Out of the BSL3 “Jail”: Simplifying SARS-CoV-2 virus neutralization tests

Determining if a patient has SARS-CoV-2 neutralizing antibodies calls for virus neutralization tests that require days of work and a BSL3 lab. In the time of a pandemic, could this be simplified?
Out of the BSL3 “Jail”: Simplifying SARS-CoV-2 virus neutralization tests

When infected by viruses, the body produces different antibodies that bind to different parts of the virus; however not all antibodies are created equal - some are able to block (i.e., neutralize) infection, while others cannot.

Traditionally, to test for such neutralizing antibodies, a conventional virus neutralization test (cVNT) using live SARS-CoV-2 is necessary. Given the pathogenicity of SARS-CoV-2, cVNTs are required to be performed in a biosafety level 3 laboratory (BSL3). BSL3 labs require special training and procedures to prevent accidental release of the pathogen. Even experienced researchers take hours to process samples, followed by days of incubation time, taking up to a week to complete the entire experiment. If your experiment does not work (our sincerest apologies), you will have to go through this process again.

Asst Prof Danielle Anderson (co-corresponding author, seated) with Mr Velraj Sivalingam (standing) working in the Duke-NUS BSL3 facility

A virus needs a receptor to enter and infect host cells. It is known since the early days of the COVID-19 outbreak that this receptor is an enzyme called ACE2. It is also known that the SARS-CoV-2 protein interacting with ACE2 is the Spike protein, more specifically, a region called the receptor-binding domain (RBD). It is well known that during coronavirus infection that the majority of neutralizing antibodies target RBD. This premise is thus used in virus neutralization tests.

To simplify cVNTs, Prof Linfa thought of applying a biochemical simulation strategy in late Feb 2020: using beads conjugated with ACE2 and RBD in the liquid phase. Neutralizing antibodies, if present, will block the interaction of ACE2 and RBD, which can then be quantified by Luminex. This assay, although multiplexable, can be expensive and requires specialist equipment. One day in early March, an idea came into view: “what if ACE2 is coated on ELISA plates instead?” And the rest was history: surrogate virus neutralization test (sVNT) was born. The commercial kit, cPass, was launched on 15 May 2020, a true “COVID-19 speed” of less than three months from conceptual invention to a commercial product!

This removes the need for live viruses, hence out of the BSL3 “jail”. This reduces the barrier for testing SARS-CoV-2 neutralizing antibodies which can be useful for resource-limited settings.

Dr Wanni Chia (co-first author) stacking a cPass kit, a commercialized sVNT by Genscript, in a video created by the lab to celebrate Prof Linfa Wang’s 60th birthday in May 2020  (“A scientist” is one of the many titles used to describe his different professional roles)

Aside from its use to determine prior infection or exposure status - which may be used in contact tracing, we envision that sVNT will be most useful during vaccine trials when we need a test to tell whether someone has responded to the vaccine (i.e. produce neutralizing antibodies). Equally important, sVNT will be a useful tool to determine longevity of neutralizing antibodies post infection or vaccination. In these cases where large scale testing is essential, we will no longer have to take the cumbersome route of BSL3-based cVNTs. Instead, sVNT enables diagnostic labs to determine neutralizing antibodies with just the usual lab equipment and shortens the turnaround time from days to hours.

Hopefully, with the shortened amount of time required to determine the presence of neutralizing antibodies, scientists and clinicians would be able to speed up COVID-19 research, vaccine approval, and up our game against COVID-19. Stay safe!

Vivian Chen and Charles Tiu contributed equally to this post.

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