JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1991, P. 1312-1316 Vol. 29, No. 70095-1137/91/071312-05$02.00/0Copyright X 1991, American Society for Microbiology

Antigenic Variation among Strains of Chlamydia pneumoniaeCAROLYN M. BLACK,'* JAMES E. JOHNSON,' CAROL E. FARSHY,1 TERESA M. BROWN,'

AND BJ0RN P. BERDAL2

Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 andInstitute of Medical Biology, University of Troms0, Troms0, Norway2

Received 1 October 1990/Accepted 8 April 1991

The antigenic profiles of six strains of Chlamydia pneumoniae were analyzed by the microimmunofluores-cence test (MIF) and immunoblotting with human serum and murine monoclonal antibody. MIF-derivedantibody titers in serum samples from culture-positive patients were four- to eightfold higher againstautologous isolate antigen than they were against the prototype antigen strain TW-183. Sera of patients withrespiratory illness that were culture negative and complement fixation positive for Chlamydia spp. producedhigher titers by MIF against a strain of C. pneumoniae isolated in the area than they did against TW-183. Fortwo of five cases, the criteria for establishing the diagnosis of acute infection were met only with use of theantigen from the local strain; TW-183 was inadequate for this purpose. Immunoblot profiles revealed antigenicdifferences between strains that varied with the human serologic response; i.e., unique antigens wererecognized by the sera of some individuals and not by the sera of others. Using the reactivity of a genus-specificmonoclonal antibody against a major outer membrane protein, we found that strain CWL-011, isolated inAtlanta, Ga., may possess a major outer membrane protein with a molecular mass between those of C.trachomatis L2 and other C. pneumoniae strains. These data provide evidence of several new and uniqueserotypes of C. pneumoniae and suggest that the serologic diagnosis of C. pneumoniae infection may require theuse of antigens from more than one strain of this species.

Chlamydia pneumoniae is a recently recognized speciesconsisting of the strains commonly referred to as TWAR(11). These strains are associated with acute respiratoryinfections in humans, especially atypical pneumonia (12). Inadult populations, the prevalence of antibody titers indica-tive of exposure to the organism has been reported to be inthe range from 50 to 78% (12, 14). Culture-confirmed diag-nosis of infection followed by propagation of clinical isolateshas proven difficult to date, and the methods for this proce-dure have not yet been standardized. Diagnosis is based onthe detection of fourfold rises in antibody titer in themicroimmunofluorescence test (MIF) or an elevated immu-noglobulin G (IgG) or IgM titer when only a single serumsample is available (12). Conventionally, the prototype strainTW-183 (Washington Research Foundation, Seattle, Wash.)is used as the only C. pneumoniae antigen in serologic tests,with additional use of Chlamydia trachomatis or Chlamydiapsittaci antigen pools, depending on the laboratory. Gray-ston and colleagues (12) at the University of Washingtonhave recently reported that all the isolates of C. pneumoniaein their collection appear to be members of a single strain,designated TWAR, on the basis of multiple parameters ofprotein and nucleic acid measurements. Several new isolatesof C. pneumoniae from cases of community-acquired pneu-monia have been characterized at the Centers for DiseaseControl, Atlanta, Ga. (6, 7). We report here an antigenicanalysis of six C. pneumoniae strains and present evidencethat these isolates represent serotypes unique from that ofthe prototype strain TW-183.

MATERIALS AND METHODS

Strains. C. pneumoniae CWL-011, CWL-029 (on depositas ATCC VR1310), CWL-050, and CM-1 were isolated at the

* Corresponding author.

Centers for Disease Control from patients at a local hospitalwith radiologic evidence of pneumonia (1). The CWL strainswere cultured from oropharyngeal swabs. Strain CM-1 wascultured from sputum. C. pneumoniae FML-10 was one offive strains isolated from nasal swabs from patients inNorway with respiratory infection associated with an out-break of adenovirus (4). C. pneumoniae TW-183 was ob-tained from the Washington Research Foundation. All C.pneumoniae strains tested were reactive with a Chlamydiagenus-specific monoclonal antibody (Kallestad Diagnostics,Austin, Tex.) and a C. pneumoniae-specific monoclonalantibody (Washington Research Foundation) but not with aC. trachomatis species-specific monoclonal antibody (SyvaDiagnostics, Palo Alto, Calif.). These strains also reactedpositively in a C. pneumoniae-specific polymerase chainreaction test (2). Preliminary studies by electron microscopyof infected HeLa cells demonstrated that elementary bodiesof strain CWL-029 lack the loose periplasmic membranedescribed by Chi et al. (10) for strain TW-183. Elementarybodies of strain TW-183 tested in parallel did exhibit thisloose periplasmic membrane; thus, strain CWL-029 maydiffer from strain TW-183 in its ultrastructural morphology.

