Immunologic response of patients with legionellosis against major protein-containing antigens of Legionella pneumophila serogroup 1 as shown by immunoblot analysis.

Major protein-containing antigens of Legionella pneumophila serogroup 1 were were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with rabbit antisera to 14 different Legionella species or serogroups. Fourteen bands were observed in immunoelectropherograms of whole-cell, sonicated cell, and heated cell preparations, seven of which appeared in the supernatant fluid from the heated cells and three of which were shown in an outer membrane fraction. Immunoblots of whole-cell antigen preparations of 14 Legionella species or serogroups revealed seven major Legionella proteins: antigens with molecular weights of 58,000, 79,000, and 154,000 were present in all Legionella sp. strains, antigens with molecular weights of 44,000 and 97,000 occurred in multiple species, and antigens with molecular weights of 14,000 and 25,000 were present only in L. pneumophila strains. All sera from 15 patients with culture-confirmed L. pneumophila serogroup 1 disease and 14 of 18 (78%) sera from serologically diagnosed patients reacted with the 58-kilodalton (kDa) common antigen. In contrast, less than one-half of the sera reacted with the L. pneumophila-specific proteins (14 and 25 kDa). Absorption of sera with Escherichia coli cells had no effect on their reactivity with the 58-kDa antigen, whereas absorption with L. pneumophila serogroup 1 cells removed reactivity. These data suggest that the 58-kDa antigen may prove useful in serodiagnostic tests for legionellosis.     

Seroepidemiological study of Legionella infection in Denmark. A 28-month retrospective survey

An indirect immunofluorescence (IF) test for Legionella antibodies has been used since 1978 at Statens Seruminstitut, Copenhagen. An increasing annual number of blood specimens from all parts of the country has been tested by IF and the number of Legionella antigens in the test was increased from 4 over 10 to 13, resulting in an ever growing number of seropositive patients over the years. We investigated the occurrence of serologically diagnosed Legionella infections from November 1982 through February 1985, a period of 28 months during which the same 13 Legionella antigens were applied in the IF test. We used CDC's criteria for the serological diagnosis of a current Legionella infection: a greater than or equal to 4-fold rise in antibody titre to greater than or equal to 128 in the IF test. In a test of more than 5,000 blood specimens from 3,374 patients, 69 were found to have diagnostic titre rises. When analysed according to serological reactions with three groups of antigens, seroconversion to a L. pneumophila antigen was found to be more frequent in patients 30-59 years old than seroconversion to a non-L. pneumophila Legionella antigen, while in the age group 60-69 this relation was reversed. Thirteen of the 69 patients had acquired their infection abroad. Twelve of these were below the age of 60, and they had all seroconverted to a L. pneumophila antigen. Clinical data were in accordance with the assumption that Legionella may have been the aetiological agent of the disease in our patients selected by serological criteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunologic diversity among serogroup 1 Legionella pneumophila urinary antigens demonstrated by monoclonal antibody enzyme-linked immunosorbent assays.

We tested urine specimens from 222 patients with serogroup 1 Legionella pneumophila pneumonia in two enzyme-linked immunosorbent assays (ELISAs) which used different monoclonal antibodies (A and B) as detector antibodies. Of 171 specimens which contained enough antigen to be detected in the ELISAs, 169 reacted in only one of the two assays. A total of 25 patients whose infections were acquired in any of three Indianapolis hospitals excreted antigen reactive with monoclonal antibody B, but 18 patients who were treated for infections acquired elsewhere reacted with monoclonal antibody A. The urinary antigen ELISA reactivity patterns correlated with the reactivity patterns of L. pneumophila isolates when a separate panel of seven monoclonal antibodies was used. The isolate patterns, in turn, correlated well with environmental isolate patterns from two of the hospitals with nosocomial cases. We conclude that at least two different epitopes exist on the antigen molecules in urine from patients with serogroup 1 L. pneumophila pneumonia and that the subtyping of urinary antigens can be useful epidemiologically.     

Agglutinating antibody titers to members of the family Legionellaceae in cystic fibrosis patients as a result of cross-reacting antibodies to Pseudomonas aeruginosa.

