The legionnaires' disease bacterium, LegioneUa pneumophila, is a facultative intracellular parasite. Its interaction with phagocytes has characteristics in common with several other intracellular parasites. Critical aspects of L. pneumophila intracellular multiplication are evasion of lysosomal host cell defenses and the presence of a nutritionally appropriate environment. Following phagocytosis, wild-type

We have examined whether a molecule that is capable of inducing immune protection, the major secretory protein (MSP) of Legionellapneumophila, is required for virulence in a guinea pig model of Legionnaires' disease. To do so, we have compared the virulence in guinea pigs of an isogenic pair of L. pneumophila, Philadelphia 1 strain, one of which produces MSP (MSP+) and one of which does not (MSP-).Both the MSP-strain and the MSP+ strain of L. pneumophila are highly virulent for guinea pigs, inducing similar signs and progression of illness. Both strains are lethal and have comparable LD50s and LD100s. Both strains multiply in the lungs of guinea pigs at a similar rate, and both strains produce indistinguishable pathological lesions in the lungs. Both strains maintain a stable phenotype with guinea pig passage, i.e., the MSP-strain does not regain the capacity to secrete MSP and the MSP+ strain retains its capacity to secrete MSP after lung passage.

We have examined the capacity of the major secretory protein (MSP) of Legionella pneumophila to induce humoral, cell-mediated, and protective immunity in a guinea pig model of Legionnaires' disease. MSP was purified to homogeneity by ammonium sulfate precipitation, molecular sieve chromatography, and ion-exchange chromatography. The purified MSP was nonlethal and nontoxic to guinea pigs upon subcutaneous administration. Guinea pigs immunized with a sublethal dose of aerosolized L. pneumophila or a subcutaneous dose of MSP developed a strong cell-mediated immune response to MSP. Such guinea pigs exhibited marked splenic lymphocyte proliferation and cutaneous delayed-type hypersensitivity to MSP in comparison with control animals. Guinea pigs immunized with MSP also developed a strong humoral immune response to MSP, as assayed by an ELISA. The median reciprocal antibody titer was 362 (range 45 to greater than 2,048) for immunized animals compared with less than 8 for controls. In contrast, guinea pigs immunized with a sublethal dose of L. pneumophila failed to develop anti-MSP antibody. Guinea pigs immunized with MSP and then challenged with a lethal aerosol dose of L. pneumophila exhibited highly significant protective immunity in each of five consecutive experiments. MSP induced protective immunity in dose-dependent fashion (40 greater than 10 greater than 2.5 greater than 0.6 micrograms MSP); vaccination with two doses of as little as 2.5 micrograms MSP induced significant protective immunity (p = 0.01, Fisher's Exact Test, two-tailed). Altogether, 21 (81%) of 26 animals immunized with 40 micrograms MSP survived challenge compared with 0 (0%) of 26 sham-immunized control animals (p = 7 x 10(-10), Fisher's Exact Test, two-tailed). MSP-immunized but not control guinea pigs were able to limit L. pneumophila multiplication in their lungs. This study demonstrates that (a) guinea pigs sublethally infected with L. pneumophila develop a strong cell-mediated immune response to MSP; (b) guinea pigs immunized with MSP develop a strong humoral and cell-mediated immune response to MSP; (c) guinea pigs immunized with MSP develop a very high level of protective immunity to lethal aerosol challenge with L. pneumophila; and (d) MSP-immunized animals are able to limit L. pneumophila multiplication in their lungs. MSP, an extracellular protein of an intracellular pathogen, has potential as a vaccine for the prevention of Legionnaires' disease. Secretory molecules of other intracellular pathogens may also have vaccine potential.

No abstract

IntroductionWe have examined the capacity of a live avirulent mutant form of the intracellular bacterial pathogen Legionella pneumophila to induce immune responses and protective immunity in guinea pigs. The mutant L. pneumophila is nonlethal to guinea pigs and does not revert to virulence with passage through guinea pigs. In contrast, exposure of guinea pigs to aerosols containing wild-type L. pneumophila induces a pneumonic illness that clinically and pathologically resembles Legionnaires' disease in humans. Guinea pigs immunized by aerosol exposure to mutant L. pneumophila developed a strong humoral immune response to wild-type L. pneumophila antigens with reciprocal antibody titers of 32-512 (median 256) by the indirect fluorescent antibody assay, compared with titers of < 2 for control (sham immunized) guinea pigs. Mutant immunized but not control guinea pigs also developed strong cell-mediated immune responses to wild-type L. pneumophila antigens, as demonstrated in assays of cutaneous delayed-type hypersensitivity and in vitro splenic lymphocyte proliferation.Mutant immunized guinea pigs developed strong protective immunity to lethal aerosol challenge with wild-type L. pneumophila. In four independent experiments 40-83% of mutant immunized guinea pigs survived compared with 0% of control guinea pigs. Overall, 13 of 21 (62%) mutant immunized guinea pigs survived compared with 0 of 21 (0%) control guinea pigs (P = 0.00002, Fisher's exact test, two-tailed). Mutant immunization induced protection comparable to wild-type immunization in these studies; 8 of 14 (57%) guinea pigs immunized by wild-type L. pneumophila survived.This study demonstrates that guinea pigs immunized with a live avirulent mutant L. pneumophila vaccine (a) develop a strong humoral immune response to wild-type L. pneumophila antigens; (b) develop a strong cell-mediated immune response to wild-type L. pneumophila antigens; and (c) develop protective immunity to lethal aerosol challenge with wild-type L. pneumophila. This study demonstrates the feasibility of a vaccine against Legionnaires' disease.