Chlamydia spp. the major surface protein may play an important

INFECTION AND IMMUNITY, Mar. 1982, p. 1024-1031 Vol. 35, No. 3 0019-9567/82/031024-08$02.00/0 Antigenic Analysis of the Major Outer Membrane Protein of Chlamydia spp. HARLAN D. CALDWELL'* AND JULIUS SCHACHTER2 Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 598401 and George W. Hooper Foundation, University of California, San Francisco, San Francisco, California 941432 Received 21 September 1981/Accepted 17 November 1981 The major outer membrane proteins (MOMPs) of several Chlamydia trachomatis serotypes (B, D, G, H, and L2) and of the C. psittaci meningopneumonitis strain were purified by preparatory sodium dodecyl sulfate-(sds)-polyacrylamide gel electrophoresis. The isolated SDS-polypeptide complexes, which varied in their apparent subunit molecular weights, were used as immunogens to raise hyperimmune rabbit antisera. The specificities of these antisera were determined both by rocket immunoelectrophoresis with the soluble SDS-polypeptide complex as antigen and by micro-immunofluorescence with whole organisms. By rocket immunoelectrophoresis, each of the soluble C. trachomatis MOMPs was immunologically related; however, no immunological cross-reactions occurred with the C. psittaci meningopneumonitis polypeptide, indicating that the MOMPs are antigenically distinct among members of these two chlamydial species. The same antisera were highly reactive with intact organisms by micro-immunfluorescence, demonstrating that at least some of the antibodies raised with SDS-polypeptide complexes reacted with native antigenic sites of these surface proteins. By microimmunofluorescence, anti-momp sera remained species specific; but, unlike the results observed by rocket immunoelectrophoresis, distinct differences in the reactivity and specificity of these antisera were observed among C. trachomatis serotypes. C. trachomatis isolates were separated into two distinct serogroups on the basis of their reactivity with anti-momp sera. B complex organisms (B, Ba, D, E, F, G, K, Li, L2, and L3) all reacted strongly with anti-momp sera of the B, D, G, and L2 serotypes. In contrast, these same antisera were poorly reactive with the C complex serotypes A, C, H, I, and J. Anti-H MOMP serum was the most serospecific, since high-antibody titers were found only against the homologous H serotype organism. These findings indicate that MOMPs of different strains of C. trachomatis are antigenically complex and that antigenic heterogeneity exists among the surface-exposed portions of the protein. Recent studies from several laboratories have reported on the identification of a quantitatively predominant (major) outer membrane protein (MOMP) associated with the cell envelopes of Chlamydia spp. Hatch et al. (8) identified MOMPs with an apparent subunit molecular weight (MW) of approximately 43,000 (43K) from the 6BC strain of Chlamydia psittaci and from two different C. trachomatis serotypes. More recently, Salari and Ward (15) found similar proteins exposed on the surfaces of 14 different C. trachomatis serotypes. These authors found that the major polypeptide varied in MW, ranging from 38 to 42K, depending on the C. trachomatis serotype studied. The distribution in size of the major polypeptide among the different serotypes correlated closely with the predominant human infections caused by the serotype, leading these authors to suggest that 1024 the major surface protein may play an important functional role in the pathogenesis of chlamydial infections. In studies by Caldwell et al. (2), a 39.5K MOMP was purified from isolated outer membranes of the C. trachomatis L2 serotype. That 39.5K MOMP accounted for as much as 60% of the total outer membrane protein of the organism and was structurally an important macromolecule that appeared to play a role in maintaining the integrity of the outer membrane and the morphology of the chlamydial elementary body (EB). These same studies showed that this structural protein was unique in that it retained immunogenicity in the presence of sodium dodecyl sulfate (SDS). Antisera raised against the SDS "denatured" polypeptide reacted with whole chlamydiae by micro-immunofluorescence (micro-if), with a specificity very similar to that found with antisera raised against

VOL. 35, 1982 whole viable L2 organisms. Those preliminary findings were very promising since they suggested that this abundant structural polypeptide might be a prominent surface antigen recognized after immunization and perhaps after infection with C. trachomatis organisms. Here, these studies are extended to more thoroughly define the antigenic properties of several MOMPs isolated from five different C. trachomatis serotypes and a C. psittaci strain. MATERIALS AND METHODS Organisms and growth conditions. C. trachomatis organisms were: lymphogranuloma venereum (LGV) strain 434/Bu, serotype L2; ocular trachoma strain TW-5/OT, serotype B; cervicitis strain UW-3/Cx, serotype D; cervicitis strain UW-57/Cx, serotype G; and cervicitis strain UW-4/Cx, serotype H. The C. psittaci meningopneumonitis (Mn) Cal-10 strain was used and was a generous gift supplied by Priscilla Wyrick, University of North Carolina. C. trachomatis organisms were grown in HeLa 229 cells, and EBs of each strain were purified by centrifugation on discontinuous Renografin