A Virus Neutralization Assay That’s Faster than the Rest

HiBiT protein tagging speeds up the process of measuring if an antiviral treatment or vaccine is working

There is an obvious problem that researchers face when trying to develop treatments for deadly viruses: the viruses are extremely dangerous for researchers to handle. To test their treatment or vaccine against the actual virus, they need adequate biosafety protection to contain the virus and ensure that it does not infect the researchers themselves. This is especially relevant to viruses like SARS-CoV-2, Ebola, and Marburg because they are incredibly contagious and lethal. Scientists working with them are obligated to take extreme measures to protect themselves in high-level biosafety level laboratories (BSL 3 or 4) designed to limit any chance of exposure in or outside the lab. There are only a small number of these higher-level laboratories in the world, severely limiting the volume of scientists who can work on them.

However, advancements in biology and virology have created powerful and flexible alternatives for working with dangerous ‘live’ viruses. For instance, scientists can carefully break the live virus into individual pieces that can be put to work in a far safer environment. With more than 6 million deaths attributed to SARS-CoV-2 thus far, and the increasing number of variants that have facilitated its spread, having tools that enable more scientists to study it is essential.

Another important set of tools in the virologists’ wheelhouse are ‘pseudoviruses’, which have proven to be a breakthrough in studying and working to stymie the infectiveness of viruses. They are made by “taking an entry protein from a virus that you are interested in and adding that onto the surface of an unrelated virus or virus particle,” says Mitchell. “Decades of [viral] research has shown the [cell] entry is very much indicative of the entry of the real virus.”

According to Jonathan Mitchell, senior research scientist at Promega, “our pseudovirus-like particles are missing some of the key components that would make them infectious… no viral RNA or DNA — no viral nucleic acid that would allow it to replicate.”

The power of PsVLP neutralization assays

The example of SARS-CoV-2 is not an arbitrary one. Promega is one of many companies that have been hard at work developing technologies, treatments and testing assays for dealing with the novel virus. (Assay is a technical term for an analytic procedure intended to qualitatively assess or quantitatively measure the presence, amount, or functional activity of a target entity.) Promega has made great strides due to the power of their PsVLP neutralization assays.

Neutralization is the ability of antibodies to bind to viral particles and prevent them from infecting cells, and SARS-CoV-2 is an especially difficult virus to neutralize. One of the keys to SARS-CoV-2’s infectiveness is its so-called ‘spike proteins’ that dot the virus’ surface and enable it to deftly invade cells. By ripping off those proteins and putting them on a virus-like particle that can’t reproduce, Promega can then test the efficacy of vaccines and treatments intended to block the spike proteins from interfacing with the cells, without the biosafety risks associated with the virus.

“We entered the virology space to offer new solutions for neutralization assays,” says Mitchell. The existing neutralization assays had significant limitations, requiring high biosafety levels and many hours of researcher time over days to render results.

“You might be waiting anywhere from a day to five days to get your answer. Whereas these assays we've developed take typically under a day or less,” says Mitchell. Using virus-like particles has eliminated these hurdles.

Promega’s PsVLP neutralization assays use ‘HiBiT protein tagging’ — taking the PsVLPs and attaching them to a split piece of a luciferase enzyme . When these HiBiT-tagged PsVLPs enter cells, the two split pieces will recombine. Which can, says Mitchell, “with the right substrate create a luminescent signal.” Neutralization blocks this entry and eliminates the luminescent signal. The result of the assay can be determined within hours by directly observing the emitted light, without the need to culture cells.

The importance of quick and accurate bioassays cannot be overstated. “Bioassays are a requirement for lot release,” explains Dr. Ulrike Herbrand, Scientific Director for Global In Vitro Bioassays for Charles River Labs. “Cell-based assays often take several days in the lab, and if they fail for whatever reason and need to be repeated, often we lose a week, maybe even more,” says Herbrand. The short turnaround of the PsVLP neutralization assays means that even if there is a failed test, a new one can be done quickly, enabling Herbrand’s teams to deliver their reports on time and reducing the risk of delay for their clients.

It’s not enough to be able to make a treatment, it’s got to be validated and tested. Biological processes have inherent variability, and testing is a continuous process. While the treatments themselves get a large amount of attention — and rightly so — the companies that make the assays and tests are no less critical to the process. Mitchell sees Promega as a helper and collaborator: “We're here to create the tools that other scientists need in order to do their work… in a way that can hopefully accelerate [scientific] research.”