We have shown that dendritic cells (DCs) genetically engineered with a recombinant adenovirus vector (Ad) to express CD40 ligand (CD40L) elicit specific humoral immunity against the Pseudomonas aeruginosa laboratory strain PAO1, without CD4(+) T cell help. In the present study, using several different strains of P. aeruginosa, we examine whether this strategy is generally applicable to enhancing clinically relevant pathogen-specific immunity. Mice immunized with DCs modified with CD40L and pulsed with heat-killed P. aeruginosa clinical strain PA514, originally isolated from the sputum of an individual with cystic fibrosis, survived lethal respiratory challenge with PA514-impregnated agar beads. Consistent with this effective in vivo protection, the immunized mice generated high levels of serum isotype-switched antibodies directed against PA514 without concomitant nonspecific elevations of total serum immunoglobulin levels. The CD40L genetically engineered DCs pulsed with seven of eight different strains of P. aeruginosa afforded significant, albeit variable, cross-protection against lethal respiratory challenge with a clinical (PA514) or laboratory (PAO1) strain of P. aeruginosa. CD40L genetically modified DCs pulsed with a clinical (PA514) or laboratory (PAO1) strain of P. aeruginosa initiated cross-reacting antibody responses against each other, but not against Escherichia coli and vice versa. These observations may be useful in developing vaccines for infectious diseases, including P. aeruginosa infection.