The objective of this study was to evaluate the prevalence and significance of antibody titers to organisms in the family Legionellaceae in 128 serum samples collected from cystic fibrosis patients at routine examinations. Antibody titers were determined for 10 antigenic types of Legionellaceae; Legionella pneumophila serogroups 1 to 6, Fluoribacter (Legionella) bozemanae, Fluoribacter (Legionella) dumoffii, Fluoribacter (Legionella) gormanii, and Tatlockia (Legionella) micdadei. The method of antibody titer determination was the microagglutination test. Elevated titers (greater than or equal to 1:64) to one or more antigens were found in 41.3% of cystic fibrosis patients but in only 9.7% of 103 normal control subjects (P less than 0.01). Titers to 8 of the 10 antigens were directly correlated with the number of Pseudomonas aeruginosa precipitating antibodies in patient sera, as determined by crossed immunoelectrophoresis (correlation coefficients, greater than or equal to 0.74). Cross-reactions between P. aeruginosa and L. pneumophila were substantiated by crossed immunoelectrophoresis of hyperimmune rabbit serum as well as patient sera against P. aeruginosa and Legionellaceae antigens. Monospecific antibody to the "common antigen" of P. aeruginosa was used to demonstrate the presence of this antigen in L. pneumophila. The presence of cross-reacting antibodies in cystic fibrosis patients chronically infected with P. aeruginosa emphasizes the need for cautious interpretation of antibody titers to members of the family Legionellaceae.     

Cross-reactions between Legionella pneumophila (serogroup 1) and twenty-eight other bacterial species, including other members of the family Legionellaceae.

Cross-reactions between Legionella pneumophila serogroup 1 and 28 other bacterial species were studied by various quantitative immunoelectrophoretic techniques. A sonicated L. pneumophila antigen and purified homologous rabbit antibody were used as a reference system. Few antigens (0 to 6) cross-reacted with non-Legionellaceae, but two were found in nearly all gram-negative bacteria tested (antigens no. 1 and 66). Antigen no. 66 of the L. pneumophila reference system was shown to be antigenically similar to the "common antigen" of Pseudomonas aeruginosa reported in many gram-negative bacteria. Greater than 85% of the antigens from L. pneumophila serogroup 1 cross-reacted with the other six serogroups of L. pneumophila. By contrast, Fluoribacter (Legionella) bozemanae, F. (L.) dumoffii, F. (L.) gormanii, and Tatlockia (Legionella) micdadei cross-reacted with only 45, 53, 39, and 43% of the reference system antigens, respectively. The antigenic relatedness of members of the Legionellaceae, expressed as a matching coefficient, is discussed in terms of its taxonomic significance. Serogroup-, genus-, and family-specific antigens are identified in the L. pneumophila reference system.     

Heterogeneity of antibody response in infection with Legionella pneumophila serogroup 1.

The antibody response of patients infected with Legionella pneumophila serogroup 1 in a common source outbreak was investigated. Heat-killed antigens from L pneumophila serogroups 1-3 and 6-10, plus several other strains of L pneumophila, together with 13 other species of legionellas were used in an indirect fluorescence antibody test. Formolised yolk sac antigens made from L pneumophila serogroups 1, 6, and 7 were also used. Although antibodies were produced to several L pneumophila serogroups or Legionella species by individuals, there was no constant pattern, suggesting that the response is a characteristic of the infected individual and not of the infecting strain of Legionella. There is evidence that heat-killed antigen made from L pneumophila serogroup 7 may give unreliable results.     

Characterization of a lipoprotein common to Legionella species as a urinary broad-spectrum antigen for diagnosis of Legionnaires' disease

We have previously identified the Legionella 19-kDa peptidoglycan-associated lipoprotein (PAL) as a species-common immunodominant antigen. We describe here for the first time the excretion and detection of the PAL antigen in infected urine specimens, which is useful for the diagnosis of Legionnaires' disease. Rabbit anti-PAL immunoglobulin G (IgG) antibody was produced by immunization with the purified, recombinant PAL of Legionella pneumophila serogroup 1 and used in the PAL antigen capture enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. A soluble-antigen capture ELISA using rabbit IgG antibodies against Legionella soluble antigens was prepared independently and used as a broad-spectrum standard test to detect soluble antigens of several Legionella species. Urine samples were obtained from guinea pigs experimentally infected with each of L. pneumophila serogroups 1, 3, and 6, and other Legionella species. The absorbance values of the PAL antigen ELISA highly correlated with those of the soluble-antigen ELISA in infected urine samples, with a correlation coefficient of 0.84 (P < 0.01). When applied to 17 infected urine samples and 67 negative controls from guinea pigs, the sensitivity and specificity of the PAL antigen capture ELISA were 88.2 and 95.5%, respectively. Compared to the commercial Biotest enzyme immunoassay, the PAL antigen ELISA was more efficient for detecting pneumophila non-serogroup 1 and nonpneumophila species. None of the 161 control human urine specimens obtained from healthy adults and patients with either non-Legionella pneumonia or urinary tract infections tested positive in the PAL antigen ELISA. The present study shows that the Legionella PAL is a very useful broad-spectrum antigen for urinary diagnostic testing. Moreover, since recombinant PAL antigen can be produced more efficiently than the soluble antigens, the development of a broad-spectrum diagnostic immunoassay based on the detection of the PAL antigen appears to be warranted.     

