Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis

Abstract

A recombinant Lactobacillus casei expressing a flagellar antigen from Salmonella enterica serovar Enteritidis was constructed and evaluated as a mucosal vaccine. Intragastric immunization of the recombinant strain conferred protective immunity against Salmonella infection in mice. This immunization did not result in antigen-specific antibody in either feces or sera but induced the release of IFN-gamma on restimulation of primed lymphocytes ex vivo. The results suggested that the protective efficacy provided by flagellin-expressing L. casei is mainly attributable to cell-mediated immune responses. In addition, an adjuvant-type effect of the antigen delivery system with L. casei was also observed.

Fig. 1

Detection of FliC production and surface-display by L. casei transformants. (a) Immunoblotting of total cell extract (CE) and culture supernatant (Sup) of L. casei carrying pLP401∷FliC (LCF) and pLPEmpty (LCN). An amount equivalent to 5 × 10⁷ cfu of bacteria was loaded into each lane, and 50, 25, 12.5, and 6.25 ng of purified FliC were included as a standard. The sizes of the molecular mass markers are shown in the left margin. (b) Flow cytometric analysis of the L. casei transformants. Ten thousand particles were analyzed and fluorescence levels (FL1-H) from bacterial cells are shown in histogram form.

Fig. 2

Protective efficacy conferred by i.g. immunization of FliC-expressing L. casei. The level of protection was compared to that provided by immunization with the same amount (10¹⁰ cfu) of LCN, 50 μg of purified FliC, and PBS. A protection index was calculated as (mean value of log 10 SE cfu from non-immunized (PBS) group) − (log₁₀ SE cfu in each individual). Bars represent the mean value of each group (n = 7). ^*P < 0.05 (vs. LCN or PBS).

Fig. 3

Detection of FliC-specific serum IgG by ELISA (n = 6). Antibody titers are given as − log₂ (dilution × 10) + standard deviation (S.D.). Clear signals from the non-immunized group were not detected (ND). The types of immunization are shown in the bottom margin. 10¹⁰:10¹⁰ cfu, 10⁹:10⁹ cfu, 50:50 μg, 5:5 μg, 0.5:0.5 μg.

Fig. 4

(a) Amount of IFN-γ released from primed cells by ex vivo FliC-restimulation (n = 3). Spleen cells from immunized mice were incubated with or without FliC and the amount of IFN-γ released was measured by ELISA. Values are given as means + S.D. (b) Intracellular cytokine (IFN-γ) staining of primed cells (n = 3). Ten thousand events were acquired and the percentage of CD4⁺ cells containing IFN-γ was analyzed. The figures represent one mouse of each group, and the values are given as the mean ± S.D.