Characterization of an intracellular ATP assay for evaluating the viability of live attenuated mycobacterial vaccine preparations.
The viability of BCG vaccine has traditionally been monitored using a colony-forming unit (CFU) assay. Despite its widespread use, results from the CFU assay can be highly variable because of the characteristic clumping of mycobacteria, their requirement for complex growth media, and the three week incubation period needed to cultivate slow-growing mycobacteria. In this study, we evaluated whether an ATP luminescence assay (which measures intracellular ATP content) could be used to rapidly estimate the viability of lyophilized and/or frozen preparations of six different BCG vaccine preparations - Danish, Tokyo, Russia, Brazil, Tice, and Pasteur - and two live attenuated mycobacterial vaccine candidates - a ΔlysAΔpanCD M. tuberculosis strain and a ΔmmaA4 BCG vaccine mutant. For every vaccine tested, a significant correlation was observed between intracellular ATP concentrations and the number of viable attenuated bacilli. However, the extractable intracellular ATP levels detected per cell among the different live vaccines varied suggesting that validated ATP luminescence assays with specific appropriate standards must be developed for each individual live attenuated vaccine preparation. Overall, these data indicate that the ATP luminescence assay is a rapid, sensitive, and reliable alternative method for quantifying the viability of varying live attenuated mycobacterial vaccine preparations.
Laboratory of Mycobacterial Diseases and Cellular Immunology, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD 20892, USA.
Kristopher
K
Kolibab
Steven C
SC
Derrick
William R
WR
Jacobs
Sheldon L
SL
Morris
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D001428:Bacterial Vaccines; D007424:Intracellular Fluid / Q000737:chemistry; D050296:Microbial Viability; D009163:Mycobacterium bovis / Q000254:growth & development / Q000276:immunology / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000254:growth & development / Q000276:immunology / Q000378:metabolism*; D032581:Tuberculosis Vaccines / Q000276:immunology / Q000378:metabolism; D014613:Vaccines, Attenuated / Q000276:immunology / Q000378:metabolism
D001428:Bacterial Vaccines; D032581:Tuberculosis Vaccines; D014613:Vaccines, Attenuated; D000255:Adenosine Triphosphate