Isolations were performed by conventional procedures byusing the HeLa 229 cell line as the host, centrifuge-assistedinfections, and cycloheximide in the medium (13, 14). Antigenfor immunoblotting was prepared from elementary bodiespurified by centrifugation through a cushion of 30% Renogra-fin-76 (Squibb Diagnostics, New Brunswick, N.J.) (5).

Sera and monoclonal antibody. Serum samples for immu-noblots were from three patients with culture-confirmed C.pneumoniae infections who presented with community-ac-quired pneumonia at a hospital in Atlanta, Ga., during a10-month period in 1987 and 1988 (6, 7). The CWL strains ofC. pneumoniae used in this study (described above) werecultured from specimens from these patients and are thusdescribed as autologous antigen in immunoblot studieswhere appropriate. An additional serum specimen included

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TABLE 1. Titers of IgG determined by MIF in serum samplesfrom patients with culture-confirmed C. pneumoniae infection

by using autologous and heterologous antigen

Patient IgG titer determined with the following antigens:Strain

srmnserum CWL-011 CWL-029 CWL-050 TW-183 CJAb

CWL-011 S1 512 32 256 32 32S2 1,024 256 512 256 128

CWL-029 S1 128 128 128 16 16S2 1,024 2,048 1,024 512 256

CWL-050C 256 128 256 64 <8

a Patients were culture positive for the indicated strains. SI and S2designate serum samples obtained approximately 4 weeks apart.

b Antigen pool of C. trachomatis serovars C, J, and A.I Patient was unavailable for collection of second blood sample.

in the immunoblot studies was collected from a patient inAtlanta with culture-confirmed C. pneumoniae infectionwho had radiographically proven pneumonia but who wasnot hospitalized. C. pneumoniae CM-1 (described above)was isolated from this patient. Serum specimens used forimmunoblotting were collected approximately 1 month fol-lowing the onset of symptoms from the patient infected withstrain CWL-029 and 9 months following the onset of symp-toms from the patient infected with strain CWL-011. Acute-phase serum specimens (collected from several days to 1week following the onset of symptoms) from patients in-fected with strains CWL-050 and CM-1 were used. Noconvalescent-phase serum specimens from these two pa-tients were available for testing.Serum samples for MIFs were from culture-negative pa-

tients in Norway with acute respiratory disease and Chla-mydia genus-positive titers by complement fixation (3). Ofthese Norwegian patients, patient 1 presented with trache-itis, patients 2 to 4 presented with pneumonia, and patient 5presented with bronchitis. Acute- and convalescent-phaseserum specimens from these patients were tested by MIF;specimen collection intervals are described in detail in Table2. A genus-specific monoclonal antibody against the majorouter membrane protein (MOMP) was kindly supplied byW. J. Newhall (Centers for Disease Control) for use inimmunoblots.MIF. Purified elementary bodies were formalinized for use

as antigen, and the MIF was performed by the method ofWang and Grayston (16). MIF antigen was standardized bymeasuring the protein concentrations of suspensions ofelementary bodies following their purification by differentialcentrifugation and density gradient centrifugation in Percoll(15). Serial twofold dilutions of serum were tested, beginningat a dilution of 1:16.

Immunoblotting. Proteins of purified chlamydial elemen-tary bodies were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting asdescribed previously (14) by using precast 10% polyacryl-amide gels with a Tris-tricine buffer system (Novex, Encini-tas, Calif.). Molecular masses were determined with low-molecular-weight standards (Integrated Separation Systems,Hyde Park, Mass.). Convalescent-phase sera from humanswith culture-confirmed C. pneumoniae infections (describedabove) were reacted overnight at 4°C with rocking. Mono-clonal antibody was reacted for 2 h at room temperature. Asa control against nonspecific binding reactions, serum sam-ples were blotted against uninfected HeLa cell antigen.

