This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.
Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay formats are currently in use worldwide so there is a need for an accurate and high-throughput method for measuring RSV NAbs. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A and can also be adapted for RSV subgroup B and different sample types. This method is highly reproducible, with inter-assay variations for the reference antiserum being less than 10%. We believe this assay can be readily established in many laboratories worldwide at relatively low cost. Development of an improved, high-throughput assay that measures RSV NAbs represents a significant step forward for the standardization of this method internationally as well as being critical for the evaluation of novel RSV vaccine candidates in the future.
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in the pediatric population worldwide1. Despite its high burden, there is still no vaccine or treatment available. Since 2013, the World Health Organization (WHO) has declared RSV vaccine development as a major research priority, with annual WHO consultation meetings2,3. The WHO has agreed on using RSV neutralizing antibody (NAb) measurement to monitor vaccine immunogenicity, as this is recognized as the major serological marker of protection4. NAbs have been shown to protect against severe RSV infection in a number of studies as well as clinical trials of the anti-RSV monoclonal antibody palivizumab, currently the only prophylactic strategy available4.
There are multiple NAb assay formats used by laboratories worldwide, including cell-based and molecular-based assays, which have made standardization efforts challenging5,6,7,8. However, the conventional plaque-reduction neutralization (PRN) assay that measures the number of reduced plaque forming units (PFU) by the presence of an RSV-specific antibody still remains the gold standard9. Here, we report an improved, simplified, and high-throughput PRN protocol that can be used on numerous cell lines, for different RSV strains and with increased assay throughput. This protocol has been tested using clinical samples from different settings as well as on samples from animal model experiments.
NOTE: All steps have to be performed in a BSL2 hood unless stated differently. Viral titration is required in advance of a PRN assay to determine the optimal RSV concentration used in the PRN assay. It is recommended to aliquot the virus stocks in a small volume that will be thawed once and used for each NAb assay. Using the same viral stock for all NAb assays performed for all samples from one study is also recommended. Make sure culture media and phosphate-buffered saline (PBS) is warmed at 37 °C before adding to cell plates.
1. RSV Viral Titration
NOTE: Depending on the number of virus stocks and the number of duplicates, the assay plate can be set up according to Figure 1. Each virus stock should be titrated in triplicate down the assay plate, starting at the highest viral concentration (i.e., 1:10). Serial titrations can be typically 1:10. A549 cell culture and maintenance as well as RSV culture procedure are done using standard procedures and are not included in this protocol.
2. RSV Neutralization Assay
The titration of a virus stock was performed from 1:10 to 1:108 dilution to determine the virus stock concentration prior to the PRN assay (representative results shown in Figure 5). From Figure 5, PFU can be counted reliably at dilutions of 1:104 and 1:105. The average number of PFU from triplicate wells at the same dilution was calculated. Since the average number of spots at 1:105 dilution was 14, the viral titer of this viral stock was determined as follows:
14 (average number of spots)/[10-5 (dilution) x 0.1 mL (volume of inoculum)] = 1.4 x 107 PFU/mL
Using the above described method, we have measured NAbs for both RSV-A and B in human samples. Figure 6 demonstrates the interpretation of well images of three representative serum samples with varying NAb titers against RSV-A. Figure 6A shows well images of sample titration replicates for each representative serum (the actual assay plate had three technical replicates). Serum dilutions as indicated are different for each sample. Virus control wells containing only virus and no serum had their average PFU calculated and used to determine the 50% neutralization cut-off, which in this example was 52. Figure 6B illustrates how the titration of the three sera was determined using this cut-off. The log2 reciprocal of the serum dilution is plotted on the x-axis and the number of PFU as a percentage of control on the y-axis. Symbol and error bars represent the mean of triplicates ± standard deviation. Fifty percent of the average spots in the virus-control wells (indicated by the horizontal dashed line) was used as a cut-off to determine the 50% neutralizing antibody titer of a serum sample. The neutralizing antibody titer is defined as the reciprocal of the dilution that would result in 50% inhibition of virus activity.
As each experiment includes the RSV reference antiserum and positive virus control wells, monitoring the inter-assay variability provides quality control between experiments. Figure 7 shows the results of the RSV-A PRN assay in two different adult cohorts from the Gambia cohort (N = 21)10 and healthy volunteers in Melbourne (N = 36). Titers of RSV NAb were variable within each population, with the Gambian adults having a lower mean NAb titer compared to the Melbourne adults. The RSV reference antiserum is also shown (N = 52 replicates), demonstrating very low variability with a coefficient of variation (CV) of 6.82%.
