CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,1071-412X/97/$04.0010

Nov. 1997, p. 700–704 Vol. 4, No. 6

Copyright © 1997, American Society for Microbiology

Reactions of Polyclonal and Neutralizing Anti-p54 MonoclonalAntibodies with an Isolated, Species-Specific 54-Kilodalton

Protein of Chlamydia pneumoniaeMARGIT WIEDMANN-AL-AHMAD,1 PETRA SCHUESSLER,2 AND HEIKE M. FREIDANK1*

Abteilung Mikrobiologie und Hygiene, Institut fur Medizinische Mikrobiologie und Hygiene, Klinikum derUniversitat Freiburg, 79008 Freiburg,1 and nanoTools, 79211 Denzlingen,2 Germany

Received 21 April 1997/Returned for modification 1 August 1997/Accepted 19 August 1997

A recently described 54-kDa protein has been detected in six type strains and three patient isolates ofChlamydia pneumoniae by immunoblotting with sera from patients positive for antibodies to C. pneumoniae bythe microimmunofluorescence test. This protein was not found in either C. trachomatis E or C. psittaci Z 432as an antigen, confirming its species specificity. The 54-kDa protein was isolated by continuous-elutionelectrophoresis and immunoglobulin G monoclonal antibodies (MAbs) against the isolated antigen wereproduced. MAb 8B11E6 reacted only with the 54-kDa band of C. pneumoniae and not with C. trachomatis E orC. psittaci in a Western immunoblot assay. This antibody was purified and tested for neutralizing activitytogether with three additional anti-p54-active MAbs (8B11E6, 8B11B4, and 10F1C1). In Buffalo green monkeycells, all of the MAbs significantly reduced the infectivity of C. pneumoniae elementary bodies, whereas noneutralizing activity could be observed with C. trachomatis E or C. psittaci Z 432. These results not only confirmthe species specificity of the 54-kDa protein but also indicate that this protein might play an important role inthe pathogenesis of C. pneumoniae infection. Furthermore, the results suggest a possible protective role ofanti-p54 antibodies in an adaptive immune response.

Chlamydia pneumoniae causes respiratory diseases like bron-chitis, pneumonia, sinusitis, and pharyngitis. Several chronicdiseases have also been shown to be associated with C. pneu-moniae infection. Thus, C. pneumoniae may be a factor in thedevelopment of asthma (7) and of reactive arthritis or Reiter’ssyndrome (1). Seroepidemiologic studies (18, 19) and morpho-logical and microbiological detection of C. pneumoniae in ath-eromatous plaques (11) suggest an association with coronaryartery disease and other atherosclerotic syndromes, althoughnegative results have also been reported (25).

C. pneumoniae has been classified according to DNA ho-mology, morphology, and biological characteristics. Addition-ally, recognition of antigenic structures by monoclonal anti-bodies (MAbs) is important for species differentiation (6, 17).However, attempts to identify the antigens recognized by theseantibodies by immunoblotting, immunoaffinity chromatogra-phy, or radioimmunoprecipitation were not successful (17).Immunoblot studies with patient sera identified several C. pneu-moniae species-specific proteins, including a 98-kDa protein(3), 43- and 46-kDa proteins (8), and proteins with a molecularmass range of 50 to 60 kDa (8, 9, 12, 16, 24). In our recentimmunoblot analysis of sera that had been previously exam-ined for antibodies to C. pneumoniae and C. trachomatis withthe microimmunofluorescence (MIF) test, a 54-kDa proteinwas recognized in C. pneumoniae TW-183 by 93% of sera con-taining anti-C. pneumoniae antibodies, whereas no such reac-tion was found with C. trachomatis antigen (4). Thus, the 54-kDa protein was proposed to be both species specific and amajor immunogen during infection.

In the present study, we examined six C. pneumoniae type

strains and three isolates from German patients by immuno-blotting with anti-C. pneumoniae antibody-positive patient serato confirm the species specificity of the 54-kDa protein. Wedescribe the isolation of the 54-kDa protein and report onimmunoblot analysis and neutralization assays with MAbsagainst this 54-kDa protein. So far, only one MAb against a76-kDa species-specific protein of C. pneumoniae has beendescribed; this antibody neutralized chlamydial infectivity incell culture and reacted in an immunoblot assay (15), but it isnot known whether this antigen plays a role in human C. pneu-moniae infections.

