media only MAI)/(virus only MAI − media only MAI). Since the measured value in a PRNT assay is the number of plaques, the minimum value is always zero (because a media only well will have zero plaques) and the maximum value is the number of plaques in a virus only well and the relative value simplifies to the fraction: (# plaques in sample)/(# plaques in control). In this way, the MAI, %R and number of plaques were used to produce the RMAI, RPR, and RPRNT, respectively, at each serial serum dilution. These three data sets (RPRNT, RMAI and RPR) were then used, via a variable slope sigmoidal equation (Hill equation) to calculate the approximate inhibitory concentration that neutralizes 50% (ID50 ) of WR-GFP infection using Graphpad (San Diego, CA) Prism Software (Version 4.00). 3. Results 3.1. Dose response of WR-GFP to determine optimal MOI and optimal duration for signal output In order to determine optimal GFP expression, adherent Vero E6 cells were infected with WR-GFP at multiplicities of infection
ranging from 0.015 to 30 and mean average intensity of GFP per well determined in the presence or absence of Ara-C (Fig. 1). Visual expression using the ArrayScan uses two channels of detection for this assay. The first detects nuclear staining (Fig. 1A) and provides a count of viable cells from which the GFP viral marker (Fig. 1B) is counted allowing direct overlap of two channels and determination of overall GFP (viral) expression as well as percent of all viable cells expressing GFP (virus) (determined by intensity comparison to a cutoff value calculated from non-infected controls). Based on these observations, two methods of viral expression determination were used. The first measured mean average cell GFP intensity per well, independently of the viable cell counts based on nuclear staining (Fig. 2A). The second measured the percentage of GFP responding cells based on viable labeling of total cells measured by Hoechst staining (Fig. 2B). Optimization of dose response indicates the benefit of using Ara-C in generating a differential range of expression at various MOIs (Fig. 2). The greatest observed differences were observed when using mean average intensity (∼18×) compared to percentage of responders (∼9×) in the absence of AraC. In addition, analysis of expression over time revealed that at MOI range of 0.059–0.937, the separation between values obtained with and without Ara-C were greatest at 21 h or more postinfection (Fig. 3). Use of lower MOI allows conservation of viral stocks while maintaining optimal dynamic range at 20–27 h postinfection while achieving similar expression levels as higher MOIs. 3.2. The HCS–GFP neutralization assay measures human vaccinia-specific immunoglobulins A sample of VIG was obtained, and the standard protocol described above was performed using a twofold serial dilution from 1:250 to 1:3,276,000. A standard PRNT assay was also performed in parallel at the same dilutions for comparison (Fig. 4). GFP assays were controlled by using virus only (VIG negative) wells to determine the upper end of GFP expression and a viral negative control to define non-expressors. Using this strategy, the upper and lower end of GFP expression was defined and relative values were calculated to 1 and 0, respectively. The effective 50% neutralization titers of VIG were 4606 (11.5 g/mL) and 6988 (7.6 g/mL) for MAI and % responder analysis, respectively (Fig. 4A and B). Standard PRNT assays indicated titers of VIG to achieve 50% reduction were 3506
Fig. 1. Vero E6 cell monolayer infected with WR-GFP and imaged via Cellomics ArrayScan HCS in the Hoechst channel (A) and the GFP channel (B) for the same visual field. Cells are identified via Hoechst nuclear staining and average GFP intensity of each cell is recorded and used to determine GFP responder (infected cell) status. Mean average GFP intensity per well (MAI) and the percentage of GFP responders (% responders) are then calculated. 18 h post-infection using Ara-C, MOI ≈ 0.1, 10× magnification.
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Fig. 2. Dose response curves for WR-GFP via both MAI (A) and % responders (B) at 21 HPI. Vero E6 cells were infected in triplicate with dilutions of purified WR-GFP in a 96-well plate. After 2 h incubation at 37 ◦ C the inoculant was aspirated and replaced either media alone, or media and Ara-C. After 21 h incubation, the media was aspirated and the cells were fixed and stained with Hoechst 33342 and read using the Cellomics ArrayScan HCS system.
(15.1 g/mL) (Fig. 4C), indicating lower dynamic range compared to the HCS–GFP assay.
tive values (RPRNT, RMAI, and RPR) also indicate a reasonable level of correlation.
