[The adjuvant effect of chimeric flagellin fliC/esat]

To determine the role of ESAT-6 chimeric flagellin in TB immunology.

The coding sequences of flagellin of Salmonella typhimurium and ESAT-6 of Mycobacterium tuberculosis were cloned by PCR and identified by sequencing, respectively. Chimeric flagellin gene fliC/esat was constructed by overlap PCR technique. The ESAT-6 coding fragment was inserted to the hypervariable region of Salmonella flagellin gene fliCi. And then prokaryotic exprssion plasmids of pET-fliC/esat, pET-fliC and pBCX-esat were constructed and transformed into E.coli BL21(DE3), followed by induction of IPTG. The expressed proteins fliC/esat and ESAT-6 were identified by Western-blot assay using specific monoclonal antibody (mAb) HYB076-08. Bone marrow dendritic cells (BMDCs) were in vitro stimulated by fliC/esat and ESAT-6 proteins, and analyzed for the expression levels of CD40, CD80, CD86 and CD54 molecules. The secreted IL-12p70 was determined by ELISA. Moreover, C57BL/6 mice were immunized intravenously with fliC/esat or ESAT-6 protein. The specific IFN-γ-secreting cells and IL-4-secreting cells from the immunized mice were detected by ELISPOT assay using an ESAT-6 peptide as a stimulus.

The results showed that the proteins of fliC/esat and ESAT-6 were expressed solubly, with the sizes of 64 kD and 39 kD respectively. Western blot analysis showed that both proteins reacted with the specific mAb against ESAT-6. BMDCs maturation was triggered by the chimeric flagellin fliC/esat. In contrast, ESAT-6 protein alone didn't activate BMDCs. IL-12p70 was also detected in the supernatants of BMDCs. The results showed that the chimeric flagellin fliC/esat induced significantly higher level of the secreted IL-12p70 than that of ESAT-6 protein. Furthermore, the chimeric flagellin fliC/esat significantly enhanced the Th1-biased immune responses against ESAT-6 in the immunized C57BL/6 mice.

The chimeric flagellin we generated exerts Th1 type adjuvant activity for ESAT-6 protein.