MATERIALS AND METHODS

Chlamydia strains and growth conditions. The following C. pneumoniae strainswere used in this study: C. pneumoniae AR-39 and TW-183 (Washington Re-search Foundation, Seattle); C. pneumoniae ATCC 1310, ATCC 1355, ATCC1356, and ATCC 1360 (American Type Culture Collection, Rockville, Md.);patient isolates FR-1, FR-2 (both isolated in our laboratory from patients withrespiratory tract infections), and HK (a patient isolate kindly provided by A.Groh and M. Hartmann, Institut fur Medizinische Mikrobiologie, Jena, Germa-ny). C. trachomatis E (American Type Culture Collection) and C. psittaci Z 432(kindly provided by C. Jantos, Institut fur Medizinische Mikrobiologie, Gießen,Germany) (20) were also used in this study.

All C. pneumoniae strains were grown in Buffalo green monkey (BGM) cellcultures for 72 h at 37°C in a 5% CO2 atmosphere, and the C. trachomatis andC. psittaci strains were grown for 48 h as previously described (5). The mediaused were: Eagle’s minimal essential medium (GIBCO) with 5% fetal calf serum(Vitromex) for the growth of the cell monolayers and Eagle’s minimal essentialmedium with antibiotics (vancomycin at 100 mg/ml, streptomycin at 50 mg/ml,nystatin at 50 IU/ml), cycloheximide at 1 mg/ml, and 10% fetal calf serum(CMGA medium) for propagation of chlamydiae after infection.

Purification of EB. The strains were harvested from BGM cell monolayersgrown in 600-ml polystyrene culture flasks (Greiner). In brief, the infected BGMcells (six flasks per strain) were detached with glass beads and the suspension wascentrifuged at 30,000 3 g for 30 min at 4°C. The pellet was resuspended inphosphate-buffered saline (PBS), and the cells were ruptured by sonication. Thissuspension was centrifuged at 500 3 g for 10 min at 4°C to remove the cell debris.The supernatant, containing the elementary bodies (EB), was pelleted over a35% gastrografin solution (Schering). The pellet was further purified by centrif-ugation over a discontinuous gastrografin gradient (3).

Staining of chlamydiae. The reticulate bodies and EB were stained, aftermethanol fixation (10 min), with a genus-specific, fluorescein-conjugated anti-

* Corresponding author. Mailing address: Abteilung Mikrobiologieund Hygiene, Institut fur Medizinische Mikrobiologie and Hygiene,Klinikum der Universitat Freiburg, Postfach 820, D-79008 Freiburg,Germany. Phone: 049 761 203 6540. Fax: 049 761 203 6562. E-mail:freidank@sun11.ukl.uni-freiburg.de.

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body (Pathfinder Chlamydia Culture Confirmation System; Kallestad Diagnos-tics, Chaska, Minn.) in accordance with the manufacturer’s instructions.

Separation of chlamydial proteins by SDS-PAGE and immunoblot analysis.The proteins of purified chlamydial EB were separated by discontinuous sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% acryl-amide by the method of Laemmli (13). Protein concentration was determined bythe method of Lowry et al. (14), with bovine serum albumin as the standard.After separation by SDS-PAGE, proteins were electrophoretically transferredonto an Immobilon polyvinylidene difluoride membrane (Millipore) by themethod of Towbin et al. (23). After transfer, the membrane was incubated inblocking solution (3% skim milk in PBS–0.05% Tween 20) for 1 h and thenincubated with C. pneumoniae antibody-positive patient sera, subjected to theMIF test (immunoglobulin G [IgG] titer, .1:16), or hybridoma supernatants. Analkaline phosphatase-conjugated IgG antibody (Dianova) was used as the sec-ondary antibody, and the membrane was developed with nitroblue tetrazoliumchloride-X phosphate solution (Schleicher & Schuell) (4).

In addition, sera from two C. pneumoniae culture-positive patients were eval-uated. Both female patients (30 and 38 years old) had suffered from respiratorytract infections with bronchitis and sinusitis. Sera obtained both at the onset ofillness and 8 weeks later were found to be negative by the MIF test (IgG titer,,1:8) and were examined by immunoblot assay.

Isolation of the 54-kDa species-specific protein of C. pneumoniae. For isolationof the 54-kDa protein, purified EBs of C. pneumoniae AR-39 were used. Theprotein was isolated by continuous-elution electrophoresis (Model 491 Prep Cell;Bio-Rad) with conventional gel electrophoresis buffer systems. The followingconditions were used for optimal resolution: 8% acrylamide, 8-cm gel length inthe small gel tube, 1 mg of total protein as the sample load, and 12 W of constantpower during the run. The separated proteins were collected in 1-ml fractions,and the 54-kDa fraction was detected by immunoblot analysis with C. pneu-moniae antibody-positive sera.

