COVID-19 tests are in short supply. The companies that make the necessary chemicals--called reagents--can't keep up with demand. But researchers at West Virginia University are developing new tests on their own to identify who has COVID-19 now and who had it in the past but recovered.
To get enough reagents to run thousands of tests of samples is a big problem--not only at WVU, not only in West Virginia and not only in the United States but around the world. When you don't have enough resources, you have to be creative. How can we develop something in house--here at WVU--so we don't have to depend on these companies?"
Ivan Martinez, Associate Professor, WVU Cancer Institute and School of Medicine
Processing numerous COVID-19 tests at once will be critical for testing large segments of the West Virginia population or students returning to WVU for the fall semester.
"We hope that our work will help the state and WVU reopen quickly and safely for business," said Peter Stoilov, an associate professor of biochemistry.
Taking apart the Legos
Martinez and Stoilov are pursuing a new diagnostic test that can recognize the novel coronavirus in a nose-swab sample.
The test works by detecting the RNA of SARS-CoV-2, the virus that causes COVID-19. RNA--a single strand of nucleic acid--contains all of the genetic instructions needed to create a SARS-CoV-2 virus. In humans, DNA--with its familiar double-helix molecular structure--does the job.
Only people currently infected with COVID-19 would test positive for SARS-CoV-2 RNA. Everyone else would test negative, including people who are infected with a different coronavirus that might cause the common cold, for example.
It is important for the test to be as sensitive as possible. There is always a chance that the amount of virus will be too low to detect. Having a sensitive test reduces the likelihood that the viral RNA will be "overlooked" and a patient misidentified as negative for COVID-19 when--in actuality--they're infected and could transmit the disease to other people.
"This is something very important that everyone getting tested should understand: we can be certain that a positive test is positive, but we can never say with absolute certainty that a negative test is negative," Stoilov said. "All we can say is that there was not enough of the virus for the test to detect it. It could be that there was no virus at all, but it is also possible that there two few viral particles for the test to produce a signal."
The test that Stoilov, Martinez and their team are developing can sense miniscule amounts of RNA in a sample. It does this by amplifying even tiny amounts of SARS-CoV-2 RNA.
"Imagine that the virus is assembled out of Legos," said Martinez, who teaches in the Department of Microbiology, Immunology and Cell Biology. "We put in this chemical--lysis buffer--and it disassembles the Legos. That way we can expose the RNA genome of the virus. Then, we use very small magnetic beads that will bind to the RNA so we can separate it from the rest of the Legos by using a bigger magnet. After that, we use special pieces of DNA--called primers--that bind to a region of that RNA. Then we use two enzymes: one that copies the RNA genome of the virus to DNA, and another enzyme that copies this little piece of DNA multiple times."
As the DNA fragments replicate exponentially--two becoming four, four becoming 16, 16 becoming 256--they make a fluorescence sensor that the scientists use shine brighter and brighter.
"After all of the cycles that you do with this enzyme--after 30 to 40 cycles--now you have trillions of copies of DNA," Martinez said. "And these trillions of copies give you trillions of signals. That's why this test is so sensitive."
Just how sensitive is it? Once someone's nose has been swabbed for the novel coronavirus, the swab is placed in a viral transport medium--a milliliter of liquid--before it's sent to a lab and tested. With WVU's test, as few as 75 copies of RNA that washed away from the swab are enough to trigger a positive result if the virus is present.
"To give you an example, the test initially used by the CDC had a limit of detection over 1,000 copies per milliliter," said Peter Perrotta, a professor and chair of the Department of Pathology, Anatomy and Laboratory Medicine and part of the test-development team. "The test developed by Drs. Martinez and Stoilov is more sensitive than many tests that are currently used in this country."
The test's accuracy isn't its only advantage. So are the number of samples it can process at a time.
