A trivalent vaccine consisting of "flagellin A+B and pilin" protects against Pseudomonas aeruginosa infection in a murine burn model.

Pseudomonas aeruginosa is a common nosocomial pathogen in burn patients, and rapidly achieves antibiotic resistance, and thus, developing an effective vaccine is critically important for combating P. aeruginosa infection. Flagella and pili play important roles in colonization of P. aeruginosa at the burn wound site and its subsequent dissemination to deeper tissue and organs. In the present study, we evaluated protective efficacy of a trivalent vaccine containing flagellins A and B (FlaA + FlaB) + pilin (PilA) in a murine burn model of infection. "FlaA + FlaB + PilA" induced greater protection in P. aeruginosa murine burn model than the single components alone, and it showed broad immune protection against P. aeruginosa strains. Immunization with "FlaA + FlaB + PilA" induced strong opsonophagocytic antibodies and resulted in reduced bacterial loads, systemic IL-12/IL-10 cytokine expression, and increased survival after challenge with three times lethal dose fifty (LD50) of P. eruginosa strains. Moreover, the protective efficacy of "FlaA + FlaB + PilA" vaccination was largely attributed to specific antibodies. Taken together, these data further confirm that the protective effects of "FlaA + FlaB + PilA" vaccine significantly enhance efficacy compared with antibodies against either mono or divalent antigen, and that the former broadens the coverage against P. eruginosa strains that express two of the three antigens.

Flagellin and pilin immunization against multi-drug resistant Pseudomonas aeruginosa protects mice in the burn wound sepsis model.

Pseudomonas aeruginosa is a formidable pathogen and a major threat to burn patients. Antimicrobial therapy is often unsuccessful because P. aeruginosa can develop multi-drug resistance; thus, immunotherapy and vaccine can be a rational alternative. Flagella and type IV pili have been identified as important virulence factors in the colonization and pathogenesis of P. aeruginosa in burn wound infections. Immunogenicity and efficacy of mixed recombinant full-length type b flagellin (r-b-flagellin) and recombinant PilA (r-PilA) as candidate vaccines were assessed by measuring humoral and cellular responses, using an experimental burned mouse model. Primary immunization with "r-b-flagellin+r-PilA" followed by two booster shots was sufficient to generate a robust humoral response, which was predominantly a Th2 response consisting mainly of subtype IgG1 and low levels of IgG2a. Analysis of the cytokine response among immunized mice showed an increased production of IL-4, INF-γ and IL-17 by splenocytes upon stimulation by "r-b-flagellin+r-PilA". Opsonophagocytosis assays confirmed the enhanced killing of bacteria by anti "r-b-flagellin+r-PilA" immune sera. These antibodies were also able to reduce bacterial load in the site of original infection into the liver and spleen of challenged mice. The reduction of systemic bacterial spread resulted in an increased survival rate of challenged immunized mice. In conclusion, immunization with "r-b-flagellin+r-PilA" proteins provides a better protective response against P. aeruginosa infection in the burn mouse model.

Immunogenicity and protective efficacy of Pseudomonas aeruginosa type a and b flagellin vaccines in a burned mouse model.

Immunogenicity and efficacy of Pseudomonas aeruginosa type a and b flagellins (hereafter, flagellins) as candidate vaccines were evaluated using an experimental burned mouse model. The protection afforded and the reduction in bacterial burden achieved by these vaccine candidates were determined. Primary immunization with flagellins followed by two booster shots generated a robust immune response. Cytokine analysis demonstrated the secretion of interleukin-4 more than interferon-γ from immunized T-cells in response to in vitro antigen stimulation. IgG response was of Th2 type, predominantly with IgG1 and lower IgG2a levels before and after challenge. In vitro opsonophagocytosis assays confirmed protective potential of immune sera via enhanced bacterial cell killing. Immune sera also inhibited P. aeruginosa motility. Serum cytokine analysis demonstrated high IL-12 and low IL-10 levels in flagellin-immunized mouse sera. Reduced systemic bacterial spread from original infection site into liver and spleen was associated with increased survival. Immunization of mice with flagellins increased the humoral immune response and protection against P. aeruginosa infection in our mouse model.

Active Immunization with Recombinant PilA protein Protects Against Pseudomonas aeruginosa Infection in a Mouse Burn Wound Model.

Pseudomonas aeruginosa as an opportunistic human pathogen that causes lethal infections in immunocompromised patients. Type IV pili are critical factors in virulence and colonization of P. aeruginosa in acute burn wound infection. The immunogenicity and efficacy of P. aeruginosa recombinant PilA (r-PilA) was evaluated in an experimental model of burn wound sepsis as a vaccine candidate. In this study, female C57BL/6 mice were divided into five groups. Mice in the experimental groups received either r-PilA vaccine alone or in combination with the alum adjuvant or complete Freund's adjuvant (CFA). Mice in the negative control group received phosphate-buffered saline (PBS). In order to characterize the response of Th1-Th2 to immunization, the cytokine profiles of spleen cells isolated from r-PilA immunized mice were investigated. Total IgG titers and isotopes were measured using ELISA method and finally, in order to study the systemic infection, bacterial titers in the liver, spleen and blood were also determined. Active immunization with r-PilA, which is followed by two booster shots, was sufficient to generate a robust immune response in mice. Cytokine analysis demonstrated the secretion of IL-4 and INF-É£ from splenocytes in response to in vitro antigen stimulation. The IgG response to r-PilA was a Th2 type response consis¬¬ting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. In conclusion, in this burned mouse model, vaccination with r-PilA can increase the humoral immunity, thereby leading to an effective protection against P. aeruginosa infection.