TABLE 2. MIF-derived antibody titers of complement fixation-positive serum samples from patients with acute-phase respiratory

infections by using C. pneumoniae TW-183 andNorwegian strain FML-10 as antigens

Antibody titer with the following

Patient Pse (days Antibody antigens:no. pstrunsetay class C. pneumoniae C. trachomatis

TW-183 FML-10 KGFL3b DE

1 SI (3) IgG 128 128 -_ -S2 (35) IgG 512 2,048 32 32

S, IgMS2 IgM -

2 S, (10) IgG 32S2 (48) IgG 512 -

S, IgMS2 IgM 32

3 S1 (14) IgG 128 512S2 (58) IgG 128 512

S1 IgM 128 512S2 IgM 128 512 - 128

4 S1 (14) IgG 512S2 (34) IgG 512 128 512 32

S IgM 32S2 IgM 32

5 S1 (7) IgGS2 (28) IgG 512 ',048 - 128

S1 IgMS2 IgM 512 2,048 128

a Patient 1 also exhibited a rise in titer of 8 to 32 in IgG against C.trachomatis serovars H and I (pooled). SI and S2 designate serum samplesobtained on separate days, as shown in parentheses.

b Antigen pools of C. trachomatis serovars C, J, A, B, L1, and L2 were alsotested and found to be negative for all patients.

c Antibody titers reported as negative (-) were <16.

Quality control procedures included pretesting of antigensby sodium dodecyl sulfate-polyacrylamide gel electrophore-sis and adjustment to equivalent concentrations by proteinprofiles. Gels were stained for protein by using the ProBluestaining system (Integrated Separation Systems), and pro-teins electrophoretically transferred to nitrocellulose werestained with the GoldBlot system (Integrated SeparationSystems). The protein profile patterns of all C. pneumoniaeantigens studied were similar. In addition, the efficienttransfer of proteins to nitrocellulose was demonstrated bystaining the protein remaining in gels following electropho-retic transfer.

RESULTS

MIF-derived titers. Serum samples from each of three C.pneumoniae culture-positive patients resulted in four- toeightfold higher titers by MIF when they were tested withthe autologous isolate antigen than when they were tested

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1314 BLACK ET AL.

A

12 3 4 5 6 7B

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FIG. 1. Immunoblots of serum from humans with culture-confirmed C. pneumoniae infection tested with autologous and heterologousisolate antigens. Sera were from patients infected with C. pneumoniae CWL-011 (A), CWL-029 (B), CWL-050 (C), and CM-1 (D). Lanes: 1,C. trachomatis L2 antigen; 2, C. pneumoniae CWL-011 antigen; 3, C. pneumoniae CWL-029 antigen; 4, C. pneumoniae CWL-050 antigen;5, C. pneumoniae CM-1 antigen; 6, C. pneumoniae TW-183 antigen; 7, uninfected HeLa control.

with TW-183 antigen (Table 1). In all but one case, titerswere consistently higher when the serum sample was testedwith C. pneumoniae antigen prepared from local Atlantaisolates (the CWL strains) than when it was tested withantigen prepared from TW-183. Similarly, sera from Norwe-gian patients with respiratory infections who were positivefor Chlamydia spp. by complement fixation tests consis-tently reacted more strongly in the MIF with a C. pneumo-niae strain isolated locally (Norway) than they did with

TW-183 (Table 2). In patient 2, an IgG titer rise to 512 and anIgM titer rise to 32 occurred only against the Norwegianstrain, and this patient's serum was negative when it wastested with strain TW-183 (Table 2). Patient 3 had stabletiters against both C. pneumoniae antigens, but these titerswere fourfold higher against the antigen from the Norwegianstrain. In patient 4, a preexisting titer of 512 against TW-183antigen was stable over time, while the IgG titer to theNorwegian strain antigen rose from negative to 128. Patient

kDa

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ANTIGENIC VARIATION IN C. PNEUMONIAE 1315

1 2 3 4

_ _ - MOMP

FIG. 2. Immunoblot of Chlamydia strains tested with genus-

specific anti-MOMP monoclonal antibody. Lanes: 1, C. trachomatisL2; 2, C. pneumoniae CWL-011; 3, C. pneumoniae CWL-029; 4, C.pneumoniae CWL-050.

5 seroconverted to both C. pneumoniae antigens, but titerswere fourfold higher against the Norwegian strain antigen.For patients 2 and 4, the diagnosis of C. pneumoniaeinfection was based on the Norwegian strain antigen andwould have been missed if only TW-183 antigen was testedin the MIF.Immunoblots. The electrophoresed antigens shown in Fig.