"We are now working to fully automate the COVID-19 test and establish it in the clinical lab so that we can start testing large number of samples," Stoilov said. "The timeline for placing the test in production depends on when the funding will become available and how quickly the vendors can deliver the equipment. In the meantime, we are testing a procedure to pool samples without losing sensitivity. Such sample pooling will allow us to increase the number of tests we can do in a day by at least tenfold."
He added that work carried out by the Centers for Disease Control and Prevention--and by the scientists behind Bio-On-Magnetic-Beads--was essential to the quick test development and he, Martinez and their colleagues achieved.
"We had free access to data on coronaviruses gathered by scientists from around the world, and especially China, because our colleagues put together equipment and supplies to get us going and because the Health Sciences Center's research office supported our effort," he said. "To put it concisely: we succeeded because we stood on the shoulders of giants. It would have taken us more than a year if we had to do everything from scratch, and this is a very, very optimistic estimate."
From antibody testing to vaccination
As crucial as diagnostic testing is for identifying current COVID-19 infections, it can't tell you who had the virus weeks or months ago and has since gotten over it. It's a snapshot of the present, not archival footage of the past.
That's the purpose of antibody testing. Antibodies are proteins produced by immune cells that the body generates in response to a virus, bacterium or other cell that it flags as an invader. Like a key fitting into a lock, the antibodies bind proteins on the surface of invading cells--called antigens--to prevent the pathogen from infiltrating healthy cells and making us sick.
"It became very clear that we needed to be able to measure antibodies that recognize SARS-CoV-2 in order to better understand the pandemic as well as facilitate vaccine development efforts," said Heath Damron, an assistant professor in the Department of Microbiology, Immunology and Cell Biology and the director of the Vaccine Development Center. He is leading WVU's effort to develop a SARS-CoV-2 antibody test. Alex Horspool, a postdoctoral fellow, has developed the test and validated it with hundreds of patient samples.
"Responses to vaccines will be different depending on if someone has been exposed to the virus or not," Damron said. "We also wanted to have a test that we were capable of producing the proteins for in house, so we would be less affected by supply chain issues."
Another benefit of the vaccine is its cost-effectiveness. Damron and his colleagues are working to automate their test with robotics, so they will be able to test more samples faster, at a lower cost.
"In many of the research projects being proposed, we expect in the thousands of samples to be able to survey the populations of interest," he said.
Whereas the diagnostic test relies on samples collected on nose swabs, the antibody test uses serum extracted from blood samples. It seeks out specific antibodies that recognize the SARS-CoV-2 "spike" protein. These antibodies are important not only for antibody testing but also for developing an eventual COVID-19 vaccine.
"Almost all vaccines in development target the spike protein because it is what enables the virus to bind the ACE2 receptor and infect the target cell," Damron said. "If a vaccine induces antibodies that bind to the spike and block its ability to engage the ACE2 receptor, then infection can be blocked."
The antibody test doesn't just show whether antibodies are present. It also shows how high the concentration of antibodies is. It's more like a dimmer switch than a standard, on/off light switch.
"It is likely that people with more severe infections will produce more antibodies," Damron said. "However, a lot more work is needed at this time. It is also not clear if people will be protected from being infected. We have so much to understand about this virus."
The antibody test that Damron and his colleagues are developing complements a commercial platform that WVU Medicine already uses to determine if a different SARS-CoV-2 antibody is present in blood plasma samples.
The researchers will soon send their new in-house test to the Food and Drug Administration for approval. In the meantime, they are using it for pre-clinical vaccine studies and other COVID-19 research projects.
"The teamwork that I have seen as so many individuals have contributed their unique talents during these past several weeks has highlighted the strength of WVU and our state," said Laura Gibson, HSC's senior associate vice president for research and graduate education, and the School of Medicine's associate dean for research. "Nobody is worried about who gets 'credit' moving forward. Everyone is entirely focused on being part of solving problems that need immediate action and thoughtful long-term strategies. Applying foundational science to solving real problems is why we are here as a scientific community--in it together."