A series of medical breakthroughs have transformed the COVID-19 landscape, radically reducing hospitalizations and deaths. Among the most impactful are effective vaccines and therapies known as monoclonal antibodies.
But in a never-ending arms race, SARS CoV-2, the virus that causes COVID-19, can outwit the body’s immune defenses. Researchers and clinicians hope to develop better means of identifying particular variants that can sidestep a given monoclonal antibody treatment, infect the body and cause disease.
In new research, Efrem Lim and his colleagues describe a technique known as digital PCR (dPCR), which can provide a rapid, accurate and low-cost method of pinpointing viral variants able to render monoclonal treatments ineffective.
Lim is a researcher with the Biodesign Center for Fundamental and Applied Microbiomics and associate professor with the School of Life Sciences at Arizona State University.
“This technology tells us if a life-saving treatment will work for you, against your specific COVID-19 variant infection,” Lim said. "The SARS CoV-2 virus constantly mutates. We need to deploy the most effective therapeutics against the most vulnerable points of the virus.”
The research appears in the current issue of the journal Microbiology Spectrum.
The method may advance personalized treatment for patients with COVID-19. Currently, the selection of treatments is often based on the predominant circulating variants and their general properties, rather than the specific characteristics of the virus in individual patients.
For example, the use of the therapeutic antibody Bebtelovimab has been halted in the U.S. because of the prevalence of Omicron subvariants resistant to this treatment. However, this broad-brush approach may deny life-saving treatments to patients infected with variants that are susceptible to these therapies. By using dPCR to identify the specific mutations in the virus from individual patients, it may be possible to tailor treatment plans to their specific needs, thus improving outcomes.
Potent, dynamic therapy
Monoclonal antibodies are laboratory-made molecules that function like human antibodies in the immune system. They are designed to bind to specific antigens, which are unique molecules present on the surface of pathogens such as bacteria and viruses. Monoclonal antibodies can be engineered to target virtually any antigen, making them a versatile tool in the treatment of various diseases.
The concept of monoclonal antibodies was first developed in the 1970s by César Milstein and Georges Köhler, who later received the Nobel Prize in 1984 for their work. They developed a method to produce identical (or monoclonal) antibodies, which would target a single site on an antigen. Antigens are various types of markers that help the body’s immune system identify whether something in your body is harmful.
Monoclonal antibodies have several characteristics that make them powerful tools in the treatment of diseases. Because they are designed to bind to a single specific antigen, they can target diseased cells while leaving healthy cells unaffected. Once bound to their target, monoclonal antibodies can recruit other parts of the immune system to destroy the marked cells. They can also block cell receptors or signaling pathways, thereby interfering with the ability of a pathogen or disease cell to function. This makes them a versatile tool in the treatment of various conditions, from cancers to autoimmune diseases to infectious diseases.
During the COVID-19 pandemic, monoclonal antibodies have been used to treat patients infected with the SARS-CoV-2 virus. They work by targeting the spike protein on the surface of the virus, which is the protein that the virus uses to enter human cells. By binding to the spike protein, the monoclonal antibodies can prevent the virus from entering cells, inhibiting its ability to replicate and cause disease. These treatments have been shown to reduce the risk of hospitalization and death in high-risk patients with mild to moderate COVID-19.
However, as the virus continues to evolve and new variants emerge, there are concerns about the effectiveness of these treatments against new strains of the virus. Research is ongoing to develop new monoclonal antibodies and to update existing ones to ensure their effectiveness against new variants of the virus.
In the current study, the team developed a set of tests using dPCR to rapidly and accurately identify mutations in the SARS-CoV-2 virus, with specific attention to mutations that allow the virus to evade the immune system and resist treatment. This research is of great importance as the virus continues to evolve new variants.
Polymerase chain reaction (PCR) is a method used to make millions of copies of a specific DNA sequence. This amplification allows researchers to detect occurrences of that sequence, even in very low amounts.
dPCR is a variation of traditional PCR. It works on the same principle of amplifying DNA, but instead of amplifying all the DNA together in one tube, dPCR first partitions the DNA sample into many individual, separate reactions, each containing either one or no target DNA sequence.
Then, PCR is performed on each of these tiny reactions independently. At the end of the process, each partition will either have a lot of copies if the starting partition contained the target DNA sequence, or none if it didn't.
By counting the number of partitions where amplification occurred, the test can precisely determine the amount of the target DNA in the original sample. This method can be very helpful for accurately quantifying low levels of DNA or RNA, detecting rare mutations and other applications where precise measurements of DNA or RNA are important.
The researchers designed multiple dPCR assays to detect singular changes in individual base pairs in the SARS CoV-2 virus' RNA — which are characteristic of different viral lineages, such as Delta and Omicron. These tests were validated using 596 saliva samples, which were also sequenced for comparison using a different technique called whole genome sequencing.
Next, the researchers developed further dPCR tests to identify specific mutations in the virus' spike protein that are associated with resistance to therapeutic monoclonal antibodies. In total, they were able to identify up to four of these mutations in a single test. Applying these tests to 81 saliva samples, they correctly identified mutations in various subvariants of the Omicron variant.
The research points the way to improved personalized diagnostic testing for COVID-19. Further, dPCR is not limited to any one virus and can be applied to any situation where detection or quantification of specific genetic sequences is needed.
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