3.3. The HCS–GFP neutralization assay can measure neutralizing antibody in vaccinee sera
3.4. The HCS–GFP neutralization assay is less time, labor, and material intensive than the PRNT
Serum from five vaccine recipients, two primary vaccinees and three individuals with two or more vaccinations (there were no secondary vaccine sera tested) were obtained. These, along with six vaccinia na¨ıve control sera and an orthopoxvirus IgG+ pool control, were tested using the HCS–GFP protocol described above using a twofold serial dilution from 1:40 to 1:1280, as well as a standard PRNT at the same dilutions (Table 1). Analysis of HCS–GFP data using relative mean average intensity or percent responders indicates a strong correlation between HCS–GFP and standard PRNT testing. Neutralization titers for these vaccinees fall within one twofold titer of the line of identity in either direction and represent the correlation between PRNT and HCS–GFP assay data (Fig. 5). The relationship between primary and tertiary or greater vaccinee titers as measured by the three different methods is especially interesting, as the titers as measured by RPR show a distinct separation (roughly one titer) that RMAI and PRNT do not; it appears that there may be a significant separation in titers between primary and tertiary vaccine recipients, although this may be an artifact caused by small sample size. Comparisons of the rela-
The overall ease of use of the HCS–GFP assay as compared to a PRNT is indicated by a direct comparison of the resource usage. Similar data may be obtained while taking only half the total incubation time and nearly one-half the laboratory man-hours. The assay uses one-tenth the sample volume as PRNT, conserving valuable serum. 4. Discussion It is possible, using the HCS ArrayScan system, to produce a precise and accurate high-throughput vaccinia virus neutralization assay that is faster and more reliable than the standard plaque reduction assay. The many advantages of the HCS–GFP neutralization assay over a PRNT are as follows: (i) higher resolution available due to the greater number of events counted (# cells vs. # plaques), (ii) greater reliability, because the data gathering step of the HCS–GFP assay is fully automated, and large amount of human error is eliminated, (iii) faster production time due to a reduction in the amount of incubation time needed in several steps, (iv) higher number of test sera per plate via the 96-well format versus 6-well
Table 1 Neutralization titers of human sera as determined by several methods Serum VIG 1.26A (primary) 1.26B (2+ previous) 1.26C (primary) 1.26D (2+ previous) 1.26E (2+ previous) IgG+ pool Donor 1 (na¨ıve) Donor 2 (na¨ıve) Donor 3 (na¨ıve) Donor 6 (na¨ıve) Donor A (na¨ıve) Donor C (na¨ıve)
PRNT50 (ID50 via vv RPRNT) 3506.3 ± 853.4 193.8 ± 31.3 260.3 ± 26.8 224.8 ± 31.2 319.7 ± 16.6 1083.1 ± 100.0 391.7 ± 90.8 <40 <40 <40 <40 <40 71.5 ± 8.5
MAI50 (ID50 via RMAI) 4606.2 ± 756.8 155.2 ± 18.4 174.2 ± 28.0 234.8 ± 43.1 449.4 ± 64.5 543.2 ± 53.7 184.7 ± 38.1 52.1 ± 19.9 63.8 ± 30.1 68.7 ± 4.8 78.1 ± 15.3 54.2 ± 14.2 220 ± 45.2
PR50 (ID50 via RPR) 6988.1 ± 771.9 335.6 ± 27.1 461 ± 35.9 260.9 ± 46.8 548.2 ± 92.0 592.8 ± 96.3 288.4 ± 30.2 <40 <40 88 ± 7.9 82.4 ± 8.5 <40 103.7 ± 28.9
The semi-automated GFP-based vaccinia neutralization assay described in the test was performed using various sera of human origin, as well as a standard PRNT, and a nonlinear regression of the resulting data was used to determine the 50% neutralization titers of the sera. The sample set included vaccinia immunoglobulin (VIG), sera from individuals with one vaccination (primary) or 2+ vaccinations, a vaccinia immunoglobulin positive pool, and sera from six vaccination na¨ıve donors. Neutralization titer was determined by a reduction in either the number of plaques, mean average GFP intensity, or the percentage of GFP responders (PRNT50 , MAI50 , or PR50 , respectively). This data is also shown in Fig. 5.
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Fig. 3. Detection of infection via mean average GFP intensity (MAI, left) and the percentage of GFP expressing cells (% responders, right) by automated microscopy (Cellomics ArrayScan HCS). Vero E6 cells were infected in triplicate with varying dilutions of purified WR-GFP in a 96-well plate. After 2 h incubation at 37 ◦ C the inoculant was aspirated and replaced with either media alone, or media and Ara-C. The plates were incubated and fixed at various times to determine optimal incubation time and MOI.
PRNTs, and (v) 10-fold reduction in the volume of valuable sera needed for analysis. The HCS–GFP assay produces neutralization data using two functionally separate values, RMAI and RPR. It was found that either of these values could be used to observe consistent, qualitative neutralization, but there was some question as to which of the two was the better measure of neutralization. RPR is related to the proportion of GFP expressing cells in the population, and therefore the percentage of infected cells. RMAI is related to the average amount of GFP produced in all the cells in a well, and is more closely related
to the average level of vaccinia gene expression and nuclear localized GFP accumulation. A reduction in either of these values, then, represents a measurable reduction in vaccinia virus activity, and the data shows that for a given sample serum, both RMAI and RPR will generally measure similar neutralization titers, although there are some subtle differences because the two measured values are as fundamentally different from each other as they are from the PRNT, which measures the reductions in plaque forming units as discrete units of neutralization. Although the exact relationship between these three values is as yet undefined, the data suggests that the
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Fig. 4. Neutralization of vaccinia virus by human immunoglobulin (VIG). Purified WR-GFP was incubated with dilutions of VIG in media. After 1.5 h at 37 ◦ C, this solution was used to inoculate adherent Vero E6 cells on a 96-well plate. After 2 h, the inoculant was aspirated and replaced with +Ara-C media. After 21 h, the media was aspirated and the cells were fixed and stained with Hoechst 33342 and read using the Cellomics ArrayScan system. The mean average object GFP intensity per well (MAI) (A) and percentage of GFP producing responder cells (%R) (B) were gated using virus positive and virus negative mock-infected cells to produce the relative values shown above (RMAI and RPR, respectively). A standard PRNT assay was also performed at similar dilutions and the relative plaque count (RPRNT) of each sample was calculated (C). A variable slope sigmoidal regression (Hill Equation, Levenberg Marquardt algorithm) was then used to estimate the serum concentration needed for 50% neutralization (ID50 ). Comparison between these methods was aided by expressing the results in relative form (RMAI, RPR and RPRNT for mean average GFP intensity, percentage of GFP responding cells, and number of plaques, respectively), which ranges from zero (value equal to the mock-infected, virus negative control) to one (value equal to the serum negative, virus positive control).
