Fireflies help kindle new tests and treatments for COVID-19

Fireflies, also known as lightning bugs, are insects in the Lampyridae family who use bioluminescence to attract potential mates and prey. Their conspicuous glow at twilight comes from an enzyme called luciferase, which can be isolated in the lab. Now, UTMB virologists are using the enzyme to develop faster and more accurate diagnostic tests for COVID-19 as well as to analyze potential therapies and gain a clearer understanding of the SARS-CoV-2 virus itself.

“We are very much interested in using basic research knowledge to develop systems … to support translational work, for example, diagnosing disease, vaccine development, medical countermeasures and therapeutic drugs—all those things that can be applied to the public health and human well-being,” said Pei-Yong Shi, Ph.D., professor of human genetics in the department of biochemistry and molecular biology at UTMB. He is leading the research by labeling viruses with glowing tags like luciferase to study them.

Prior to the SARS-CoV-2 outbreak, Shi’s team had been focused on flaviviruses, including West Nile virus, Zika and dengue. His lab developed the first infectious clones of the epidemic strain of both West Nile and Zika and have established new pathways for flavivirus vaccine and drug discovery.

But as the COVID-19 pandemic swept the globe, Shi’s lab adapted by altering research techniques to address the SARS-CoV-2 virus.

First, researchers harnessed luciferase to develop faster diagnostic testing through innovative assays—investigative procedures that measure the activity or amount of a substance. The lab can now visually confirm the presence of antibodies that can block a SARS-CoV-2 infection earlier than through previous methods.

“The great thing about luciferase, and the one that we specifically use which is nanoluciferase, is that it’s really bright,” said Coleman Baker, a fourth-year graduate student in the department of microbiology and immunology at UTMB who works in the Shi lab. “We found that these detection assays can be read earlier than most people thought. Most people would do these 24 to 48 hours after infection and we found that you can read them as early as 4 hours post-infection.”

Not only does the enhanced brightness provided by luciferase decrease turnaround time for a diagnosis, but it also helps speed up the process of vaccine development.

“For vaccine studies, you need to find out how much of a response somebody has to a potential vaccine,” Baker said. “So, instead of the traditional way—which could take days—we can get results in four hours after infection.”

Shi said his team has been collaborating with leading pharmaceutical companies to help them evaluate their vaccine candidates, specifically by measuring the immune response in humans in clinical trials. His lab’s system—based on reporter viruses in which the luciferase enzyme is inserted into the virus’ genome to make it easy to follow—measures the concentration of neutralizing antibodies while also allowing for a much higher throughput. According to Xuping Xie, Ph.D., assistant professor in the department of biochemistry and molecular biology at UTMB, this way of measuring the immune response is more accurate than traditional methods.

“The sensitivity and the dynamics of the assay, because of the luciferase, can really allow us to discern any small changes that the conventional way would not,” said Xie. “It’s much more sensitive, much more accurate, when you test antiviral compounds.”

Below, additional information about Luciferase from the FDA, ScienceDirect and Wikipedia

Luciferase Immunoprecipitation System (LIPS) assay is a rapid, simple, and sensitive test to detect antibody response to respiratory syncytial virus | FDA

SNIP
To develop the assay, the FDA scientists genetically engineered RSV G proteins from both subgroups. They tagged each G protein with luciferase, an enzyme that interacts with a protein called luciferin to release light.

The luciferase-tagged G proteins (RSV-GA and RSV-GB) acted as “bait” to antibodies against the G proteins in samples of human serum (the clear fluid of blood without red or white cells), causing them to bind to the tagged proteins. The scientists added special protein beads that bind the antibodies with the luciferase-tagged G proteins to the bottom of a plastic test well. Then they added luciferin and measured the amount of light released when it interacted with luciferase, which enabled them to calculate how strong the antibody response was to the G proteins.

Luciferases

Luciferases are enzymes that produce light when they oxidize their substrate. The gene for the most common luciferase comes from the firefly, but luciferases from other animals such as the sea pansy Renilla reniformis, the copepod Gaussia princeps, and the ostracod Cypridina noctiluca are also used as reporters. When a luciferase is fused to a protein of interest, its expression can be measured very accurately using a luminometer. The firefly luciferase reaction requires its substrate luciferin, plus adenosine triphosphate (ATP), O₂, and Mg²⁺. The Renilla and Gaussia luciferases use coelenterazine as their substrates; Cypridina uses its own luciferin as a substrate. Luciferases are commonly used to report the expression level of proteins to which they are fused.



Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words luciferin and luciferase, for the and enzyme, respectively.^[1] Both words are derived from the Latin word lucifer, meaning "lightbearer", which in turn is derived from the Latin words for "light" (lux) and "to bring or carry" (ferre).^[2]

Applications[edit]

Luciferases can be produced in the lab through genetic engineering for a number of purposes. Luciferase genes can be synthesized and inserted into organisms or transfected into cells. As of 2002, mice, silkworms, and potatoes are just a few of the organisms that have already been engineered to produce the protein.^[14]

In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate. Photon emission can be detected by light sensitive apparatus such as a luminometer or modified optical microscopes. This allows observation of biological processes.^[15] Since light excitation is not needed for luciferase bioluminescence, there is minimal autofluorescence and therefore virtually background-free fluorescence.^[16] Therefore, as little as 0.02 pg can still be accurately measured using a standard scintillation counter.^[17]

In biological research, luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest.^[18] Additionally, proluminescent molecules that are converted to luciferin upon activity of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays. Such substrates have been used to detect caspase activity and cytochrome P450 activity, among others.^[15]^[18]

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