Identification of Protective B Cell Antigens of Legionella pneumophila

Abstract

Abs confer protection from secondary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires’ disease. In this study, we demonstrate that Ab-mediated protection is effective across L. pneumophila serogroups, suggesting that Abs specific for conserved protein Ags are sufficient to mediate this protective effect. We used two independent methods to identify immunogenic L. pneumophila protein Ags, namely, the screening of a λ phage library representing the complete L. pneumophila genome and two-dimensional gel electrophoresis combined with Western blot analysis and protein spot identification by mass spectrometry. A total of 30 novel L. pneumophila B cell Ags were identified, the majority of which are located in or associated with the bacterial membrane, where they are accessible for Abs and, therefore, likely to be relevant for Ab-mediated protection against L. pneumophila. Selected B cell Ags were recombinantly expressed and tested in a vaccination protocol. Mice immunized with either single-protein Ags or an Ag combination showed reduced bacterial titers in bronchoalveolar lavage and lung after L. pneumophila challenge. To determine the clinical relevance of these findings, we tested Legionnaires’ disease patient sera for reactivity with the identified L. pneumophila Ags. The recognized Ags were indeed conserved across host species, because Abs specific for all three selected Ags could be detected in patient sera, rendering the identified protein Ags potential vaccine candidates.

Dissecting the Contribution of IgG Subclasses in Restricting Airway Infection with Legionella pneumophila

“…The L. pneumophila strains used in this study were the laboratory wildtype strain JR32 (Philadelphia-1, serogroup [sg] 1) (12) and an aflagellated mutant of JR32 (DflaA) (5). Additionally, we used isolates of sg4, sg6, and sg10 (Swiss National Reference Center for Legionella, Bellinzona, Switzerland) (13) as well as wild-type Legionella strains Corby (sg1) (14) and AA100 (sg1) (15) and Escherichia coli and Salmonella enterica serovar Typhimurium to determine Ab specificities.…”

“…with 5 3 10 6 CFU L. pneumophila JR32 to harvest immune serum 4 wk after the last boost. Similarly, mice were vaccinated three times s.c. with a mixture of four recombinantly expressed B cell protein Ags (Lpg1362, Lpg2271, Lpg0688, and Lpg2025, 10 mg each, emulsified in IFA supplemented with 5 mg CpG) or with PBS (emulsified in IFA with CpG s.c.) (5). For passive immunizations, mice were injected i.v.…”

“…infection (5). To this end, we immunized mice s.c. with a pool of four recombinant B cell Ags (Lpg1362, Lpg2271, Lpg0688, and Lpg2025, emulsified in IFA supplemented with CpG) or infected mice with live L. pneumophila (i.v.)…”

“…Recently, we were able to identify protein B cell Ags of L. pneumophila that we successfully used to vaccinate mice (5). During these vaccination studies, we found that the vaccination regimen and adjuvants employed highly contributed to the efficacy of the induced L. pneumophila-specific Abs in restricting infection.…”

Two sequential layers of antibody‐mediated control of Legionella pneumophila infection

“…The Lpn strains used in this study were the laboratory wildtype strain JR32 (Philadelphia‐1, sg1) and an aflagellated mutant of JR32 (Δ flaA ) . Lpn was grown for 3 days at 37°C on charcoal yeast extract (CYE) agar plates before use.…”