Potent T-cell mediated immune response against Legionella pneumophila in mice following vaccination with detoxified lipopolysaccharide non-covalently combined with recombinant flagellin A and peptidoglycan-associated lipoprotein.

BACKGROUND: Legionella pneumophila is a Gram-negative intracellular bacterium and the cause of an atypical pneumonia in humans - legionnaire's disease. Immunological assessment of bacterial antigens clarifies the way that host may develop protection against the pathogen. Lipopolysaccharide (LPS) is the main antigen of Gram-negative bacteria but is less studied because of its carbohydrate nature. Here, we immunized mice with detoxified LPS in combination with immunogenic proteins and looked into the result of bacterial challenge. METHODS: LPS of L. pneumophila was extracted by hot phenol-water method. Purified LPS was detoxified by sodium hydroxide alkaline procedure. BALB/c mice were immunized mainly with non-covalent combination of detoxified LPS (dLPS) and either of recombinant FlaA or PAL separately. Afterwards, specific serum IgG was assessed by ELISA. Mice were challenged intravenously with sublethal dose of L. pneumpphila then splenocytes were cultured. Cytokine responses of splenocytes were analyzed by ELISA. RESULTS: Polysaccharide antigen did not elicit significant serum IgG. Combination of the dLPS with recombinant FlaA and PAL led to risen IgG and its subclasses (IgG1, IgG2a and IgG2b) against polysaccharide. Mice immunized with combination of the dLPS and recombinant proteins showed significant elevation of cytokine responses in splenocyte culture after being challenged with L. pneumophila. CONCLUSIONS: Our results suggest that combination of polysaccharide antigen derived from Legionella LPS may confer raised cell-mediated responses against the pathogen when combined with Th-1 stimulating protein antigens. Although not covalently bond, Legionella detoxified LPS combination with recombinant FlaA and PAL effectively elicited Th-1 type cytokines and humoral responses against L. pneumophila in BALB/c mice.

Investigating the role of L. pnuemophila LPS derivatives in formation of specific cell-mediated immune responses against the pathogen.

Legionella pneumophila is a Gram-negative intracellular bacterium and causes legionnaire's disease an -atypical pneumonia in humans. Lipopolysaccharide (LPS) is the main antigen of Gram-negative bacteria but is less studied because of its carbohydrate nature. Here, we immunized mice with detoxified LPS and O-antigen polysaccharide in combination with bovine serum albumin (BSA) and explored the immunological responses of mice to the bacterial infection. LPS of L. pneumophila was extracted by hot phenol-water method. Purified LPS was detoxified by sodium hydroxide alkaline procedure. O-polysaccharide antigen (OPS) obtained by acetic acid treatment of LPS. BALB/c mice were immunized mainly with non-covalent combination of detoxified LPS (dLPS) or OPS with BSA separately. Pure polysaccharide antigens did not elicit significant serum IgG against LPS. Combination of the dLPS and OPS with BSA resulted in risen IgG and its subclasses (IgG1 and IgG2a) against lipopolysaccharide. Mice were challenged intravenously with sublethal dose of L. pneumpphila. Then, splenocytes were cultured and cytokine responses of splenocytes to pathogenic Legionella was studied by ELISA. Mice immunized with combination of the dLPS or OPS and BSA showed significant elevation of cytokine responses to pathogenic L. pneumophila. Our results suggest that combination of the polysaccharide antigen derived from Legionella LPS may confer raised cell-mediated responses against the pathogen when combined with a protein antigen which is capable of eliciting cell-mediated responses. Although not covalently bond, Legionella polysaccharides combined with BSA effectively elicited Th-1 type cytokines and humoral responses against L. pneumophila in BALB/c mice.