differences are an inherent quality of both the assays themselves and the serum being tested. In practice, RPR came to be the preferred measure because the sample deviation was slightly lower than that in RMAI, and it was conceptually more relatable to the PRNT. The fact that the HCS–GFP assay is capable of interpreting virus neutralization data simultaneously via these two different methods (RMAI and RPR) aids in assay quality control and adds to the overall data obtained per sample. It may be possible, by carefully comparing long-term differences between RPR, RMAI and PRNT assays, to discern vital or interesting aspects of the biological systems at play.
On a day-to-day basis, this neutralization assay protocol has potential to replace the PRNT as a lab-standard clinical sample neutralization assay due to the speed and reliability with which data is produced. In the event of an orthopoxvirus outbreak, the speed and high-throughput nature of the assay may prove extremely valuable. This high-throughput may also be further enhanced with the use of an automated plate-stacking robot for the ArrayScan, and through further streamlining of the sample preparation process. This current assay utilizes only two fluorescent channels: the cell identifying Hoechst channel, and the vv gene expression GFP channel. It might be possible later to utilize the four additional fluorescent channels available to identify additional pertinent
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The use of adherent cells negates the need for high-maintenance suspension cultures, and the highly automated nature of the instrument greatly increases throughput. In addition, adherent cell assays may arise from the HCS–GFP assay to adapt to fluorescent reading equipment more readily available. With respect to sensitivity, the HCS–GFP assay (VIG titer of 7.6 g/mL) compares favorably to flow cytometer assays that used the same GFP vaccinia construct (VIG titre of 6.7 g/mL) (Earl et al., 2003; Lustig et al., 2005), and also an ELISA colorimetric assay using a different vaccinia recombinant (VIG titer of 21.6 g/mL) (Manischewitz et al., 2003). In conclusion, it will be valuable to compare the HCS–GFP assay to PRNT in more detail as part of a large-scale vaccination study. Although PRNT, RMAI, and RPR are all viable measures of neutralization, the variables measured are different intrinsically, and a comparison of the different neutralization titers measured by these methods may be valuable, as well as a comparison to IgG ELISA data. In addition, the Cellomics system could possibly lend itself to use in further serological and diagnostic assays. Acknowledgements The authors would like to thank Jack Bennink, Patricia L. Earl, and Jon Yewdell of the Laboratory of Viral Diseases, NIAID, NIH, for producing and supplying WR-GFP, and Cody Clemmons (CDC poxvirus program) for initial ArrayScan tests. References
Fig. 5. Scatter plots of 50% neutralization titers via regression of mean average GFP intensity (MAI50 ) (A) or percentage of GFP responders (PR50 ) (B) data vs. 50% neutralization titer via PRNT (PRNT50 ). The solid black line is a 1:1 identity line, and the dotted lines represent one twofold titer in either direction. Data from sera with titers outside the bounds of these assays (PRNT50 , MAI50 or PR50 less than 40) included for illustrative purposes only.
functions and entities involved in orthopoxvirus neutralization. This might be done by fluorescently tagged antibodies (which, additionally, may be used to substitute for WR-GFP entirely, making it possible to break free from a purely WR vaccinia-based assay) that bind to CEV, EEV, IMV, cell surface attachment ligands, other key viral proteins, etc. Considering that each channel has two usable, distinct outputs (RMAI and RPR), this could potentially provide six simultaneous and distinct measures of the systematic, possibly multi-step neutralization of orthopoxvirus particles. It may also be possible to produce viable neutralization assays using other GFP producing orthopoxvirus (or other virus) recombinants. One potential disadvantage of the HCS–GFP assay is that few laboratories may have this instrument available. However, this disadvantage is mitigated by the versatility, reliability, and ease of use of the ArrayScan. The images from each plate run are stored on a dedicated SQL server and may be reanalyzed using different computational methods months, or even years, after the initial run.
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A rapid, high-throughput vaccinia virus neutralization assay for testing smallpox vaccine efficacy based on detection of green fluorescent protein Matthew C. Johnson, Inger K. Damon, Kevin L. Karem ∗ Poxvirus Program, Division of Viral and Rickettsial Diseases, US Centers for Disease Control and Prevention, 1600 Clifton Road, Mail Stop G-43, Atlanta, GA, United States
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Article history: Received 9 November 2007 Received in revised form 14 February 2008 Accepted 21 February 2008 Available online 2 April 2008 Keywords: Vaccinia Neutralization Smallpox GFP Hoechst
a b s t r a c t Virus neutralization remains a vital tool in assessment of vaccine efficacy for smallpox in the absence of animal smallpox models. In this regard, development of a rapid, sensitive, and high-throughput vaccinia neutralization assay has been sought for evaluating alternative smallpox vaccines, use in bridging studies, as well as understanding the effects of anti-viral immunotherapeutic regimes. The most frequently used method of measuring vaccinia virus neutralization by plaque reduction is time, labor, and material intensive, and therefore limiting in its utility for large scale, high-throughput analysis. Recent advances provide alternative methods that are less labor intensive and higher throughput but with limitations in reagents needed and ease of use. An innovative neutralization assay is described based on a modified Western Reserve vaccinia vector expressing green fluorescent protein (WR-GFP) and an adherent cell monolayer in multi-well plate format. The assay is quick, accurate, provides a large dynamic range and is well suited for large-scale vaccination studies using standard adherent cell lines. Published by Elsevier B.V.