Production of anti-54 kDa MAb. Female BALB/c mice (8 weeks old) wereimmunized subcutaneously with p54 (5 mg per animal and injection) four timesat 14-day intervals; the first injection was in complete Freund’s adjuvant, and thefollowing injections were in incomplete Freund’s adjuvant. Two weeks later, theanimals were given booster injections (5 mg of p54 intraperitoneally in PBS) onthree consecutive days. One day after the last injection, the animals were sacri-ficed and the spleens were excised. The spleen cells were fused with nonproducermyeloma cells by the method of Kohler and Milstein (10). For cloning, the cellsuspension was distributed among the wells of 10 microtiter plates. Hybrid cloneswere screened for antibody production by Western blot assay. The antibodyclasses were determined with a mouse hybridoma subtyping kit (BoehringerGmbH, Mannheim, Germany) in accordance with the manufacturer’s instruc-tions.

Neutralization assay. Neutralization studies were done with BGM cell culturesas described by Byrne et al. (2), with minor modifications. Briefly, the hybridomasupernatants (10F1C1, 8B11B4, and 8B11E6) were diluted in sucrose-phos-phate-glutamine (SPG) medium to obtain antibody dilutions of 1:2, 1:4, 1:6, 1:10,and 1:20. Antibody 8B11E6 was purified by hydrophobic-interaction chromatog-raphy with phenyl-Sepharose (Pharmacia, Freiburg, Germany) in accordancewith the manufacturer’s instructions. The purified antibody stock solution wasdiluted with SPG medium to obtain final concentrations of 0.01, 0.1, 1, 10, and100 mg/ml. As a control, a hybridoma supernatant with anti-epidermal growthfactor antibody was used in the same dilutions as the other hybridoma superna-tants. The EB stock solution (C. pneumoniae AR-39, C. trachomatis E, or C.psittaci Z 432) was diluted in SPG medium to contain approximately 2 3 104

inclusion-forming units/ml, and a 90-ml inoculum was added to 90-ml of eachantibody dilution. In parallel, tests were prepared with 90 ml of SPG medium and90 ml of EB, with 90 ml of heat-inactivated hybridoma supernatant or antibody(56°C, 30 min) and 90 ml of EB, and with 90 ml of heat-inactivated hybridoma

supernatant or antibody in the presence of guinea pig serum (Virion GmbH,Wurzburg, Germany) as a source of complement at a dilution of 1:40 and 90 mlof EB. The mixtures were incubated for 30 min at 37°C on a slowly rockingplatform before the BGM cell monolayers, prepared in 96-well flat-bottommicrotiter plates, were inoculated with 50 ml of this mixture (in triplicate for eachdilution of each antibody). The plates were centrifuged (1,960 3 g, 1 h, 37°C), thesupernatant was removed, and 200 ml of CMGA medium was added to each well.The plates were incubated for 72 h at 37°C in 5% CO2. After 3 days of incuba-tion, the cells were fixed with methanol and the Chlamydia inclusions werestained with a genus-specific fluorescein-conjugated MAb (Pathfinder ChlamydiaCulture Confirmation System; Kallestad Diagnostics). The number of inclusionsper 1603 field was determined, and at least five fields per well were viewed.Neutralization was defined as a 50% reduction in the inclusion count comparedto control wells without antibody (2).

RESULTS

Immunoblotting with patient sera. The 54-kDa protein wasdetected in all nine different C. pneumoniae strains from Tai-wan, the United States, and Germany by immunoblotting withC. pneumoniae antibody-positive patient sera (MIF titers, .1:16). No such reaction was seen with either C. trachomatis E orC. psittaci Z 432 (Fig. 1). Additionally, a protein of about 45kDa was detected in all of the C. pneumoniae strains examinedin this study (Fig. 1).

FIG. 2. Immunoblot of C. pneumoniae AR-39 antigen with early and conva-lescent-phase (8 weeks after the beginning of the infection) sera from twoculture-positive patients with an MIF titer of ,1:8. As controls, C. pneumoniaeMIF antibody-positive sera and MAb supernatant 8B11E6 were used. Lanes: 1,C. pneumoniae MIF antibody-positive serum 10; 2, supernatant 8B11E6; 3, earlyserum from culture-positive patient A; 4, convalescent-phase serum from cul-ture-positive patient A; 5, early serum from culture-positive patient B; 6, con-valescent-phase serum from culture-positive patient B; 7, C. pneumoniae MIFantibody-positive serum 11. Lane M contained low-molecular-weight markers(Pharmacia). Molecular sizes in kilodaltons are shown on the sides.

