With any assay or technique, scaling up of sample sizes can pose multiple problems and necessitate numerous procedural changes and refinements. Fortunately, the original protocol was highly amenable to being scaled up. Most of the assay materials can be manipulated in a 96-well format with multichannel pipettes. One of the largest bottlenecks in the procedure was the transfer of serum from individual microcentrifuge storage tubes to the 96-well plate for dilution. Initial serum storage in 96-well plates would result in considerable time savings. Without any automation, we were able to readily test up to 20 96-well plates per operator per assay while remaining within the time constraints of the various protocol steps. The use of robotic workstations would dramatically increase this number. Another of the larger bottlenecks was the time and effort required for growing, maintaining, and expanding the HeLa cell cultures (we required ~4 × 109 cells each week). Laboratories utilizing this assay for a large numbers of samples are encouraged to invest in appropriate bulk cell culture systems.
We also found that acceptable levels of virus activity, MOI, and duration of infection may differ between laboratories. Care should be taken to optimize these variables within each laboratory before large-scale testing is performed. In spite of these differences, the inclusion of the VIG standards should still allow results to be compared between laboratories. The development of comprehensive sets of assay standards would allow direct comparisons between laboratories and serve to address this concern.
Our results showed that the range of ID50
values was quite wide (15.7 to 1,314.2), with 100% of our subjects demonstrating detectable levels of humoral immunity higher than those found with a small panel of vaccinia virus-naïve subjects (average ID50
= 1.11). Our study inclusion criteria included a documented “take.” Historically, this take has been considered evidence of successful vaccination and protection from disease (17
). Recent studies have confirmed that the vaccine take is associated with seroconversion and positive vaccinia virus-specific T- and B-cell responses (2
), although in the absence of endemic disease, we can only estimate correlates of protection. Historically, titers of 1:20 or 1:32 were assumed to represent protective levels (34
), and while a direct comparison of our results with historical estimates of protection must be interpreted with caution, it is interesting that, in spite of the presence of a documented vaccine “take,” a small number of individuals (22/1,076 [2.0%]) had ID50
values of <32. Further investigation of the immune responses (both humoral and cellular) is warranted to understand this apparent disconnect between the presence of a vaccine take and the neutralizing Ab titer seen with these individuals.
In our study cohort, the assay was easily able to accurately and reproducibly detect serum titers as low as 1:15 and as high as 1:1,300, and VIG titers were routinely between 1:2,735 and 1:5,537 (IQR). The range of threefold dilutions routinely used in the assay (1:20 to 1:540) sufficed for most subjects, and the use of a quadratic trend fit to the data allowed us to accurately determine ID50
values even when the dilutions did not all fall within the linear range of the assay. For those individuals with ID50
values well above 540, it was necessary use a higher dilution range (1:60 to 1:1,620). The need for repetition could be easily remedied by expanding the range of dilutions initially tested, for example (to a range of 1:20 to 1:1,620). Recent studies examining long-term persistence of smallpox immunity have found that humoral responses initially decline in the 5 to 10 years following vaccination and then remain stable for decades. For individuals vaccinated within 1 decade of serologic testing, these reports cite 50% neutralization titers of between 10 and 100 (10
), 64 and 256 (19
), and 64 and 128 (50
), values that are quite similar to the range of titers (median ID50
, 132.2; IQR, 78.8 to 205.6) seen in our population tested within 4 years of vaccination.
Vaccinia virus produces two main infectious forms, intracellular mature virus (IMV) and extracellular enveloped virus (EEV) (33
). EEV contains an additional membrane and several unique viral proteins and mediates long-range dissemination in vivo (43
). Full protection against poxvirus infection or disease is believed to require antibodies against both IMV and EEV (7
). The vast majority of viral particles produced are IMV, and EEV particles are extremely fragile; consequently, most poxvirus-specific serological tests, including the assay discussed here, measure Ab responses to IMV. Comet reduction assays are typically used to measure anti-EEV activity; however, these assays suffer from the same limitations as PRNTs. Alternatively, one can modify PRNT assays to focus on EEV-specific antibodies by using freshly made stocks of EEV supplemented by anti-IMV antibodies to reduce IMV activity and to remove EEV virions with disrupted membranes (30
). Collection of vSC56 EEV virions from the supernatant of infected cells and the inclusion of anti-IMV Abs would be relatively straightforward modifications to the protocol and would allow investigators to measure EEV-specific neutralizing Ab titers. The need to continually prepare fresh EEV stocks, the differing viral titers of these stocks, and the need to ensure a steady supply of anti-IMV antibodies may limit the high-throughput nature of the assay, but the trade-off may well be worth it for certain study designs.
The reproducibility of this method, as well as its ability to provide reliable estimates of each individual's ID50, is further highlighted by the final results. The within-person CVs of the log (ID50) values were small, with the mean CV being equal to 6.9% and with 95% of the CVs falling between 1.0% and 16.7%. Likewise, the intraclass correlation coefficient of 0.74 is quite high, indicating that the measurement error of the assay is quite small relative to the person-to-person differences.
In conclusion, this assay has many benefits over traditional PRNTs. The β-Gal assay requires a low volume of clinical sample, is easily adapted to a 96-well format or even automated handling for high-throughput applications, is rapid, and provides an objective, automated readout, with high quality control confidence. This assay performed extremely well in our hands, and even lacking robotic workstations for automated sample handling, we were able to assay over 3,000 specimens. This assay should prove useful in clinical trials of next-generation smallpox vaccines or to monitor changes in immune status over time. It could also be used as a screen to determine which individuals within a given population should be revaccinated to boost waning immunity. Given the large number of recombinant viruses expressing β-Gal or other reporter genes, this assay could also be easily adapted to other viral systems.