1. Introduction Successful eradication of smallpox in the last century occurred due to the efficacy of vaccinia as a vaccine, but ironically in the absence of good indicators of correlates of protective immunity. A “take” at the vaccination site remains the most reliable indicator of successful vaccination. Virus neutralization provides another measure of inferred protective immunity. Traditional orthopoxvirus limiting-dilution neutralization methods are laborious, particularly for large numbers of test samples and take up to 48–72 h for plaque formation (Katz, 1987). Analysis of results takes additional time and data output may be subjective since plaques are counted manually. Manual counting based on visual readout represents a source of non-standardized error (user based) and can weaken the interpretation in number of events counted per sample (usually <50) and can also induce subjective differences if plaque size variation is observed. Nevertheless, these assays are vital for both clinical diagnosis of viral infection and research into the humoral immune response to infection. These limitations in traditional plaque reduction assays provide an impetus for development of high-throughput, automated neutralization assays that do not rely on plaque formation, and subsequent manual counting, as a measurement of anti-virus activity. In recent years there have been several assays developed that are high-throughput, semi-automated, and that do not rely on
∗ Corresponding author. Tel.: +1 404 639 1598; fax: +1 404 639 1060. E-mail address: [email protected] (K.L. Karem). 0166-0934/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jviromet.2008.02.009
plaque formation and manual counts. Some of these detect aggregate cell infection as indicated by enzyme immunoassay (Eyal et al., 2005) or the expression of recombinant reporter genes such as galactosidase (BGZ), green fluorescent protein (GFP) and luciferase (Chakrabarti et al., 1985; Dominguez et al., 1998; Manischewitz et al., 2003; Rodriguez et al., 1988), while others utilize recombinant GFP expression coupled with flow cytometry to produce an individual cell-based readout as an additional advance in sensitivity (Cosma et al., 2003; Earl et al., 2003). Perceived difficulties of these assays may include the use of cell suspension cultures for GFP assays, which may be laborious to maintain, and a lower dynamic range observed with enzymatic (BGZ) assays. Described here is an alternative GFP fluorescent assay in a high-throughput format that uses adherent cells (monolayer) and provides exceptional dynamic range without the use of flow cytometry and single cell suspensions.
2. Materials and methods 2.1. Virus Western Reserve vaccinia vector expressing green fluorescent protein (WR-GFP) (VV.NP-S-EGFP) was kindly provided by Patricia Earl (Laboratory of Viral Diseases, NIAID, NIH). This recombinant vaccinia virus is referred to as WR-GFP to reflect that it was derived from the Western Reserve strain, and contains a chimeric gene that encodes the influenza virus nucleoprotein, ovalbumin SIINFEKL peptide, and enhanced GFP regulated by the P7.5 early–late
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´ et al., 1999; Mackett et al., 1984; Norbury et al., promoter (Anton 2002). Nuclear localization of GFP expression and accumulation has been observed. The virus was propagated in BSC-40 cells and was purified before storage at −80 ◦ C until needed. 2.2. VIG and human sera Vaccinia immunoglobulin (VIG) (Cangene VIGIV, Product 172, Lot#1730403, 2006, 53 mg/mL) was obtained for use in vaccine efficacy studies using traditional plaque reduction assays (Wittek, 2005). Human serum samples were obtained as part of CDC vaccine study and public health response efforts. Assay control sera were derived for ELISA-based serodiagnostics and represent na¨ıve donors and vaccine recipients. 2.3. ArrayScan HCS Reader and target acquisition software from Cellomics Inc. The ArrayScan system is an automated fluorescent microscope coupled with image-processing and analytical software that can autonomously record the size, location, and fluorescent intensity (in several channels) of a large number of objects on a 96-well plate (Cellomics Inc., Pittsburgh, PA). For this assay, cells are identified by Hoechst 33342 nuclear stain and the average GFP intensity per cell is observed in order to determine the level of vaccinia virus gene expression (through both mean average GFP intensity, and the percentage of GFP expressing cells). The instrument has the capability to record images in six separate fluorescent channels: Hoechst, FITC (GFP), TRITC, Cy5, YFP, and TexasRed. The protocol described below relies on a two-channel scan with nuclear (Hoechst) staining in channel 1 and GFP in channel 2. The Cellomics target acquisition software identifies objects in the visual field via channel 1 and records features such as location, size, and shape in order to identify cell-like objects. Multichannel (Hoechst and GFP) features such as average fluorescent intensity (by object area) and object responder status (as determined by comparing object average intensity to a cutoff value determined by controls) are recorded, as well. Well level features, which include mean average GFP intensity and the percentage of GFP responders, are then calculated from the object level values.