Figure 1: Viral titration plate layout for quantitation of RSV stocks. v = stock of RSV virus (v1, stock of viral batch 1; v2, stock of viral batch 2; v3, stock of viral batch 3), N = negative control well, X = media added. Color coding is only to aid visualization of plate layout. Please click here to view a larger version of this figure.
Figure 2: Example count settings used for the spots reader. Screenshot of the parameters typically used for counting virus plaques. Please click here to view a larger version of this figure.
Figure 3: Examples of artefact and disrupted cell monolayers. (A) Artefacts. (B) Disrupted cell monolayers. Please click here to view a larger version of this figure.
Figure 4: Plate template for RSV PRN assay. V = viral positive control wells, N = negative control wells, X = media added, S = reference antiserum with all dilutions (from 1:100 to 1:12,800) in column 12. Color coding is only to aid visualization of plate layout. Please click here to view a larger version of this figure.
Figure 5: An example of an RSV viral titration result. Representative wells from an assay performed in triplicate are shown. The numbers in each well refer to the number of plaques counted using the spots reader. TMC = too many to count. Please click here to view a larger version of this figure.
Figure 6: Results from a representative RSV PRN assay. (A) The number of PFU counted for three individual serum samples according to different dilution ranges. The numbers in each well image refer to the number of plaques counted. (B) NAb titer interpretation from the well images in panel A for the three representative human serum samples. The data is calculated on the basis of the 50% cut-off from the virus control wells. Horizontal bars represent mean ± SD. Please click here to view a larger version of this figure.
Figure 7: RSV neutralizing antibody titers in Melbourne (N = 21) and Gambian (N = 36) adults. The human RSV reference serum is also shown for comparison (N = 52 replicates). Symbols represent individual titers and horizontal bars represent geometric mean titer ± 95% CI. The N = 52 results are based on three technicians over a two-month period. Please click here to view a larger version of this figure.
Supplementary Figure 1: Comparison of NAb titers for RSV-A. (Left) NAb titers calculated using the linear and non-linear regression models (N = 152 assays). (Right) Correlation between RSV-A NAb titers using both linear and non-linear regression models. Please click here to download this file.
We have developed and optimized a simple and efficient RSV micro-neutralization assay that can be readily adapted in most laboratories. This assay is able to measure viral infection ability as well as measuring the inhibition of viral infection by NAb at the cellular level using computerized image scanning. The use of an imaging-based platform and specific antibody-based systems has increased the specificity and sensitivity of spot detection compared to traditional plaque detection methods6,7. This allows characterization of RSV strains with low infectivity and also provides an accurate quantification of RSV concentration prior to use in the PRN assay.
It is recommended that the same batch of viral stock is used for each study in order to minimize any batch-to-batch variations between RSV stocks. Other important aspects of the assay include the consistency of virus input concentration, the integrity of the A549 cell monolayer, and using same count settings to ensure assay consistency and accuracy. The amount of virus added to the plate can be verified again in a separate plaque viral titration. However, monitoring the positive control wells of the PRN assay also provides an indication of actual viral titer and is also worthwhile to monitor across assays. Moreover, including the reference serum in every plate is another quality control measure to ensure that assays are reproducible. Our experiments did not use the recently validated RSV international standard antiserum from the NIBSC, as it was not available at the time of our study11. Using this RSV international standard antiserum should be recommended for future studies measuring RSV NAb so that results across laboratories can be compared with confidence.
Development of a relatively simple, high-throughput assay would facilitate global standardization efforts for the use of RSV NAbs as more laboratories worldwide would be able to use this method. This is particularly important given the number of RSV vaccine candidates in development, with some currently in phase 3 trials. It is critical that the results of future clinical studies of RSV vaccines be comparable, and therefore a standardized assay represents a key aspect to this goal. While a number of low- and middle-income countries may not be able to directly support acquisition of the equipment needed, linkages through international collaborations and/or capacity building programs will be critical over the medium to long term.
This NAb assay protocol is flexible in being able to be adapted for different cell lines (A549, Hep2, and Vero) and can be used for different RSV strains. We have also adapted this method for RSV-B successfully. As the assay platform has been developed for a 96-well plate format, this provides a cost-effective and high-throughput alternative with high accuracy because it allows more replicates and the inclusion of the reference serum control and negative control on every plate. Using the same reference serum control is also recommended as this is critical to ensure quality control and quality assurance. In our hands, we observed very low CVs of 6.82% for the reference serum which is much lower than reported CVs for other biological assays in the range of 30-40%5.
