Chimeric flagellin expressed by Salmonella typhimurium induces an ESAT-6-specific Th1-type immune response and CTL effects following intranasal immunization

Abstract

The flagellin component FliC of Salmonella typhimurium is capable of activating the innate immune system via specific interactions with TLR5 and can also act as a carrier of foreign antigen to elicit antigen-specific immune responses. Thus, we constructed an attenuated Salmonella strain SL5928(fliC/esat) expressing chimeric flagellin that contained the ESAT-6 antigen coding sequence of Mycobacterium tuberculosis inserted into the highly variable region of the Salmonella flagellin coding gene fliC(i). The chimeric flagellin functioned normally, as demonstrated using a flagella swarming assay and electron microscopy. To analyze the effects of chimeric flagellin, the cell-mediated immune response and cytotoxic T lymphocyte (CTL) effects specific for ESAT-6 antigen were tested after intranasal immunization of mice with flagellated Salmonella SL5928(fliC/esat). The results showed that SL5928(fliC/esat) intranasal immunization can strongly elicit an ESAT-6-specific T helper (Th) 1-type immune response in mucosal lymphoid tissues, such as nasopharynx-associated lymph nodes, lung and Peyer's patches, and a Th1/Th2 response was elicited in spleen and mesenteric lymph nodes. Furthermore, intranasal immunization of SL5928(fliC/esat) produced efficient CTL effects, as demonstrated using a 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. Thus, our study revealed that Salmonella flagellin acts as a carrier for foreign antigen and triggers strong Th1 and CTL responses during intranasal immunization. Chimeric flagellin is potentially an effective strategy for the development of novel vaccines against tuberculosis in humans and animals.

Figure 1

Diagram showing construction of the chimeric fliC/esat fragment.

Figure 2

Western blot analysis. Flagellin of SL5928(fliC) and chimeric flagellin of SL5928(fliC/esat) were extracted by acid treatment followed by ammonium sulfate precipitation. (a) The crude extract of fliC protein and chimeric flagellin fliC/esat were subjected to SDS–PAGE (M, standard molecular weight markers in kDa). Lane 1, crude extract of flagellin of SL5928(fliC); lane 2, crude extract of chimeric flagellin of SL5928(fliC/esat). (b) The crude extract of fliC protein and chimeric flagellin fliC/esat were transferred onto nitrocellulose and treated with anti-ESAT-6 antibodies (mAb HYB 076–08). A prominent 52 kDa band was observed (lane 4). Lane 3, crude extract of flagellin of SL5928(fliC); lane 4, crude extract of chimeric flagellin of SL5928(fliC/esat). mAb, monoclonal antibody; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Figure 3

Observed motility phenotypes for the transduced cells: (a) SL5928(fliC); (b) SL5928(fliC/esat); (c) SL5928.

Figure 4

Electron microscopy of transduced cells: (a) SL5928(fliC/esat); (b) SL1438.

Figure 5

Cytokine secretion of flagellated Salmonella SL5928(fliC/esat) through intranasal immunization. C57BL/6 mice were immunized intranasally with SL5928(fliC/esat), SL5928(fliC) or PBS. IFN-γ-producing and IL-4-producing cells in the lung, PPs, NALTs, spleen and MLNs were evaluated by ELISPOT assay with ESAT-6 peptide as the stimulus. Results were expressed as the mean number of spots per 10⁶ cells (six mice per group). **P<0.01 for the SL5928(fliC)-immunized and PBS-immunized mice using a Student's t-test. IFN, interferon; MLN, mesenteric lymph node; NALT, nasopharynx-associated lymph node; PBS, phosphate-buffered saline; PP, Peyer's patch.

Figure 6

Characterization of ESAT-6-specific CTL activity in vivo in immunized SL5928(fliC/esat) mice. The histogram shows CFSE^low untreated and CFSE^high ESAT-6 peptide-pulsed target cells in the spleens of SL5928(fliC) and SL5928(fliC/esat) mice infected 15 h post-transfer, respectively. Results were representative of three experiments, each with five mice per group. CFSE, carboxyfluorescein diacetate succinimidyl ester; CTL, cytotoxic T lymphocyte.