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inhibitor, was added to stop viral replication, and to inhibit late gene expression. This would permit only one round of viral gene expression, in an effort to achieve greater reproducibility. With Ara-C, it was found that the average GFP intensity per well and the overall number of GFP expressing cells reached a maximum value around 20 h post-infection (HPI), and stayed constant until roughly 68 HPI, when cell death began to occur, due to media depletion. Based on these initial studies, a general use protocol was developed as follows. (i) Vero E6 cells were diluted with 10% DMEM from a stock cell suspension to 2.5E5 cells/mL, and 200 L of the resulting suspension was added to each well on a Costar 3606 96well flat bottom assay plate. The plates were rocked gently for 15 min before being placed in a 37 ◦ C CO2 incubator for 24 h. (ii) Serial dilutions of heat-inactivated serum were diluted in an 8 × 12 rack of Titertek microtubes with 2% DMEM such that each tube then contained 180 L of diluted serum. Each microtube rack also contained 12 tubes containing 180 L of media only, for infection without serum, and 12 tubes containing 360 L media, for uninfected wells. (iii) Previously titered stock WR-GFP was thawed on ice for 1 h before sonication at 40% power for three bursts of 60 s, with 20 s on ice between bursts. The virus was then diluted in 2% DMEM and 180 L viral suspension was added to the necessary tubes and mixed by pipette. The microtube rack was rocked briefly and placed in a 37 ◦ C CO2 incubator for 2 h, rocking every 30 min. (iv) The media on the 96-well plates was carefully aspirated and replaced with the contents of the microtubes, 100 L per well, three wells per tube. The plates were rocked briefly and placed in a 37 ◦ C CO2 incubator for 2 h, and rocked every 30 min. (v) The inoculant was then aspirated, and each well received 100 L 2%DMEM with 46 g/mL Ara-C before being placed back in a 37 ◦ C CO2 incubator for 21–26 h. (vi) The cells were fixed by adding 25 L 10% formalin to each well, rocking briefly, and incubating for 20 min. The plates were aspirated as before and 100 L of Hoechst 33342 (10 g/mL in DDI H2 O) was added to each well before rocking briefly and incubating an additional 20 min, aspirating a final time, and adding 100 L PBS to each well before being sealed. The plates were then either scanned immediately, or wrapped in aluminum foil and stored at 4 ◦ C. 2.5. Traditional plaque reduction neutralization titer assay (PRNT)
2.4. HCS–GFP neutralization assay Initial efforts using HCS–GFP attempted to identify virus presence in cells within a few cycles to increase the sensitivity of an assay that measures abundance of virus in cells after some limited number of rounds of viral replication, before cell lysis. This would enhance signal generation, hopefully prior to reaching saturation kinetics (due to unlimited bursts of viral replication), which would confound the observation to be measured: is the virus neutralized by the sera—readout being ability to infect a cell. This initial assay strategy was capable of observing qualitatively serum neutralization of vaccinia virus, however the well to well levels of measurable GFP were found to vary considerably in the virus only controls, presumably due to variations in the number of lysed cells per well, making it impossible to produce an accurate and sensitive measure of neutralization. It was found that the accuracy of the assay could be greatly improved by fixing cells 6 h post-infection and thus ensuring that viral replication and subsequent cell lysis did not progress, but this increase in accuracy brought about an extreme decrease in sensitivity due to the low levels of GFP production at this early time point (only one cycle of viral replication). Subsequent infections and fixing of cells were tested from 6 to 45 h to determine GFP intensity levels at varying time points. In addition to testing various time points for GFP expression, cytosine arabinoside (Ara-C), an antimetabolic DNA replication
The following protocol was adapted from a standard lab PRNT protocol (Newman et al., 2003). (i) Vero E6 cells were diluted from a stock suspension in 10% DMEM to 2.2E5 cells/mL, and 2 mL of the resulting suspension was added to each well on a Costar 3506 6well plate. The plates were rocked gently for 15 min before being placed in a 37 ◦ C CO2 incubator for 48 h. (ii) Six twofold serial dilutions of heat-inactivated serum were prepared in 5 mL Falcon tubes with 2% DMEM such that each tube then contained 1.2 mL. There was also a tube contained 1.8 mL media only, for infection without serum present, and one tube containing 3.6 mL media only, for uninfected wells. (iii) Previously titered stock WR-GFP was thawed on ice for 1 h before sonication at 40% power for three bursts of 60 s, with 20 s on ice between bursts. The virus was then diluted in 2% DMEM to 100 PFUs per mL and 1.2 mL was added to every tube except for those for uninfected wells. The tubes were then vortexed briefly and placed in a 37 ◦ C CO2 incubator for 2 h, pausing every 30 min to vortex briefly. (iv) The media in the plates was carefully aspirated and replaced with the contents of the tubes, 1 mL per well, two wells per tube, such that each non-serum containing well received 50 PFUs. The plates were rocked briefly and placed in a 37 ◦ C CO2 incubator for 2 h, pausing every 30 min to rock briefly. (v) The inoculant was then aspirated, and each well received 2 mL 2% DMEM. The plates were then incubated in a 37 ◦ C CO2 incubator for 48 h. (vi) The wells were fixed and stained by adding 2 mL crystal
