Integrated Automation Yields Less Time, More Throughput during Dengue Virus Vaccine Development (Frontiers in Immunology original research article)
Discover how an automated work cell boosted productivity when developing a vaccine against a harmful virus.
Key Takeaways
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Dengue virus infections are rapidly gaining prevalence across the world, especially in tropical and sub-tropical climates. Without current targeted dengue therapies, prevention is an essential healthcare strategy to protect individuals and communities.
Researchers at Merck recently detailed their efforts to develop a highly effective vaccine that would prevent dengue infection in the original research article, “Fully automated high-throughput immuno-µPlaque assay for live-attenuated tetravalent dengue vaccine development”, published by Frontiers in Immunology.
The vaccine candidate is a multivalent live virus vaccine (LVV). This means that it is made from live, weakened viruses that cannot cause disease but can stimulate an immune response. It also means that the vaccine is intended to offer broad protection against multiple dengue virus strains; in this dengue vaccine study the tetravalent vaccine protects against four dengue virus strains.
TESTING FOR CRITICAL QUALITY ATTRIBUTES (CQAs)
CQAs are properties or key characteristics of a drug or vaccine candidate that must fall within appropriate limits to ensure safety and quality in the final product. As an important CQA in vaccine development, potency includes extensive studies to measure vaccine sample immunogenicity along with vaccine product effectiveness, safety, and consistency.
Potency methods can be grouped into relative potency, antigen mass, and infectivity categories. The latter is sub-categorized into traditional plaque assays, tissue culture infectious dose 50 (TCID50), cell culture infectious dose 50 (CCID50), animal lethal dose (LD50) and focus-forming assays (FFA). Regarding this final sub-category, the Merck researchers determined that an automated system would significantly benefit the challenges inherent to the multivalent vaccine analytical demand.
WHAT IS A FOCUS-FORMING ASSAY?
In a broad overview of a microplate-based FFA method, cells are grown in monolayers, virus is added, the plate is incubated. After media overlay is added, the plates are again incubated to allow for viral spread. The cells are then fixed, immunostained, and imaged using a microplate reader. Each identified cluster of infected cells is called a focus, and multiple clusters are called foci.
Unlike traditional plaque assays, focus-forming assays can detect foci before a plaque forms and grows large enough for visual detection. Smaller detectable focus-forming units (FFU) and immunostaining enable the FFA method to be miniaturized into microplate format for high-throughput processing. In turn, high-throughput processing helps to reduce overall vaccine development time.
HOW AN AUTOMATED SYSTEM CAN BENEFIT MULTIVALENT VACCINE DEVELOPMENT
Automated laboratory systems broadly benefit distinct scientific domains. With highly consistent and repeatable operation over time, automated work cells can strengthen reproducibility and process safety compared to manual methods. Lab automation enables assay miniaturization to increase throughput while reducing related reagent and consumable costs.
The hands-free approach reduces active labor time so that users can focus on higher-value tasks. Without the variability risk that arises when individuals manually perform tasks, it’s easier to compare results over time and across multiple laboratories or facilities.
When developing potency methods for the LVV candidate, the Merck team set out to develop and automate a high-throughput FFA method that could manage the complexity and additional optimization steps needed for a multivalent vaccine compared to a monovalent vaccine while also achieving the above-mentioned automation benefits.
THE AUTOMATED µPLAQUE ASSAY
The researchers developed a high-throughput FFA, dubbed the µPlaque assay, in 96-well plate format to measure dengue sample absolute titer. The assay workflow (Figure 1A) proceeds using a cell culture system and an integrated robotic system (Figure 1B) that is fully enclosed within a Class II, Type B2 environment. This automated work cell system, designed and integrated by HighRes Biosolutions, includes a live cell plate incubator, AmbiStore random access sample storage carousel, automated liquid handler, microplate dispenser, plate washer, plate hotel, PlateOrient labware orientation device, and LidValet high-speed delidding hotel. All labware transfers between devices were managed by an ACell robotic arm mounted on a linear rail.
Figure 1. The Merck µPlaque assay. A. Assay workflow overview from cell plating through plate imaging. B. Automated work cell used for hands-free assay processing. Image from original research article authored by Wang, Y., et al, and published in Frontiers in Immunology.
CONFIRMING AUTOMATED ASSAY PERFORMANCE
The team optimized the automated µPlaque assay for the tetravalent nature of the vaccine and confirmed that repeatability and precision were acceptable for dengue viral titer testing.
They also performed a concordance study between the automated µPlaque FFA assay and the traditional manual plaque assay. Results demonstrated that the automated method significantly improved throughput, with 384 samples processed per run compared to 32 samples in the manual method.
Automated assay duration was 4-5 days, depending on serotype, and involved one user, compared to the manual assay that lasted for 8 days and required two users. Data suggest no significant proportional bias (PFU/mL) between the manual and automated assay.
In addition to yielding robust data, the laboratory automated system can capture all information regarding the samples as they progress through the workflow. This analytical and meta information can be stored and output in a standardized format for inclusion in submission reports or used to support further assay optimizations.
CONCLUSION
The µPlaque assay method, processed on a fully contained HighRes automated system, yields comparable results to manual assays while providing significant benefits in throughput, time, and effort. Next steps for the researchers include incorporating deep learning to further enhance the plaque or foci counting method and multiplexing the µPlaque assay for further sample processing throughput and other time-reducing efficiencies.
Learn more about this study, the first of its kind to be published, including how the HighRes lab automated system aided accelerated vaccine development, at doi.org/10.3389/fimmu.2024.1356600.
If you seek ways to improve efficiencies, save time, and increase throughput in your workflow, contact your local HighRes expert.