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Firefly Chemistry Used to Evaluate Tuberculosis Vaccines
The results of a study by Food and Drug Administration (FDA) scientists suggest that a laboratory test based on firefly chemistry could accurately and rapidly evaluate the potency of tuberculosis vaccines in triggering immune responses.
The findings are important because the new test might be able to replace the existing one, which is time-consuming, labor intensive, and often does not yield reproducible results. Additional studies are underway to further evaluate the findings to determine the adoptability of this test. This test has the potential to facilitate the production process of current tuberculosis vaccines while enabling development of newer vaccines now being studied in the laboratory.
The main quality control test currently used to evaluate commercially available BCG vaccines is called the colony-forming unit assay (CFU). The CFU assay measures the number of viable M. bovis bacteria that grow on the surface of a Petri dish from samples taken from the manufactured vaccine. Since the vaccine must be able to generate live organisms in the body to trigger optimal protective immune responses, this test is used to determine vaccine potency. The CFU assay takes up to four weeks to complete, the bacteria require complicated growth conditions, and repeated tests don’t always yield the same results. In contrast, the ATP test yielded reproducible results and can be completed within one day.
The new FDA test triggers luminescence (release of tiny packets of light) that indicates the presence of energy-rich molecules called ATP that all cells need to function properly. The FDA scientists modified an existing version of the test to make it more rapid and reliable.
The key to the reaction is the interaction of the enzyme luciferase with the molecule luciferin. In the presence of ATP, this reaction releases light, whether it occurs in the firefly or in the laboratory test. Since living cells need ATP in order to function, the intensity of light is an accurate reflection of the amount of ATP—and therefore, the number of living bacteria--growing from a sample bacteria comprising the vaccines.
The scientists used the test to evaluate six vaccines composed of lyophilized (“freeze-dried”) or frozen tuberculosis bacteria, Mycobacterium bovis BCG. The bacteria became active after being reconstituted in a special culture liquid, and then were grown in Petri dishes. The scientists also evaluated two live, attenuated (weakened) populations of genetically engineered M. bovis that are now being studied as potential new tuberculosis vaccines. For all the vaccines, the study found a significant correlation between the ATP concentrations in the bacterial cells and the number of attenuated bacteria that were still viable, and therefore, likely to trigger an effective immune response.
“Characterization of an intracellular ATP assay for evaluating the viability of live attenuated mycobacterial vaccine preparations”
J Microbiological Methods
90 (2012) 245-249
Kristopher Kolibaba, Steven C. Derricka, William R. Jacobsb, Sheldon L. Morrisa,*
a Laboratory of Mycobacterial Diseases and Cellular Immunology. Center for Biologics
Evaluation and Research, US Food and Drug Administration, Bethesda, MD
b Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, NY