1A to D were prepared at equivalent protein concentrations,and the nitrocellulose strips were cut from the same nitro-cellulose paper or from two pieces of paper prepared fromgels run in parallel in an identical manner in the same

apparatus. Immunoblot analysis of the reactivities of sera

from culture-positive patients with autologous and heterolo-gous isolates revealed antigenic differences between strainsof C. pneumoniae (Fig. 1A to D). The blot profiles showedthat antigenic differences exist among the strains isolated atthe Centers for Disease Control as well as when these strainswere compared as a group with strain TW-183. Serum fromthe patient infected with C. pneumoniae CWL-011 reactedweakly with strain CWL-029 and formed unique patterns ofbands for each strain of C. pneumoniae tested (Fig. 1A).Serum from the patient infected with CWL-029 reacted with65- to 70-kDa proteins in strains CM-1 and TW-183 but notwith the proteins in the three CWL strains (Fig. 1B). Serumspecimens from this patient also distinguished among theCWL strains by reacting with an -42-kDa band in strainsCWL-011 and CWL-050 but not CWL-029 and reacting withan -24-kDa band in strain CWL-050 but not CWL-011 or

CWL-029 (Fig. 1B). Serum from the patient infected withCWL-050 reacted weakly with strains CWL-029 and CM-1 toform only a single 50-kDa band, but it formed patterns ofbands when it was reacted with antigen from strains CWL-011, CWL-050, and TW-183 (Fig. 1C). For each of thepatients infected with the CWL strains, the immunodomi-nant antigen of 58 to 62 kDa showed genus-wide reactivity(Fig. 1A to C). In most cases, no or weak reactivity was

observed with the MOMP. In contrast, serum specimensfrom the patient infected with C. pneumoniae CM-1 (isolatedindependently from the CWL strains) recognized a predom-inant band at a molecular mass of 15 kDa, but only in strainTW-183 (Fig. 1D). Serum from this patient also reactedstrongly with a 50-kDa antigen that was present only instrains TW-183 and CWL-011.

Reactivity of a genus-specific anti-MOMP monoclonalantibody (Fig. 2) revealed that strain CWL-011 appears topossess a MOMP with a molecular mass between those of C.trachomatis L2 and the other C. pneumoniae strains (CWL-029 and CWL-050).

DISCUSSION

C. pneumoniae was established as a separate species onthe basis of serologic, DNA, and structural analyses (11).Grayston and colleagues (12) have studied isolates of thisspecies from several different countries and so far haveidentified only a single strain or serovar, called TWAR, inrespect to the traditional name used for this agent. Althoughit has not been well defined, it appears that the evidence ofa single serotype is based largely on the similarity of theisolates in protein profiles (data not shown), genomic DNArestriction patterns, and reactivity of patient serum in theMIF with the commonly used antigen from strain TW-183.Campbell et al. (8, 9) recently reported results of an immu-noblot analysis of the serologic response to C. pneumoniaeinfection and found patterns of common reactivity similar tothose reported in the present study. However, to our knowl-edge, a comparison of reactivity profiles to different C.pneumoniae strains in the MIF, immunoblot, or any othertest has not been done. In contrast to what has previouslybeen thought, the data presented here provide evidence ofseveral new and unique serotypes of C. pneumoniae andemphasize the need for careful interpretation in the serologicdiagnosis of C. pneumoniae infection. Reliance on MIF forthe diagnosis of infection may require use of antigens frommore than one strain of this species, perhaps in the form ofantigen pools like those used in the MIF for C. trachomatisantibodies (15).

Antigenic diversity among strains of C. pneumoniae ap-pears to reside within several different proteins, particularlythe 65- to 70-kDa proteins. Although strain variation can alsoinvolve the MOMP, this protein is not commonly recognizedby serum from infected patients in an immunoblot. Theimportance of antigenic variation in the MOMP or otherproteins when the MIF is used to diagnose C. pneumoniaeinfection is therefore not known. The potential clinicalsignificance of variation between strains of C. pneumoniaewarrants further investigation but will require the study of alarger collection of clinical isolates supported by completeepidemiologic data. Studies investigating the genetic basesfor strain variation are under way in our laboratory.A separate, yet important, issue that emerges secondarily

from our data is that of potential cross-reactivity in the MIFbetween Chlamydia species antigens. Data from other labo-ratories have been disparate and remain controversial. Ourstudy of MIF titers in serum samples from complementfixation-positive patients against different C. pneumoniaeantigens included several conventional pools of C. tracho-matis antigens (Table 2). Rises in antibody titer were ob-served against the C. trachomatis antigen pools DE andKGFL3 in concert with C. pneumoniae antigen in four of fivepatients studied. Rises in antibody titer occurred against theDE pool in the sera of all four of these patients, whereas atiter rise against the KGFL3 pool occurred in the sera of twoof four patients reactive with C. trachomatis. Serum samplesfrom all patients were negative when tested with other C.trachomatis antigen pools (see footnote b of Table 2). Risesin titer to the DE and KGFL3 pools also involved IgM inpatients 3 and 5. Although the possibility of concurrent C.trachomatis infection has not been ruled out, the facts that

