Previous studies from this laboratory have suggested a role for cell-mediated immunity in host defense against Legionella pneumophila. In this paper, cell-mediated immunity to L. pneumophila in patients recovered from Legionnaires' disease was studied by examining patient mononuclear cell responses to L. pneumophila antigens. Patient mononuclear cells were assayed both for their capacity to respond to L. pneumophila antigens with the production of cytokines that activate monocytes, as measured by monocyte inhibition of L. pneumophila multiplication, and for their capacity to respond with proliferation, as measured by [(3)H]thymidine incorporation. Patient mononuclear cells incubated with formalin-killed L. pneumophila generated cytokines (supernatants) that were capable of activating in vitro freshly explanted monocytes from a person without historical or serological evidence of L. pneumophila infection (nonpatient). Such activated nonpatient monocytes inhibited the intracellular multiplication of L. pneumophila, and the degree of inhibition was proportional to the concentration of supernatant added. Patient mononuclear cells incubated with 5 x 10(6) - 5 x 10(8) formalin-killed L. pneumophila/ml for 4 d produced maximally potent supernatants; supernatants generated in flat-bottomed wells were equivalent in potency to supernatants generated in cone-shaped wells. Patient L. pneumophila-induced mononuclear cell supernatants were less potent than patient concanavalin A-induced mononuclear cell supernatants. Patient mononuclear cells also responded to formalin-killed L. pneumophila with proliferation (lymphoproliferation). Patient mononuclear cells responded more strongly to L. pneumophila antigens than mononuclear cells of age- and sex-matched nonpatients, as measured by both assays; responses to concanavalin A were comparable. Mononuclear cells from patients recovered from Legionnaires' disease responded more strongly to L. pneumophila than to Mycobacterium leprae antigens, whereas mononuclear cells from patients with tuberculoid leprosy responded more strongly to M. leprae antigens. These findings indicate that cell-mediated immunity to L. pneumophila develops in patients with Legionnaires' disease and, taken together with previously reported findings, that cell-mediated immunity plays a major role in host defense against L. pneumophila. The monocyte activation assay described in this paper has general applicability for the study of monocyte and mononuclear cell effector functions in selected patients. The assay may be used to study (a) the capacity of a patient's monocytes to be activated to an antimicrobial potential by a standard preparation of cytokines and (b) the capacity of a patient's mononuclear cells to generate such monocyte-activating cytokines in response to a mitogen or antigen.

Phagocytosis of Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in human mononuclear phagocytes and causes Legionnaires' disease, occurs by a novel mechanism. A phagocyte pseudopod coils around the bacterium as the organism is internalized. Human monocytes, alveolar macrophages, and polymorphonuclear leukocytes all phagocytize L. pneumophila by this unusual process, termed "coiling phagocytosis," and these leukocytes phagocytize not only live L. pneumophila in this way, but also formalin-killed, glutaraldehyde-killed, and heat-killed L. pneumophila. In contrast, under the same experimental conditions, monocytes phagocytize Streptococcus pneumoniae, encapsulated and unencapsulated E. coli, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Neisseria gonorrhoeae, and Neisseria meningitidis by conventional phagocytosis. Treatment of L. pneumophila with high-titer anti-L. pneumophila antibody abolishes coiling phagocytosis; such bacteria are internalized by conventional phagocytosis. These experiments raise the possibility that a surface component of L. pneumophila mediates the unusual response by the phagocyte. Such a component, if elaborated in vivo, might be responsible for extrapulmonary manifestations of Legionnaires' disease suspected of being toxin-mediated.

Tuberculosis remains one of humankind's greatest killers, and new therapeutic strategies are needed to combat the causative agent, Mycobacterium tuberculosis, which is rapidly developing resistance to conventional antibiotics. Using the highly demanding guinea pig model of pulmonary tuberculosis, we have investigated the feasibility of inhibiting M. tuberculosis glutamine synthetase (GS), an enzyme that plays a key role in both nitrogen metabolism and cell wall biosynthesis, as a novel antibiotic strategy. In guinea pigs challenged by aerosol with the highly virulent Erdman strain of M. tuberculosis, the GS inhibitor L-methionine-SR-sulfoximine (MSO) protected the animals against weight loss, a hallmark of tuberculosis, and against the growth of M. tuberculosis in the lungs and spleen; MSO reduced the CFU of M. tuberculosis at 10 weeks after challenge by approximately 0.7 log unit compared with that in control animals. MSO acted synergistically with isoniazid in protecting animals against weight loss and bacterial growth, reducing the CFU in the lungs and spleen by approximately 1.5 log units below the level seen with isoniazid alone. In the presence of ascorbate, which allows treatment with a higher dose, MSO was highly efficacious, reducing the CFU in the lungs and spleen by 2.5 log units compared with that in control animals. This study demonstrates that inhibition of M. tuberculosis GS is a feasible therapeutic strategy against this pathogen and supports the concept that M. tuberculosis enzymes involved in cell wall biosynthesis, including major secretory proteins, have potential as antibiotic targets.

Francisella tularensis is the causative agent of tularemia and a Tier I bioterrorism agent. In the 1900s, several vaccines were developed against tularemia including the killed "Foshay" vaccine, subunit vaccines comprising F. tularensis protein(s) or lipoproteins(s) in an adjuvant formulation, and the F. tularensis Live Vaccine Strain (LVS); none were licensed in the U.S.A. or European Union. The LVS vaccine retains toxicity in humans and animals-especially mice-but has demonstrated efficacy in humans, and thus serves as the current gold standard for vaccine efficacy studies. The U.S.A. 2001 anthrax bioterrorism attack spawned renewed interest in vaccines against potential biowarfare agents including F. tularensis. Since live attenuated-but not killed or subunit-vaccines have shown promising efficacy and since vaccine efficacy against respiratory challenge with less virulent subspecies holarctica or F. novicida, or against non-respiratory challenge with virulent subsp. tularensis (Type A) does not reliably predict vaccine efficacy against respiratory challenge with virulent subsp. tularensis, the route of transmission and species of greatest concern in a bioterrorist attack, in this review, we focus on live attenuated tularemia vaccine candidates tested against respiratory challenge with virulent Type A strains, including homologous vaccines derived from mutants of subsp. holarctica, F. novicida, and subsp. tularensis, and heterologous vaccines developed using viral or bacterial vectors to express F. tularensis immunoprotective antigens. We compare the virulence and efficacy of these vaccine candidates with that of LVS and discuss factors that can significantly impact the development and evaluation of live attenuated tularemia vaccines. Several vaccines meet what we would consider the minimum criteria for vaccines to go forward into clinical development-safety greater than LVS and efficacy at least as great as LVS, and of these, several meet the higher standard of having efficacy ≥LVS in the demanding mouse model of tularemia. These latter include LVS with deletions in purMCD, sodBFt , capB or wzy; LVS ΔcapB that also overexpresses Type VI Secretion System (T6SS) proteins; FSC200 with a deletion in clpB; the single deletional purMCD mutant of F. tularensis SCHU S4, and a heterologous prime-boost vaccine comprising LVS ΔcapB and Listeria monocytogenes expressing T6SS proteins.