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violet to each well. After 20 min, the plates were rinsed and allowed to air-dry before manually counting the plaques. 2.6. Data analysis Three output-measured values were obtained. The HCS–GFP neutralization assay recorded the mean average GFP intensity (MAI) per well, and the percentage of GFP responders (%R) per well, while the PRNT assay measured the number of plaques per well. MAI and %R were both observed due to similarities to the assays developed by Manischewitz et al. (MAI) and Earl et al. (%R). To compare results between assays, these values were gated using their respective minimum control (mock-infected wells) and maximum control (infected, serum-negative wells) values. This was done by calculating a “relative” value, defined as the fraction between the controls: relative value = (measured value − minimum control)/(maximum control − minimum control). The relative value represents a given variable on a range from zero (same as minimum) to one (same as maximum); linearity in this case is assumed due to the low MOI. For the MAI, for instance, this fraction is represented as: RMAI = (sample MAI − media only MAI)/(virus only MAI − media only MAI). Since the measured value in a PRNT assay is the number of plaques, the minimum value is always zero (because a media only well will have zero plaques) and the maximum value is the number of plaques in a virus only well and the relative value simplifies to the fraction: (# plaques in sample)/(# plaques in control). In this way, the MAI, %R and number of plaques were used to produce the RMAI, RPR, and RPRNT, respectively, at each serial serum dilution. These three data sets (RPRNT, RMAI and RPR) were then used, via a variable slope sigmoidal equation (Hill equation) to calculate the approximate inhibitory concentration that neutralizes 50% (ID50 ) of WR-GFP infection using Graphpad (San Diego, CA) Prism Software (Version 4.00). 3. Results 3.1. Dose response of WR-GFP to determine optimal MOI and optimal duration for signal output In order to determine optimal GFP expression, adherent Vero E6 cells were infected with WR-GFP at multiplicities of infection
ranging from 0.015 to 30 and mean average intensity of GFP per well determined in the presence or absence of Ara-C (Fig. 1). Visual expression using the ArrayScan uses two channels of detection for this assay. The first detects nuclear staining (Fig. 1A) and provides a count of viable cells from which the GFP viral marker (Fig. 1B) is counted allowing direct overlap of two channels and determination of overall GFP (viral) expression as well as percent of all viable cells expressing GFP (virus) (determined by intensity comparison to a cutoff value calculated from non-infected controls). Based on these observations, two methods of viral expression determination were used. The first measured mean average cell GFP intensity per well, independently of the viable cell counts based on nuclear staining (Fig. 2A). The second measured the percentage of GFP responding cells based on viable labeling of total cells measured by Hoechst staining (Fig. 2B). Optimization of dose response indicates the benefit of using Ara-C in generating a differential range of expression at various MOIs (Fig. 2). The greatest observed differences were observed when using mean average intensity (∼18×) compared to percentage of responders (∼9×) in the absence of AraC. In addition, analysis of expression over time revealed that at MOI range of 0.059–0.937, the separation between values obtained with and without Ara-C were greatest at 21 h or more postinfection (Fig. 3). Use of lower MOI allows conservation of viral stocks while maintaining optimal dynamic range at 20–27 h postinfection while achieving similar expression levels as higher MOIs. 3.2. The HCS–GFP neutralization assay measures human vaccinia-specific immunoglobulins A sample of VIG was obtained, and the standard protocol described above was performed using a twofold serial dilution from 1:250 to 1:3,276,000. A standard PRNT assay was also performed in parallel at the same dilutions for comparison (Fig. 4). GFP assays were controlled by using virus only (VIG negative) wells to determine the upper end of GFP expression and a viral negative control to define non-expressors. Using this strategy, the upper and lower end of GFP expression was defined and relative values were calculated to 1 and 0, respectively. The effective 50% neutralization titers of VIG were 4606 (11.5 g/mL) and 6988 (7.6 g/mL) for MAI and % responder analysis, respectively (Fig. 4A and B). Standard PRNT assays indicated titers of VIG to achieve 50% reduction were 3506
Fig. 1. Vero E6 cell monolayer infected with WR-GFP and imaged via Cellomics ArrayScan HCS in the Hoechst channel (A) and the GFP channel (B) for the same visual field. Cells are identified via Hoechst nuclear staining and average GFP intensity of each cell is recorded and used to determine GFP responder (infected cell) status. Mean average GFP intensity per well (MAI) and the percentage of GFP responders (% responders) are then calculated. 18 h post-infection using Ara-C, MOI ≈ 0.1, 10× magnification.
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Fig. 2. Dose response curves for WR-GFP via both MAI (A) and % responders (B) at 21 HPI. Vero E6 cells were infected in triplicate with dilutions of purified WR-GFP in a 96-well plate. After 2 h incubation at 37 ◦ C the inoculant was aspirated and replaced either media alone, or media and Ara-C. After 21 h incubation, the media was aspirated and the cells were fixed and stained with Hoechst 33342 and read using the Cellomics ArrayScan HCS system.
(15.1 g/mL) (Fig. 4C), indicating lower dynamic range compared to the HCS–GFP assay.
tive values (RPRNT, RMAI, and RPR) also indicate a reasonable level of correlation.