Cloning and expression of Legionella pneumophila antigens in Escherichia coli.

To isolate and characterize Legionella pneumophila antigens, we constructed a genomic library of L. pneumophila serogroup 1 (strain 130b). L, pneumophila DNA fragments (2.5 to 7.5 megadaltons) obtained by partial digestion with Sau 3A endonuclease and size fractionation on a sucrose density gradient were inserted into the dephosphorylated BamHI site of vector pBR322; CaCl2-treated Escherichia coli cells of strain HB101 were transformed with hybrid plasmids. To detect expression of antigens, 2,559 ampicillin-resistant transformants were transferred to nitrocellulose paper, lysed in situ, and screened by enzyme immunoassay (EIA) with E. coli-absorbed rabbit anti-L. pneumophila sera. A total of 77 (3%) of the colonies were reactive by EIA; 31 (1.2%) were strongly reactive, and 6 were strongly reactive by EIA without colony lysis. Analysis of 29 stable, strongly reactive clones by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting showed antigenic bands in 18 clones by EIA with E. coli-absorbed antisera. Absorption of antisera with heat- and Formalin-killed L. pneumophila antigen eliminated or diminished the reactivity of the antigenic bands in representative clones. These studies confirm that several L. pneumophila antigens can be cloned and expressed in E. coli.     

De novo synthesis of Legionella pneumophila antigens during intracellular growth in phagocytic cells.

Legionella pneumophilia is a gram-negative rod which is able to multiply within phagocytic cells. The process of phagocytosis leads to a rapid environmental change that might require a coordinate regulation of gene expression to ensure intracellular survival. Since there is little information on up- and downregulation of genes during the early phases of phagocytosis, we radiolabeled intracellular L. pneumophila at different times after phagocytosis by macrophages of the Mono Mac 6 cell line and immunoprecipitated antigens with antilegionella sera or monoclonal antibodies. We could identify two antigens which were upregulated, one of which was the Mip protein, three antigens which were downregulated, and three antigens which were not detectable in extracellularly grown L. pneumophila. The Mip protein was stained most intensively 4 to 8 h after intracellular infection, suggesting that it is needed during intracellular multiplication rather than initiation of infection. A 44-kDa antigen which was not detectable during extracellular growth was most prominent from 2 to 4 h postinfection when Mono Mac 6 cells were used as phagocytic cells. The 44-kDa antigen was also expressed during growth with Acanthamoeba castelanii, MRC-5, and U937 cells but with different kinetics. Synthesis of this antigen was not dependent on protein synthesis of the host cell. Since the 44-kDa antigen could be precipitated by an antiserum produced against a recombinant Escherichia coli harboring a plasmid with an L. pneumophila insert which also codes for the mip gene, we believe that the corresponding gene is within the vicinity of the mip gene. We named this protein legionella intracellular growth antigen (LIGA), since it could be found exclusively in intracellularly grown L. pneumophila.     

Detection of Legionella pneumophila antigens in patients' sera using monoclonal antibodies

Three monoclonal antibodies (McAb) were produced against soluble antigens of Legionella pneumophila serogroup 1 which was cultured on BCYE agar. The McAbs were all of the IgM isotype. The McAbs were used in the McAb-based ELISA for detection of circulating L. pneumophila antigens in 186 sera collected from patients with symptoms and signs suggestive of atypical pneumonia. The normal reference optical (OD) density value of each of the McAbs was determined using 44 sera collected from healthy blood donors. The antigen positivity rates for the McAbs 1C7.2B, 2B2.10F and 2B2.11E were 11.3%, 7.7% and 22.2% respectively. Antigen positivity of the McAb 2B2.10F was significantly higher in the younger age group (p < 0.05). There is no significant association between the antigen positivity with age and sex for all the McAbs. There was no cross-reaction demonstrated between the McAbs with other bacterial antigens.     

Agar microdroplet assay for delayed hypersensitivity to Legionella pneumophila serogroup 1.