Moreover, the image data and the count data (raw data) generated from this protocol can be stored indefinitely for future reference and/or analysis. Maintaining records of raw data is an essential requirement for clinical trials, particularly those involving future RSV vaccines. Our worksheet (see Supplementary Worksheet) suggested in this protocol can help to record all aspects of the experiment for the purpose of quality control and assurance control. The worksheet provides the calculation of the 50% neutralizing titer using a simple linear model as this has the advantage of not requiring any specialized software. However, the raw data from our NAb assay can be analyzed using the non-linear regression model that has also been used in the vaccine research for calculating 50% neutralizing titers, although this requires other software packages12,13. Using our experimental data, the results obtained using these two methods were similar, giving confidence to titer values obtained using the simplified linear model (see Supplementary Figure 1). The cost for setting up this assay is affordable for many laboratories with perhaps the most expensive item being the spots reader (approximately USD $50K). However, the spots reader has the advantage of being able to be used for other applications such as the enzyme-linked immunospot (ELISpot) for cytokine and/or antibody-secreting cell measurement, which makes it a valuable component in vaccine trial evaluation.
In conclusion, this improved, high-throughput RSV PRN assay can be easily implemented in many laboratories and will support the international harmonization effort of WHO for RSV NAb assays. This will be critical for the evaluation of novel RSV vaccine candidates in the future.
The authors have nothing to disclose.
The authors thank all the participants involved. We acknowledge the Victorian Government’s Operational Infrastructure Support Program. PVL is a NHMRC Career Development Fellowship recipient.
Cell line | |||
A549 | ATCC | CCL-185 | provided by Dr Keith Chappell, University of Queensland |
Viral strains | |||
RSV A2 | ATCC | VR-1540 | lot number 60430286 |
Reagents | |||
Acetone | Merck | 1000142511 | |
Alexa-Fluor donkey anti-goat IgG (stored at 4 °C) | Life Technologies | A11055 | |
CMC sodium salt powder | Sigma-Aldrich | C5678-500G | |
DMEM (no serum, 3.7 g/L NaHC, P/S) (stored at 4 °C) | Scientific Services – Tissue Culture | MCRI in house supply | |
Foetal calf Serum (stored in 50ml aliquots at -20 °C) | Interpath | SFBS-F | |
Goat X RSV antibody | Merck | AB1128 | |
human polyclonal antiserum to respiratory syncytial virus (RSV) (stored in 45 µL aliquots at -20 °C) | BEI Resources | NR-4022 | Free order through BEI Resources upon registration. This serum belong to a panel of human antiserum and immune globulin to RSV (NR-32832) |
M199 powder | Life Technologies | 31100035 | |
Milk diluent blocking solution (stored at 4 °C) | Australian Biosearch | 50-82-01 | |
Penicillin/Streptomycin (stored in 6mL aliquots at -20 °C) | Life Technologies | 15140122 | |
s.d.H2O from Milli-Q dispenser | Merck | In-house dispensation | |
Sterile 1X PBS for culture (stored at 4°C) | Scientific Services – Tissue Culture | MCRI in house supply | |
Tween 20 polysorbate | Sigma-Aldrich | 9005-64-5 | |
General Consumables | |||
Conical Falcon tubes (50 mL) | Invitro Technologies | FAL352070 | |
Filter unit 0.22um (500 mL) | Thermo Fisher | NAL5660020 | |
Sterile Eppendorf tubes (1.5 mL) | Australia PL | AM12400 | |
Sterile flat-bottom plates (96-well with lid) | Interpath | 655180 | |
Sterile U-bottom plates (96-well with lid) | Interpath | 650180 | |
5ml serological pipette | Sigma-Aldrich | CLS4487-200EA | |
10 mL serological pipette | Interpath | 607180 | |
25 mL serological pipette | Sigma-Aldrich | CLS4251-200EA | |
Tip Pipette 1-200 µL Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 96TIPS PKG960 | Fisher Biotec | TF-200-L-R-S | |
Tip Pipette 5-20 µL Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 96TIPS PKG960 | Fisher Biotec | TF-20-L-R-S | |
Tip Pipette 100-1000 µLClear Maxymum Recovery Racked Pre-sterilized 10RACKS x 100TIPS PKG1000 | Fisher Biotec | TF-1000-L-R-S | |
Tip Pipette 1-10 µL Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 100TIPS PKG1001 | Fisher Biotec | TXLF-10-L-R-S | |
Equipments and softwares | |||
ELISpot reader system | AID iSpot, Autoimmun Diagnostika GmbH, Strasburg, Germany | ||
AID ELISpot software version 5.0 | AID iSpot, Autoimmun Diagnostika GmbH, Strasburg, Germany | ||
Microsoft Excel 2007 |