3.3. The HCS–GFP neutralization assay can measure neutralizing antibody in vaccinee sera
3.4. The HCS–GFP neutralization assay is less time, labor, and material intensive than the PRNT
Serum from five vaccine recipients, two primary vaccinees and three individuals with two or more vaccinations (there were no secondary vaccine sera tested) were obtained. These, along with six vaccinia na¨ıve control sera and an orthopoxvirus IgG+ pool control, were tested using the HCS–GFP protocol described above using a twofold serial dilution from 1:40 to 1:1280, as well as a standard PRNT at the same dilutions (Table 1). Analysis of HCS–GFP data using relative mean average intensity or percent responders indicates a strong correlation between HCS–GFP and standard PRNT testing. Neutralization titers for these vaccinees fall within one twofold titer of the line of identity in either direction and represent the correlation between PRNT and HCS–GFP assay data (Fig. 5). The relationship between primary and tertiary or greater vaccinee titers as measured by the three different methods is especially interesting, as the titers as measured by RPR show a distinct separation (roughly one titer) that RMAI and PRNT do not; it appears that there may be a significant separation in titers between primary and tertiary vaccine recipients, although this may be an artifact caused by small sample size. Comparisons of the rela-
The overall ease of use of the HCS–GFP assay as compared to a PRNT is indicated by a direct comparison of the resource usage. Similar data may be obtained while taking only half the total incubation time and nearly one-half the laboratory man-hours. The assay uses one-tenth the sample volume as PRNT, conserving valuable serum. 4. Discussion It is possible, using the HCS ArrayScan system, to produce a precise and accurate high-throughput vaccinia virus neutralization assay that is faster and more reliable than the standard plaque reduction assay. The many advantages of the HCS–GFP neutralization assay over a PRNT are as follows: (i) higher resolution available due to the greater number of events counted (# cells vs. # plaques), (ii) greater reliability, because the data gathering step of the HCS–GFP assay is fully automated, and large amount of human error is eliminated, (iii) faster production time due to a reduction in the amount of incubation time needed in several steps, (iv) higher number of test sera per plate via the 96-well format versus 6-well
Table 1 Neutralization titers of human sera as determined by several methods Serum VIG 1.26A (primary) 1.26B (2+ previous) 1.26C (primary) 1.26D (2+ previous) 1.26E (2+ previous) IgG+ pool Donor 1 (na¨ıve) Donor 2 (na¨ıve) Donor 3 (na¨ıve) Donor 6 (na¨ıve) Donor A (na¨ıve) Donor C (na¨ıve)
PRNT50 (ID50 via vv RPRNT) 3506.3 ± 853.4 193.8 ± 31.3 260.3 ± 26.8 224.8 ± 31.2 319.7 ± 16.6 1083.1 ± 100.0 391.7 ± 90.8 <40 <40 <40 <40 <40 71.5 ± 8.5
MAI50 (ID50 via RMAI) 4606.2 ± 756.8 155.2 ± 18.4 174.2 ± 28.0 234.8 ± 43.1 449.4 ± 64.5 543.2 ± 53.7 184.7 ± 38.1 52.1 ± 19.9 63.8 ± 30.1 68.7 ± 4.8 78.1 ± 15.3 54.2 ± 14.2 220 ± 45.2
PR50 (ID50 via RPR) 6988.1 ± 771.9 335.6 ± 27.1 461 ± 35.9 260.9 ± 46.8 548.2 ± 92.0 592.8 ± 96.3 288.4 ± 30.2 <40 <40 88 ± 7.9 82.4 ± 8.5 <40 103.7 ± 28.9
The semi-automated GFP-based vaccinia neutralization assay described in the test was performed using various sera of human origin, as well as a standard PRNT, and a nonlinear regression of the resulting data was used to determine the 50% neutralization titers of the sera. The sample set included vaccinia immunoglobulin (VIG), sera from individuals with one vaccination (primary) or 2+ vaccinations, a vaccinia immunoglobulin positive pool, and sera from six vaccination na¨ıve donors. Neutralization titer was determined by a reduction in either the number of plaques, mean average GFP intensity, or the percentage of GFP responders (PRNT50 , MAI50 , or PR50 , respectively). This data is also shown in Fig. 5.
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Fig. 3. Detection of infection via mean average GFP intensity (MAI, left) and the percentage of GFP expressing cells (% responders, right) by automated microscopy (Cellomics ArrayScan HCS). Vero E6 cells were infected in triplicate with varying dilutions of purified WR-GFP in a 96-well plate. After 2 h incubation at 37 ◦ C the inoculant was aspirated and replaced with either media alone, or media and Ara-C. The plates were incubated and fixed at various times to determine optimal incubation time and MOI.
