Serological tests, also known as “antibody tests,” are one of the foundations of clinical medicine, with a particularly important role in infectious disease. They can detect active infections, but can also identify past exposures to infectious agents. These are both important pieces of information that can guide clinical care, and help explain why some people experience mild disease whereas others are much more severely affected.
During pandemics and epidemics, public health officials must identify individuals who are actively infected or have previously been infected, both to control the spread of disease and, in the case of vaccine-preventable illnesses, to prioritize individuals for vaccination. The SARS-CoV-2 pandemic has shone a bright light on the crucial need for accurate serological tests, especially now that vaccines are beginning to be deployed worldwide.
Now scientists led by Colin Zamecnik, PhD, a research scientist at UCSF and the Chan Zuckerberg Biohub, and UCSF Assistant Professor of Medicine Jayant Rajan, MD, PhD, have developed ReScan, an innovative new serological test that employs a specialized version of the technique known as phage display.
Building Phage Libraries
In research honored with a Nobel Prize in 2018, researchers showed that phage – a type of virus that attacks bacteria – can be made to incorporate chunks of a diverse range of genetic material and then “display” a corresponding protein on their surface. This technique has attracted considerable interest among scientists who study infectious diseases and autoimmune diseases, because it allows for the rapid development of massive protein libraries that can be used to perform fast, unbiased diagnostic screens of clinical samples from patients.
For example, by using phage to display protein fragments from the approximately 3,000 viruses known to infect humans, a UCSF/CZ Biohub research team led by Michael Wilson, MD, associate professor of neurology, showed that a mysterious polio-like illness that affects children is probably caused by a common viral agent. In another application, by creating a phage library of 700,000 human proteins, Wilson and Joe DeRisi, PhD, UCSF professor of biochemistry and biophysics and CZ Biohub co-president, led a team that identified a new autoimmune disease that affects men with testicular cancer.
In the new work, first authors Zamecnik and Rajan joined Wilson, DeRisi and colleagues, using the complete genome sequence of the SARS-CoV-2 virus to create a phage library representing all of the virus’s proteins, which they were able to deploy in a clinical diagnostic format in just a matter of weeks.
“The versatility and quick turnaround of phage display libraries make them a great tool for fast-moving public health emergencies,” said Zamecnik, “and we were very pleased with ReScan’s performance in this environment.”
A key advantage of using phage display in this context, Zamecnik said, is that in addition to detecting COVID patients’ immune response to SARS-CoV-2 infection, the scientists could precisely identify exactly which parts of the virus are being most strongly targeted by the immune system.
Rapid, Adaptable Technology
As reported Oct. 20 in Cell Reports Medicine, to create ReScan, Zamecnik repurposed a special type of liquid-transfer robot called an ECHO to print test strips with hundreds of spots, each made up of phage displaying various SARS-CoV-2 proteins. These tests strips are a fresh take on the microarray technology originally developed by DeRisi to confirm that a coronavirus now known as SARS-CoV-1 was responsible for the first SARS outbreak in Asia in the early 2000s.
When individual patients’ blood samples are applied to the ReScan strips, antibodies in the blood bind to the phage making up the spots on the strips, each of which correspond to a particular part of the virus. By comparing results from many strips, researchers can determine which spots are most commonly bound by antibodies across many patients, and use this information to decide which viral proteins are the best candidates to incorporate into further tests to make the most accurate diagnoses.
But one problem remained. “Our printing process for the strips was super-fast – 10 to 100 times faster than commercially available printers – but analyzing them was slow and tedious,” said Zamecnik. “What we really needed was a high-throughput image-processing system to scan lots of strips in parallel and identify the best ‘hot spots’ to concentrate on.”
Last November, Zamecnik described this bottleneck to bioengineer and image-analysis expert Kevin Yamauchi, PhD, a former Biohub scientist who is now a postdoctoral researcher at Biozentrum in Switzerland. Zamecnik and Yamauchi quickly drew up some specifications, and Yamauchi arrived at an elegant solution in the form of “dotblotr,” an open-source software package that quickly analyzes ReScan strips and reports the best SARS-CoV-2 phage to use.
With this information in hand, scientists can go back to the stock from which the strips were printed and easily make more tests that are focused just on the spots that elicited the strongest immune response. Phage can make copies of themselves, so they are an inexhaustible test resource – with an infinitesimal amount of the original phage, trillions more can be grown in a matter of hours with no complex procedures required.
The best diagnostic proteins identified by ReScan can be packaged into more standard lab tests, and in the Cell Reports Medicine paper, the authors report that the performance of one such test was similar to or better than other commercially available serology platforms.
“Phage display, especially when combined with the printable microarray strips we’ve developed in ReScan, is a rapid and highly adaptable technology that can be easily retooled to meet the challenges posed by emerging infectious diseases of the future,” Zamecnik said.