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the sera of these patients had no C. trachomatis antibodies atthe time of presentation, that the patients had no symptomsof genital infection, and that the sera of all patients reactedwith one or both of the same two C. trachomatis antigenpools support the likelihood that these rises in antibody titerto C. trachomatis antigens were due to cross-reactivity withC. pneumoniae antigens, especially in the case of the DEantigen pool. Further study is needed to distinguish, char-acterize, and assess the importance of potential cross-reac-tivity between species in the MIF to ensure the accuratediagnosis of Chlamydia infections.

ACKNOWLEDGMENT

We are indebted to W. Jim Newhall for invaluable assistance andadvice.

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J. F. Campbell. 1989. Characteristics of atypical chlamydialagents isolated from pneumonia patients in Atlanta, abstr.D-105, p. 100. Abstr. 89th Annu. Meet. Am. Soc. Microbiol.1989. American Society for Microbiology, Washington, D.C.

2. Berdal, B. P., C. M. Black, J. S. Lewis, L. Palmer, and P. I.Fields. Unpublished data.

3. Berdal, B. P., P. I. Fields, and H. Melbye. Scand. J. Infect. Dis.,in press.

4. Berdal, B. P., P. I. Fields, S. H. Mitchell, and G. Hoddevik. 1990.Isolation of Chlamydia pneumoniae during an adenovirus out-break, p. 445-448. In W. R. Bowie, H. D. Caldwell, R. P. Jones,et al. (ed.), Chlamydial infections. Proceedings of the 7thInternational Symposium on Human Chlamydial Infections.Cambridge University Press, Cambridge.

5. Caldwell, H. D., J. Kromhout, and J. Schachter. 1981. Purifica-tion and partial characterization of the major outer membraneprotein of Chlamydia trachomatis. Infect. Immun. 31:1161-

1176.6. Campbell, J. F., R. C. Barnes, P. E. Kozarsky, and J. S. Spika.

Submitted for publication.7. Campbell, J. F., R. C. Barnes, P. E. Kozarsky, and J. S. Spika.

1989. Program Abstr. 29th Intersci. Conf. Antimicrob. AgentsChemother., abstr. 267.

8. Campbell, L. A., C.-C. Kuo, and J. T. Grayston. 1990. Struc-tural and antigenic analysis of Chlamydia pneumoniae. Infect.Immun. 58:93-97.

9. Campbell, L. A., C.-C. Kuo, S.-P. Wang, and J. T. Grayston.1990. Serological response to Chlamydia pneumoniae infection.J. Clin. Microbiol. 28:1261-1264.

10. Chi, E. Y., C.-C. Kuo, and J. T. Grayston. 1987. Uniqueultrastructure in the elementary body of Chlamydia sp. strainTWAR. J. Bacteriol. 169:3757-3763.

11. Grayston, J. T., C.-C. Kuo, L. A. Campbell, and S. P. Wang.1989. Chlamydia pneumoniae sp. nov. for Chlamydia sp. strainTWAR. Int. J. Syst. Bacteriol. 39:88-90.

12. Grayston, J. T., S.-P. Wang, C.-C. Kuo, and L. A. Campbell.1989. Current knowledge on Chlamydia pneumoniae, strainTWAR, an important cause of pneumonia and other acuterespiratory diseases. Eur. J. Clin. Microbiol. 8:191-202.

13. Kuo, C.-C., S.-P. Wang, and J. T. Grayston. 1977. Growth oftrachoma organisms in HeLa 229 cell culture, p. 328-336. In D.Hobson and K. K. Holmes (ed.), Nongonococcal urethritis andrelated infections. American Society for Microbiology, Wash-ington, D.C.

14. Ladany, S., C. M. Black, C. E. Farshy, J. M. Ossewaarde, andR. C. Barnes. 1989. Enzyme immunoassay to determine expo-sure to Chlamydia pneumoniae (strain TWAR). J. Clin. Micro-biol. 27:2778-2783.

15. Newhall, W. J., B. Batteiger, and R. B. Jones. 1982. Analysis ofthe human serological response to proteins of Chiamydia tra-chomatis. Infect. Immun. 38:1181-1189.

16. Wang, S.-P., and J. T. Grayston. 1974. Human serology inChlamydia trachomatis infection with microimmunofluores-cence. J. Infect. Dis. 130:388-397.

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