FIG. 1. Immunoblots of different C. pneumoniae strains with anti-C. pneumoniae antibody-positive sera. (A) Lane groups: 1, C. pneumoniae AR-39 tested with sera1 to 9; 2, strain TW-183 tested with sera 1 to 6; 3, strain ATCC 1355 tested with sera 1 to 3; 4, strain ATCC 1310 tested with sera 1 to 3. (B) Lane groups: 1, strainHK; 2, strain FR-1; 3, strain FR-2; 4, strain ATCC 1356; 5, strain ATCC 1360; 6, C. trachomatis E; 7, C. psittaci Z 432. All seven strains were tested with patient sera10 and 11. Lanes M contained low-molecular-weight markers (Pharmacia). Molecular sizes in kilodaltons are shown beside the gels.

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The sera of two C. pneumoniae-infected patients with acuterespiratory tract infections who were culture positive for C.pneumoniae but remained C. pneumoniae antibody negative(MIF titers, ,1:8) did not react with the 54-kDa protein eitherat the onset of infection or 8 weeks later, during infection (Fig.2).

MAbs against the isolated 54-kDa protein. Eight anti-p54chlamydial MAbs, all of them of the IgG1 isotype, were pro-duced and were evaluated by immunoblot analysis with the C.pneumoniae AR-39 antigen. The supernatants of the eight hy-bridomas reacted strongly and exclusively with the 54-kDaprotein (Fig. 3). One of these MAbs (8B11E6) was used forfurther immunoblot testing. Immunoblot assays were per-formed with all of the C. pneumoniae type strains, the patientisolates, C. trachomatis E, C. psittaci, and BGM cells. MAb8B11E6 recognized the 54-kDa protein in all of the C. pneu-moniae strains examined, whereas it did not react with unin-

fected BGM cells or C. trachomatis or C. psittaci antigen (Fig.4).

Neutralization assay. Three hybridoma supernatants (10F1C1,8B11E6, and 8B11B4) and purified MAb 8B11E6 were used ina neutralization assay. All three anti-54-kDa protein superna-tants exhibited C. pneumoniae AR-39 infectivity-neutralizingactivity in cell culture, and the antibody dilution required for a50% reduction of chlamydial infectivity was in the range of1:100 to 1:150 (Fig. 5). Neither heat inactivation of the super-natants nor addition of complement (1:40) influenced the neu-tralizing capacity. The purified MAb reduced chlamydial in-fectivity by 50% at an antibody concentration of about 0.09mg/ml. No significant differences were observed among theuntreated antibody, the heat-inactivated antibody, and theheat-inactivated antibody plus complement, respectively. Theneutralizing effect of 8B11E6 was limited to C. pneumoniaeinfection, while it could not be observed with C. trachomatis Eor C. psittaci Z 432.

Addition of the anti-54-kDa protein supernatants and thepurified MAb reduced the number of inclusions but had noinfluence on the morphology of the inclusions. No neutralizingactivity was seen in the control culture with an anti-epidermalgrowth factor hybridoma supernatant without antichlamydialantibodies.

DISCUSSIONThe immunodominant proteins of C. pneumoniae have pre-

viously been analyzed by immunoblotting of type strain TW-183 with sera from C. pneumoniae antibody-positive patients.These sera reacted strongly with a 54-kDa protein of C. pneu-moniae TW-183 but not with C. trachomatis antigen, which ledto the assumption that the 54-kDa protein was species specific(4).

The results of the present study indicate that this 54-kDaprotein is expressed not only by the type strain TW-183 but byall of the different type strains and isolates of C. pneumoniaetested. Furthermore, it could not be detected in C. trachomatisor C. psittaci by using specific MAbs, confirming its speciesspecificity. The strong, regular reaction of the 54-kDa proteinof all of the strains tested with patient sera suggests strongimmunogenicity during infection. Other authors have de-scribed a 53- or 55-kDa protein as an immunodominantC. pneumoniae-specific antigen recognized during human in-fection (8, 9, 12, 16, 24). These two proteins may be identical

FIG. 3. Immunoblot of C. pneumoniae AR-39 antigen with eight anti-p54MAbs. The blotted membrane was incubated with hybridoma supernatant at adilution of 1:10 (lanes 1 to 8) or with an anti-C. pneumoniae-positive serum at adilution of 1:100 (lane 9) as a positive control, respectively. Lane M containedlow-molecular-weight markers (Pharmacia). Molecular sizes in kilodaltons areshown on the sides.

