Flagellin fusion proteins as adjuvants or vaccines induce specific immune responses

Abstract

Vaccination is the most efficient prophylaxis against a variety of infectious diseases. New vaccination strategies rely on the incorporation of effective adjuvants, which stimulate the innate immune response and, in turn, activate the adaptive immune response. It is well established that flagellin induces inflammatory responses through the activation of antigen-presenting cells (APCs). In order to evaluate whether flagellin can serve as a carrier for the development of adjuvants or vaccines, we prepared a flagellin-enhanced green fluorescent protein (EGFP) fusion protein. Our results demonstrate that a flagellin-EGFP fusion protein is capable of stimulating APCs, resulting in the maturation of these cells and secretion of proinflammatory cytokines. Furthermore, APCs pulsed with the flagellin-EGFP fusion protein effectively process and present EGFP antigens. More importantly, animals immunized with the flagellin-EGFP fusion protein developed specific anti-EGFP T-cell responses. In contrast, recombinant EGFP was not able to stimulate APCs, nor did it induce a T-cell response. Thus, recombinant-flagellin fusion proteins may be suitable carriers as adjuvants or vaccines for the development of new vaccination strategies to induce and boost immune responses against infectious diseases and cancer.

FIG. 1.

Schematic representation of EGFP and flagellin-EGFP fusion protein. EGFP and flagellin-EGFP were synthesized as described in Materials and Methods. (A) Diagram of EGFP and flagellin-EGFP proteins. a.a., amino acids. (B) An aliquot of each protein was loaded onto a 10% polyacrylamide gel and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular weight markers (M.W.M) are indicated in thousands.

FIG. 2.

APCs are bound by flagellin-EGFP but not by EGFP. Bone marrow-derived APCs were incubated for 1 h with flagellin-EGFP or EGFP (thick lines). Thin lines represent APCs left untreated, as controls. Cells were analyzed by flow cytometry for fluorescence in the FLH-1 channel. Numbers within the plots are the percentage of APCs that showed increased fluorescence. Experiments were repeated three times with similar results. One representative experiment is shown.

FIG. 3.

Maturation of APCs is induced by LPS and by flagellin-EGFP, but not by EGFP. Freshly isolated APCs were incubated for 24 h with LPS (10 μg/ml), flagellin-EGFP, or EGFP (each at 10 μg/ml). APCs were stained to detect B7.1 (thick line) and class II MHC (broken line) molecules, or were left unstained as control (solid line), and analyzed by flow cytometry. Experiments were repeated three times with similar results. One representative experiment is shown.

FIG. 4.

A flagellin-EGFP fusion protein induces a proinflammatory response. Cultured APCs were incubated with a 10-μg/ml concentration of LPS, flagellin-EGFP, or EGFP or were left unstimulated (control). After 24 h the cell supernatants were collected and TNF-α and NO secretion were measured. (A) TNF-α production in each culture was measured by ELISA. (B) The same supernatants were used to analyze NO production, as measured by the Griess reaction. Data are the means of three independent experiments (performed with duplicates) (error bars, standard deviations). A significant (P ≤ 0.001 [Student's t test]) difference was found between the control group and the flagellin-EGFP group.

FIG. 5.

APCs pulsed with flagellin-EGFP process and present EGFP. APCs cells were pulsed for 18 h with Ad-EGFP, flagellin-EGFP, or EGFP or were unpulsed. The cytotoxic activity of an anti-EGFP-specific CD8⁺-T-cell line was analyzed against the pulsed APCs in a 6-h ⁵¹Cr release assay at the indicated ratios of effector to target cells (E:T). Data are the means of two independent experiments (performed with triplicates) (error bars, standard deviations). A significant (P ≤ 0.001 [Student's t test]) difference was found between the control group and the flagellin-EGFP group.

FIG. 6.

Induction of specific CD8⁺-T-cell responses by flagellin-EGFP fusion protein. BALB/c mice were immunized with adenovirus-EGFP (A), flagellin-EGFP (B) and EGFP (C). After 2 weeks spleens were removed, and T cells were stimulated in vitro for 5 days. Cytotoxic activity was tested against BM-185-w.t, BM-185-w.t. pulsed with the H2-K^d-EGFP-peptide, and BM-185-EGFP cells in a 6-h ⁵¹Cr release assay at the indicated ratio of effector cells to target cells (E:T). Data represent the means of results for three individually analyzed mice per group in two independent experiments (error bars, standard deviations). A significant (P ≤ 0.001 [Student's t test]) difference was found between the control group and mice immunized with flagellin-EGFP.

FIG. 7.

Activation of specific CD4⁺ T cells by flagellin-EGFP fusion protein. BALB/c mice were immunized with flagellin-EGFP or EGFP. After 2 weeks spleens were removed, and CD4⁺ T cells were enriched. CD4⁺ T cells were incubated for 4 days with APCs pulsed with adenovirus-EGFP or empty adenovirus. After 3 days of incubation, tritiated thymidine (1 μCi/well for the last 18 h) was added, and thymidine incorporation was measured. Data show the means of results for three individually analyzed mice per group in two independent experiments (error bars, standard deviations). A significant (P ≤ 0.001 [Student's t test]) difference was found between the control group and mice immunized with flagellin-EGFP.