An agarose microdroplet technique was utilized to assess the cellular immunity of guinea pig lymphoid cells to Legionella pneumophila antigen in vitro. Both direct and indirect migration inhibition procedures were shown to be capable of detecting sensitization of guinea pigs to L. pneumophila antigens. Animals injected with adjuvant alone or unrelated antigens did not yield spleen cells responsive to L. pneumophila, indicating the specificity of the response. Migration inhibition factor induction by Legionella antigen in vitro correlated well with skin test responses in vivo. The positive reaction detected by migration inhibition occurred at times similar to that of skin reactivity but later than that of the earliest serum antibody titers. The assay appears to be useful for monitoring sensitization to Legionella and may be applicable to the study of cell-mediated immunity to this bacterium in infected individuals.     

Comparison and evaluation of four commercial kits relative to an in-house immunofluorescence test for detection of antibodies against <Emphasis Type="Italic">Legionella pneumophila</Emphasis>

Four commercially available kits from (1) Focus Diagnostics, (2) SERION, (3) Zeus and (4) Vircell for detection of antibodies to Legionella pneumophila were evaluated with panels of sera from patients with proven Legionella infection (n = 81) and/or other bacterial infections (n = 75). An in-house indirect Legionella immunofluorescence antibody test (IF test) was used as reference. All sera from the laboratory-proven Legionella pneumophila cases [culture, urinary antigen test and/or polymerase chain reaction] of Legionella infection were found to be positive by the in-house IF test. The relative sensitivity for Focus Diagnostics, SERION, Zeus and Vircell kits was 81.5, 76.5, 68.8 and 62.5%, respectively, and the false-positive rate was 16.0, 5.6, 29.0 and 2.7%, respectively. The in-house IF test had a false-positive rate of 4.0%. It was found that none of the four commercial kits were as sensitive and specific as the in-house IF test.     

Detection of mip gene by PCR for diagnosis of Legionnaires' disease.

The mip gene of Legionella pneumophila was demonstrated by PCR and probing in paired acute-phase and convalescent-phase sera from five patients with Legionnaires' disease but not in the acute-phase sera of 100 patients with pneumonia that showed no serological evidence of Legionella infection. PCR may help in cases presenting diagnostic difficulty.     

Identification of a novel adhesion molecule involved in the virulence of Legionella pneumophila

Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045 and may play a role in Legionnaires' disease in humans.     

The simultaneous direct detection of Mycoplasma pneumoniae and Legionella pneumophila antigens in sputum specimens by a monoclonal antibody immunoblot assay

An immunoblot (Western) assay was developed employing a species-specific monoclonal antibody to a 43 kDa Mycoplasma pneumoniae membrane polypeptide and a species-specific monoclonal antibody to 29 kDa Legionella pneumophila outer membrane protein. This assay could simultaneously detect these two different antigens directly in sputum. The 43 kDa M. pneumoniae antigen was detected by this assay in each of three M. pneumoniae culture-confirmed sputum specimens. In addition, the 29 kDa L. pneumophila antigen was detected in three of three L. pneumophila culture-confirmed sputum specimens. Neither of these two specific antigens were detected in induced sputum specimens from ten normal individuals.     

Crossed immunoelectrophoretic analysis of Legionella pneumophila serogroup 1 antigens.

By crossed immunoelectrophoresis, 85 different antigens were demonstrated in sonicated preparations of Legionella pneumophila serogroup 1 (Lp1). The precipitin patterns of 82 anodic-migrating antigens were numbered and were designated the Lp1 reference system. Eleven antigens were stable to boiling, and seven of these were shown to be surface antigens. One heat-stable surface antigen (antigen no. 61) was highly reactive with limulus amoebocyte lysates and formed a precipitin resembling lipopolysaccharide. Serum from an isolation confirmed case of Lp1 infection and serogroup-specific rabbit antiserum reacted specifically with antigen no. 61, which was designated the serogroup-specific antigen. Normal human and rabbit sera commonly had antibodies to antigen no. 66 of the Lp1 reference system. This antigen is antigenically related to the "common antigen" of Pseudomonas aeruginosa.     

Broad-spectrum enzyme-linked immunosorbent assay for detection of Legionella soluble antigens.