FIG. 4. Immunoblot analysis of nine C. pneumoniae strains with an anti-C. pneumoniae antibody-positive serum (odd-numbered lanes) or with hybridomasupernatant 8B11E6, which recognizes the 54-kDa protein (even-numbered lanes). Antigens from C. trachomatis and C. psittaci and BGM cells were used as controls.(A) Lanes: 1 and 2, C. pneumoniae AR-39; 3 and 4, strain TW-183; 5 and 6, strain ATCC 1310; 7 and 8, strain ATCC 1355; 9 and 10, strain ATCC 1356; 11 and 12,strain ATCC 1360. (B) Lanes: 1 and 2, strain HK; 3 and 4, strain FR-1; 5 and 6, strain FR-2; 7 and 8, C. trachomatis E; 9 and 10, C. psittaci Z 432; 11 and 12, BGMcells. Lanes M contained low-molecular-weight markers (Pharmacia). Molecular sizes in kilodaltons are shown beside the gels.

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to the 54-kDa protein examined in our studies; comparison ofsequence data, where available, will answer this question. Inaddition, another previously reported species-specific proteinwith a molecular mass of about 45 kDa was also detected inthis study (4, 8).

As in both of our culture-positive patients, acute primaryinfections with C. pneumoniae in adults can lead to clinicalmanifestations like bronchitis or sinusitis (22), which may bemore severe than acute reinfections in adults. Interestingly, notall patients seem to develop antibodies against the 54-kDa pro-tein, despite culture-proven C. pneumoniae infection. The rea-son might be the effect of prompt antimicrobial treatment onthe adaptive immune response, or these sera from patients maynot have recognized the epitopes of the specific antigen. Thelatter event would possibly explain the severe and long-lastingcourse of infection observed in these two patients. Further-more, it might indicate a possible protective role of anti-p54antibodies in the immune response. The hybridoma superna-tants and the purified MAb were able to neutralize the C.pneumoniae infection in cell culture in a complement-indepen-dent manner. The results of neutralization assays with C. tra-chomatis have been shown to depend on the cell line used (2).Therefore, C. pneumoniae infection and neutralization withMAbs was also studied in HL (human lung) cells, which deliv-ered the same results as BGM cells (data not shown). Theability of the anti-p54 MAb to neutralize infectivity in vitrosuggests that this protein is involved in the infection processand that it might be a potential candidate for development ofa vaccine. In addition, the results of the neutralization assaysupport previous results indicating that the 54-kDa protein issurface located (4). It might play an important role either as anadhesin in attachment, as proposed for the major outer mem-brane protein of C. trachomatis (21), or in the process of EBingestion by the host cell.

In contrast to the situation with our MAbs, previously re-ported C. pneumoniae-specific MAbs (TT-205 and RR-402)did not react in an immunoblot assay although they wereshown to neutralize infectivity (17). The reason might be theuse of different antigens for immunization: Puolakkainen et al.(17) used whole EB, thereby presenting many conformational

epitopes, whereas in our study an isolated, denatured proteinexhibiting predominantly linear epitopes was used. The MAbsestablished against the 54-kDa protein of C. pneumoniae mightprovide a new diagnostic tool for the rapid detection of C.pneumoniae in patient specimens. This would be of interest, asthe reported association of C. pneumoniae with atherosclerosisand coronary heart disease has increased the need for reliableand fast diagnostic methods. Moreover, the MAbs might beused as ligand for affinity purification of the 54-kDa protein,thereby facilitating sequence analysis, aiding in the develop-ment of immunoassays for antigen and antibody detection.Further studies with the MAbs in cell culture systems shouldprovide interesting and important information about the local-ization and function of the 54-kDa protein.

ACKNOWLEDGMENTS

We thank Heike Vogele for excellent technical assistance.This study was supported in part by grant AZ Fr. 1085/1-1 from the

Deutsche Forschungsgemeinschaft.

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FIG. 5. Neutralization assay of C. pneumoniae AR-39 infection in cell culturewith the hybridoma supernatants 10F1C2, 8B11E6, and 8B11B4 (directed againstthe 54-kDa protein). The results are expressed as percent reduction of inclusion-containing cells compared to the control culture without antibody. The inclusioncount without antibody averaged 152 per 1603 field. The dashed line indicatesa 50% reduction of inclusion counts.

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