T-cell-receptor Vβ8 for detection of biologically active streptococcal pyrogenic exotoxin type C.

Streptococcus pyogenes is an important human pathogen, commonly spread by airborne droplets but also by ingestion of contaminated food. Apart from causing infection, this pathogen produces 13 distinct types of streptococcal pyrogenic exotoxins (SPEs). The current method for detection cannot distinguish between the biologically active form of SPEs that has been reported to cause foodborne outbreaks and the inactivated toxin which poses no health risk. To measure the biological activity of SPE-C, one such toxin that was linked to foodborne outbreaks associated with milk and milk products, we developed a cell-based assay that can discern between biologically active and inactive SPE-C. To the best of our knowledge, this is the first showing that SPE-C activates T-cells expressing Vβ8. With this finding, we used a T-cell line natively expressing Vβ8 that was genetically engineered to also express the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE) in combination with a B-cell line to present the rSPE-C toxin via MHC class II to the Vβ8 TCR in an assay to detect and to discern between biologically active and inactive rSPE-C. By using this system, we demonstrated that SPE-C induced significant IL-2 secretion after 72 h and visible light emission after only 5 h, doubling by 24 h. We utilize this finding to assess the specificity of the assay and the effect of pasteurization on SPE-C activity. We observed no cross reactivity with SPE-B and significant loss of SPE-C biologically activity in spiked PBS while SPE-C spiked into milk is heat stable. Once SPE-C has formed, it is infeasible to eliminate it from milk by thermal treatment.