An enzyme-linked immunosorbent assay (ELISA) was developed which detected soluble antigens from culture extracts of Legionella pneumophila serogroups 1 to 8, L. micdadei, L. bozemanii serogroups 1 and 2, L. dumoffii, L. gormanii, L. longbeachae serogroups 1 and 2, L. wadsworthii, L. oakridgensis, L. anisa, L. feeleii serogroup 1, and L. jordanis. The assay was approximately 10-fold more sensitive for the eight L. pneumophila serogroups than for the other Legionella species tested. The ELISA detected Legionella antigens in the urine specimens of 25 of 35 patients with L. pneumophila serogroup 1, 3, 4, 6, and 8; L. micdadei; and L. longbeachae serogroup 1 infections. None of the 334 urine specimens from patients with either non-Legionella pneumonia or urinary tract infections was positive. For 10 patients from whom sequential urine specimens were available, Legionella antigens were not detectable from 7 to 19 days after laboratory diagnosis. Test sensitivity was not affected by heavy bacterial contamination. This ELISA offers the detection of a broad spectrum of Legionella antigens by a single test.     

Processing and major histocompatibility complex class II presentation of Legionella pneumophila antigens by infected macrophages

To better understand interactions between the intracellular pathogen Legionella pneumophila and macrophages (Mphis), host and bacterial determinants important for presentation of antigens on major histocompatibility complex class II molecules (MHC-II) were investigated. It was determined that immune CD4 T-cell responses to murine bone marrow-derived Mphis (BMphis) infected with wild-type L. pneumophila were higher than the responses to avirulent dotA mutant bacteria. Although this enhanced response by immune T cells required modulation of vacuole transport mediated by the Dot/Icm system, it did not require intracellular replication of L. pneumophila. Intracellular cytokine staining identified a population of immune CD4 T cells that produced gamma interferon upon incubation with BMphis infected with wild-type L. pneumophila that did not respond to Mphi infection with dotA mutant bacteria. Endocytic processing was required for presentation of L. pneumophila antigens on MHC-II as determined by a defect in CD4 T-cell responses when the pH of BMphi endosomes was neutralized with chloroquine. Investigation of MHC-II presentation of antigens by BMphis infected with L. pneumophila icmR, icmW, and icmS mutants indicated that these mutants have an intermediate presentation phenotype relative to those of wild-type and dotA mutant bacteria. In addition, it was found that antigens from dot and icm mutants are presented earlier than antigens from wild-type L. pneumophila. Although immune CD4 T-cell responses to proteins secreted by the L. pneumophila Lsp system were not detected, it was found that the Lsp system is important for priming L. pneumophila-specific T cells in vivo. These data indicate that optimal antigen processing and MHC-II presentation to immune CD4 T cells involves synthesis of L. pneumophila proteins in an endoplasmic reticulum-derived compartment followed by transport to lysosomes.     

Coinfections of Mycoplasma pneumoniae and Legionella pneumophila with influenza A virus.

Serum samples from patients with documented influenza A virus infections were examined for antibodies to Legionella pneumophila and Mycoplasma pneumoniae to determine whether simultaneous or sequential infections with L. pneumophila and M. pneumoniae were complicating factors in influenza. When the frequency of copositivity of sera to influenza A virus and L. pneumophila was compared with the expected frequency for each infection alone, the difference was not statistically significant. However, when the frequency of copositivity of sera to influenza A virus and M. pneumoniae was compared with the expected frequency for each infection alone, there was a statistically significant (P less than 0.005) absence of coincident titers. Seasonal variations and differences in relative age frequencies for the two infections may partially explain the absence of coinfections. These data also suggest that in patients with either M. pneumoniae or influenza A virus infection, some type of protective mechanism which prevents coinfections with these organisms is present.     

Characteristics of anti-Legionella antibodies in patients infected with Legionella pneumophila serogroups 1, 6, and 10.

Nosocomial infections with Legionella pneumophila serogroups 1 and 10 in the Leiden University Hospital and infections with L. pneumophila serogroup 6 in neighboring hospitals gave us an opportunity to study the development of opsonizing antibodies against L. pneumophila serogroups 1, 6, and 10 in the serum of 13 patients. Seven of these patients were infected with L. pneumophila serogroup 1, two were infected with serogroup 6, and four were infected with serogroup 10. The opsonic cross-reactivity of antibodies against these serogroups of L. pneumophila and complement involvement in opsonization were also investigated. Convalescent-phase sera from patients infected with L. pneumophila serogroup 1 or 6 were able to promote ingestion of these serogroups by polymorphonuclear leukocytes, whereas ingestion of L. pneumophila serogroup 10 was enhanced only in the presence of convalescent-phase sera from patients infected with this serogroup. Opsonization of L. pneumophila serogroups 1, 6, and 10 was complement dependent.