Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60.

We studied the immune responses of guinea pigs and humans to two Legionella pneumophila antigens. Guinea pigs surviving a lethal intraperitoneal challenge dose of virulent L. pneumophila exhibited strong cutaneous delayed-type hypersensitivity (DTH) reactions to purified OmpS (28-kDa major outer membrane protein) and Hsp60 (heat shock protein or common antigen), while weak DTH reactions were noted for extracellular protease (major secretory protein [MSP] [ProA]) and no reaction was observed with an ovalbumin (OA) control. Lymphocyte proliferation responses (LPRs) were measured for peripheral blood and spleen lymphocytes from guinea pigs surviving sublethal and lethal challenge doses of L. pneumophila. Lymphocytes from uninfected animals showed no proliferation to Hsp60 or OmpS, while lymphocytes from sublethally and lethally challenged animals exhibited strong proliferative responses to Hsp60 and OmpS. Guinea pigs vaccinated with purified OmpS exhibited low antibody titers and strong DTH and LPRs to OmpS, whereas lymphocytes from animals vaccinated with Hsp60 exhibited weak DTH responses and high antibody titers to Hsp60. All guinea pigs immunized with OmpS survived experimental challenge with L. pneumophila (two of two in a pilot study and seven of seven in trial 2) versus zero of seven OA-immunized controls (P = 0.006 by Fisher's exact test). In three vaccine trials in which animals were vaccinated with Hsp60, only 1 guinea pig of 15 survived lethal challenge. Peripheral blood lymphocytes (PBLs) from humans with legionellosis showed stronger LPRs to OmpS than PBLs from humans with no history of legionellosis (P = 0.0002 by Mann-Whitney test). PBLs of humans surviving legionellosis exhibited a lower but highly significant proliferative response to Hsp60 (P < 0.0001 compared with controls by Mann-Whitney test). These studies indicate that OmpS and Hsp60 are important antigens associated with the development of protective cellular immunity. However, as determined in vaccine trial studies in the guinea pig model for legionellosis, the species-specific antigen OmpS proved much more effective than the genus-common Hsp60 antigen.

Azithromycin pharmacokinetics and intracellular concentrations in Legionella pneumophila-infected and uninfected guinea pigs and their alveolar macrophages.

Azithromycin pharmacokinetics in Legionella pneumophila-infected and uninfected guinea pigs were assessed by measuring the drug concentration in whole lungs or the drug content in bronchoalveolar lavage (BAL) fluid in separate experiments. Azithromycin concentrations were measured by using a bioassay. The mean azithromycin content in the BAL fluid of infected guinea pigs was higher than that in controls at 10 h (0.87 versus 0.39 microgram; P = 0.05), 24 h (1.10 versus 0.37 microgram; P = 0.003), and 48 h (1.21 versus 0.28 microgram; P = 0.05) after a single intraperitoneal injection of drug (15 mg/kg). The mean peak lung azithromycin concentration was higher in control animals than in infected animals (15.8 versus 13.4 micrograms/ml). The mean lung azithromycin concentration in infected animals was significantly higher than that in controls 48 h after dosing (12.7 versus 10.4 micrograms/g; P = 0.04). There were no significant differences between infected and uninfected animals in serum azithromycin levels. Complementary experiments assessed intracellular/extracellular concentration ratios of azithromycin and erythromycin in L. pneumophila-infected and control guinea pig alveolar macrophages. Azithromycin was highly concentrated in alveolar macrophages, and the intracellular/extracellular concentration ratios for infected cells were significantly higher (P < 0.0001) than those observed in controls after 4 h (127 versus 119), 24 h (481 versus 361), and 48 h (582 versus 520) of incubation. Erythromycin was also preferentially concentrated in infected cells (P < 0.0001). AZ intracellular concentrations were at least fivefold higher than those measured for erythromycin, and this differential increased with incubation time. Thus, azithromycin recovery from BAL fluid, and from guinea pig lungs at the 48-h time point, was higher in the presence of experimental Legionnaires' disease. This likely results from recruitment of phagocytes, including macrophages, that have an enhanced capacity to highly concentrate the drug.