Pseudomonas aeruginosa is a serious pathogen in hospitalized, immunocompromised, and cystic fibrosis (CF) patients. P. aeruginosa is motile via a single polar flagellum made of polymerized flagellin proteins differentiated into two major serotypes: a and b. Antibodies to flagella delay onset of infection in CF patients, but whether immunity to polymeric flagella and that to monomeric flagellin are comparable has not been addressed, nor has the question of whether such antibodies might negatively impact Toll-like receptor 5 (TLR5) activation, an important component of innate immunity to P. aeruginosa. We compared immunization with flagella and that with flagellin for in vitro effects on motility, opsonic killing, and protective efficacy using a mouse pneumonia model. Antibodies to flagella were superior to antibodies to flagellin at inhibiting motility, promoting opsonic killing, and mediating protection against P. aeruginosa pneumonia in mice. Protection against the flagellar type strains PAK and PA01 was maximal, but it was only marginal against motile clinical isolates from flagellum-immunized CF patients who nonetheless became colonized with P. aeruginosa. Purified flagellin was a more potent activator of TLR5 than were flagella and also elicited higher TLR5-neutralizing antibodies than did immunization with flagella. Antibody to type a but not type b flagella or flagellin inhibited TLR5 activation by whole bacterial cells. Overall, intact flagella appear to be superior for generating immunity to P. aeruginosa, and flagellin monomers might induce antibodies capable of neutralizing innate immunity due to TLR5 activation, but solid immunity to P. aeruginosa based on flagellar antigens may require additional components beyond type a and type b proteins from prototype strains.Pseudomonas aeruginosa is an opportunistic pathogen responsible for a large proportion of ventilator-associated, hospital acquired pneumonia and is also a major cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa is motile via a single polar flagellum that has the added structural feature of being glycosylated (39). Flagellin is the primary protein component of the flagellar filament, and it can be classified into two serotypes, types a and b. Flagella carry out many functions, such as motility and attachment of bacteria to host cells, and can also elicit the activation of the host inflammatory response via Toll-like receptor 5 (TLR5) (6,15,29,31). Importantly, promising results in terms of prevention of the acquisition of P. aeruginosa infection in CF patients immunized with a bivalent type a and b flagellum vaccine have been published (12).Several animal studies have not only demonstrated the importance of flagella as a virulence factor in P. aeruginosa but also validated them, or their flagellin component, as target antigens for vaccination. In the burned-mouse model of infection, chemically mutagenized or genetically produced flagellum-negative strains were less virulent than flagellum-positive strains (5, 26...

Defective expression or function of the cystic fibrosis transmembrane conductance regulator (CFTR) underlies the hypersusceptibility of cystic fibrosis (CF) patients to chronic airway infections, particularly with Pseudomonas aeruginosa. CFTR is involved in the specific recognition of P. aeruginosa, thereby contributing to effective innate immunity and proper hydration of the airway surface layer (ASL). In CF, the airway epithelium fails to initiate an appropriate innate immune response, allowing the microbe to bind to mucus plugs that are then not properly cleared because of the dehydrated ASL. Recent studies have identified numerous CFTR-dependent factors that are recruited to the epithelial plasma membrane in response to infection and that are needed for bacterial clearance, a process that is defective in CF patients hypersusceptible to infection with this organism. Pseudomonas aeruginosa in cystic fibrosisCystic fibrosis (CF) is unique among human genetic disorders in that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a protein whose primary function described to date has been to regulate conductance of ions in and out of cells and intracellular vacuoles, lead to hypersusceptibility to chronic lung infection. Human diseases wherein individuals homozygous for mutant genes are predisposed to infections serve as a firm foundation for understanding the molecular, cellular, physiologic and immunologic aspects of normal and compromised resistance to infection. CF is not just a prime example of how we can learn about normal and pathologic states; it could, in fact, be the only known human genetically determined disease wherein infection with a single pathogen -Pseudomonas aeruginosa -drives the vast majority of morbidity, clinical deterioration and ultimately life-shortening mortality encountered in this disease. In humans with significant compromises to their immune system, such as advanced AIDS, we would normally expect to see a plethora of pathogens take advantage of this debilitated state, but this is not the case in CF.CF is characterized by the emergence and persistence of (and, ultimately, the inability to clear) chronic infection with a variant of P. aeruginosa (mucoid P. aeruginosa) that overproduces a surface polysaccharide known as alginate. The organism undergoes other significant genetic adaptations and selections within the CF lung, and it is the emergence of mucoid P. aeruginosa that is the primary determinant of the clinical course of this disease [1,2]. In those individuals with either an uncommon but fortuitous protective immune response to specific P. aeruginosa antigens [3,4] Histopathologic basis for defining relevant interactions between P.aeruginosa and the CF airway epitheliumAlthough CF is a multisystem disease characterized by GI and nutritional abnormalities, salt loss syndromes and male urogenital abnormalities, the major clinical problem for CF patients is chronic sinopulmonary disease. Therefore, relying on observations made fr...

Vaccines that could effectively prevent Pseudomonas aeruginosa pulmonary infections in the settings of cystic fibrosis (CF) and nosocomial pneumonia could be exceedingly useful, but to date no effective immunotherapy targeting this pathogen has been successfully developed for routine use in humans. Evaluations using animals and limited human trials of vaccines and their associated immune effectors against different P. aeruginosa antigens have suggested that antibody to the conserved surface polysaccharide alginate, as well as the flagellar proteins, often give high levels of protection. However, alginate itself does not elicit protective antibody in humans, and flagellar vaccines containing the two predominant serotypes of this antigen may not provide sufficient coverage against variant flagellar types. To evaluate if combining these antigens in a conjugate vaccine would be potentially efficacious, we conjugated polymannuronic acid (PMA), containing the blocks of mannuronic acid conserved in all P. aeruginosa alginates, to type a flagellin (FLA) and evaluated immunogenicity, opsonic killing activity, and passive protective efficacy in mice. The PMA-FLA conjugate was highly immunogenic in mice and rabbits and elicited opsonic antibodies against mucoid but not nonmucoid P. aeruginosa, but nonetheless rabbit antibody to PMA-FLA showed evidence of protective efficacy against both types of this organism in a mouse lung infection model. Importantly, the PMA-FLA conjugate vaccine did not elicit antibodies that neutralized the Toll-like receptor 5 (TLR5)-activating activity of flagellin, an important part of innate immunity to flagellated microbial pathogens. Conjugation of PMA to FLA appears to be a promising path for developing a broadly protective vaccine against P. aeruginosa.