PRNTs, and (v) 10-fold reduction in the volume of valuable sera needed for analysis. The HCS–GFP assay produces neutralization data using two functionally separate values, RMAI and RPR. It was found that either of these values could be used to observe consistent, qualitative neutralization, but there was some question as to which of the two was the better measure of neutralization. RPR is related to the proportion of GFP expressing cells in the population, and therefore the percentage of infected cells. RMAI is related to the average amount of GFP produced in all the cells in a well, and is more closely related
to the average level of vaccinia gene expression and nuclear localized GFP accumulation. A reduction in either of these values, then, represents a measurable reduction in vaccinia virus activity, and the data shows that for a given sample serum, both RMAI and RPR will generally measure similar neutralization titers, although there are some subtle differences because the two measured values are as fundamentally different from each other as they are from the PRNT, which measures the reductions in plaque forming units as discrete units of neutralization. Although the exact relationship between these three values is as yet undefined, the data suggests that the
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Fig. 4. Neutralization of vaccinia virus by human immunoglobulin (VIG). Purified WR-GFP was incubated with dilutions of VIG in media. After 1.5 h at 37 ◦ C, this solution was used to inoculate adherent Vero E6 cells on a 96-well plate. After 2 h, the inoculant was aspirated and replaced with +Ara-C media. After 21 h, the media was aspirated and the cells were fixed and stained with Hoechst 33342 and read using the Cellomics ArrayScan system. The mean average object GFP intensity per well (MAI) (A) and percentage of GFP producing responder cells (%R) (B) were gated using virus positive and virus negative mock-infected cells to produce the relative values shown above (RMAI and RPR, respectively). A standard PRNT assay was also performed at similar dilutions and the relative plaque count (RPRNT) of each sample was calculated (C). A variable slope sigmoidal regression (Hill Equation, Levenberg Marquardt algorithm) was then used to estimate the serum concentration needed for 50% neutralization (ID50 ). Comparison between these methods was aided by expressing the results in relative form (RMAI, RPR and RPRNT for mean average GFP intensity, percentage of GFP responding cells, and number of plaques, respectively), which ranges from zero (value equal to the mock-infected, virus negative control) to one (value equal to the serum negative, virus positive control).
differences are an inherent quality of both the assays themselves and the serum being tested. In practice, RPR came to be the preferred measure because the sample deviation was slightly lower than that in RMAI, and it was conceptually more relatable to the PRNT. The fact that the HCS–GFP assay is capable of interpreting virus neutralization data simultaneously via these two different methods (RMAI and RPR) aids in assay quality control and adds to the overall data obtained per sample. It may be possible, by carefully comparing long-term differences between RPR, RMAI and PRNT assays, to discern vital or interesting aspects of the biological systems at play.
On a day-to-day basis, this neutralization assay protocol has potential to replace the PRNT as a lab-standard clinical sample neutralization assay due to the speed and reliability with which data is produced. In the event of an orthopoxvirus outbreak, the speed and high-throughput nature of the assay may prove extremely valuable. This high-throughput may also be further enhanced with the use of an automated plate-stacking robot for the ArrayScan, and through further streamlining of the sample preparation process. This current assay utilizes only two fluorescent channels: the cell identifying Hoechst channel, and the vv gene expression GFP channel. It might be possible later to utilize the four additional fluorescent channels available to identify additional pertinent
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The use of adherent cells negates the need for high-maintenance suspension cultures, and the highly automated nature of the instrument greatly increases throughput. In addition, adherent cell assays may arise from the HCS–GFP assay to adapt to fluorescent reading equipment more readily available. With respect to sensitivity, the HCS–GFP assay (VIG titer of 7.6 g/mL) compares favorably to flow cytometer assays that used the same GFP vaccinia construct (VIG titre of 6.7 g/mL) (Earl et al., 2003; Lustig et al., 2005), and also an ELISA colorimetric assay using a different vaccinia recombinant (VIG titer of 21.6 g/mL) (Manischewitz et al., 2003). In conclusion, it will be valuable to compare the HCS–GFP assay to PRNT in more detail as part of a large-scale vaccination study. Although PRNT, RMAI, and RPR are all viable measures of neutralization, the variables measured are different intrinsically, and a comparison of the different neutralization titers measured by these methods may be valuable, as well as a comparison to IgG ELISA data. In addition, the Cellomics system could possibly lend itself to use in further serological and diagnostic assays. Acknowledgements The authors would like to thank Jack Bennink, Patricia L. Earl, and Jon Yewdell of the Laboratory of Viral Diseases, NIAID, NIH, for producing and supplying WR-GFP, and Cody Clemmons (CDC poxvirus program) for initial ArrayScan tests. References
Fig. 5. Scatter plots of 50% neutralization titers via regression of mean average GFP intensity (MAI50 ) (A) or percentage of GFP responders (PR50 ) (B) data vs. 50% neutralization titer via PRNT (PRNT50 ). The solid black line is a 1:1 identity line, and the dotted lines represent one twofold titer in either direction. Data from sera with titers outside the bounds of these assays (PRNT50 , MAI50 or PR50 less than 40) included for illustrative purposes only.
functions and entities involved in orthopoxvirus neutralization. This might be done by fluorescently tagged antibodies (which, additionally, may be used to substitute for WR-GFP entirely, making it possible to break free from a purely WR vaccinia-based assay) that bind to CEV, EEV, IMV, cell surface attachment ligands, other key viral proteins, etc. Considering that each channel has two usable, distinct outputs (RMAI and RPR), this could potentially provide six simultaneous and distinct measures of the systematic, possibly multi-step neutralization of orthopoxvirus particles. It may also be possible to produce viable neutralization assays using other GFP producing orthopoxvirus (or other virus) recombinants. One potential disadvantage of the HCS–GFP assay is that few laboratories may have this instrument available. However, this disadvantage is mitigated by the versatility, reliability, and ease of use of the ArrayScan. The images from each plate run are stored on a dedicated SQL server and may be reanalyzed using different computational methods months, or even years, after the initial run.
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