density gradients (E. R. Squibb & Sons, Princeton, N.J.) as previously described (2, 4). The C. psittaci strain was grown in L-929 cells, and the EB were purified by centrifugation through discontinuous Renografin density gradients (2). Isolation of outer membranes. Outer membranes of Chlamydia spp. were prepared by Sarkosyl extraction of purified EBs as described by Caldwell et al. (2). The MOMPs were quantitatively extracted from the Sarkosyl-insoluble outer membrane material with 2% SDS (British Drug House, Poole, England) in 10 mm sodium phosphate buffer, ph 8.0, containing 1.5 mm EDTA at 60 C for 1 h. The suspension was centrifuged at 30,000 x g for 30 min at 22 C. The supernatant fraction, enriched with the MOMP, was retained and used as starting material for preparatory SDS-polyacrylamide gel electrophoresis (PAGE). Protein concentrations were determined by the method of Lowry (12), with bovine serum albumin as a standard. SDS-PAGE. PAGE was done with the discontinuous buffer system of Laemmli (11). Slab gels were used, and the procedures for treatment of samples, electrophoresis, and staining of gels have been previously described (2). Protein standards for estimating subunit MWs were phosphorylase B (94K); bovine serum albumin (68K); ovalbumin (43K); carbonic anhydrase (30K); soybean trypsin inhibitor (21K); and lysozyme (14.3K) (Bio-Rad Laboratories, Richmond, Calif.). Preparatory SDS-PAGE. One milliliter (600 Fig to 1.2 mg of protein) of the supernatant fraction of SDStreated outer membranes was electrophoresed on slab gels (9 by 14 by 0.15 cm) containing 12.5% acrylamide. A 1- by 14- by 0.15-cm stacking gel was used. Gels were electrophoresed for approximately 3.5 h at a 25- ma constant current. After electrophoresis, gels were stained for 2 min with 0.25% Coomassie brilliant blue R-250 in 50%o methanol and 7% acetic acid and immediately immersed and washed in several changes of distilled water. The faintly stained MOMP band was excised with a razor blade and cut into approximately 3- to 4-mm fragments. Gel fragments were emulsified in 1 ml of 0.1% SDS in 0.0125 M Tris-0.2 M glycine, CHLAMYDIAL MAJOR OUTER MEMBRANE PROTEIN 1025 ph 8.6, by repeated passage through a double-hub 18- gauge needle. The homogenized slurry was transferred to an ISCO electrophoretic concentrator Model 1750 (Instrumentation Specialities Co., Lincoln, Nebr.). The protein was electrophoretically eluted from the homogenized acrylamide gel material at 2.5 V/cm for 4 h with 0.025 M Tris-0.2 M glycine, ph 8.6, containing 0.1% SDS. This procedure resulted in the recovery of between 60 to 80% of the electrophoresed protein. The recovered proteins had mobilities on SDS-polyacrylamide gel complexes identical to the original protein bands. In some experiments, polypeptides were recovered directly from the homogenized acrylamide by diffusion without electrophoretic elution. Crossed immunoelectrophoresis of SDS-polyacrylamide gel protein complexes. The procedure used was that of Chua and Blomberg (6) with the following modifications. In this study, smaller glass support slides (83 by 100 by 1.5 mm) were used; therefore the volumes of agarose-detergent or agarose-antibody solutions added to the glass support slide were proportionately smaller. The intermediate gel of the crossed immunoelectrophoresis phase contained 1.5% Triton X-100 (Sigma Chemical Corp., St. Louis, Mo.) instead of Lubral PX. Polyethylene glycol was omitted from the antibody gel. The agarose-antibody gel contained 15% (vol/vol) rabbit antiserum. Electrophoresis was performed at a constant voltage of 2.5 V/cm for 18 h at 15 C. Polyacrylamide gel strips were soaked in electrophoresis buffer for 30 min before placing them on the glass slide for electrophoresis into agarose-containing antiserum. Rocket immunoelectrophoresis. The procedures used were those described by Caldwell et al. (4, 5). The concentration of Triton X-100 in the agarose-buffer solution was increased to 1.5% to complex free SDS present in the MOMP antigen samples. Electrophoresis was performed at 2.5 V/cm for 16 to 18 h at 15 C. Preparation of antisera against purified MOMPs and intact chlamydiae. Antibodies against purified MOMPs and viable L2 EB were raised in female New Zealand white rabbits. For the preparation of anti-momp sera, each rabbit was given a primary immunization of 100 to 150,ug of purified MOMP in 1 ml of either Trisglycine buffer containing 0.1% SDS or 0.1% SDS alone mixed with an equal volume of Freund complete adjuvant (Difco Laboratories, Detroit, Mich.). One milliliter of this emulsion was given intramuscularly into both hind legs (day 0). The same concentration of protein mixed with Freund incomplete adjuvant was given subcutaneously in the neck of the rabbit on days 30 and 45. Rabbits were bled 7 to 10 days after the last subcutaneous injection. Antiserum against whole L2 EBs was produced by immunizing rabbits intramuscularly with 2 ml of purified EB (3 x 1010 organisms per ml) emulsified in 2 ml of Freund incomplete adjuvant. After 21 days, a series of five intravenous injections of an aqueous EB suspension (3 x 1010 organisms per ml) was given at 3-day intervals in progressively increasing doses of 0.1, 0.2, 0.3, and 1.0 ml, respectively. Rabbits were bled 10 days after the last intravenous injection. Indirect immunofluorescence. The micro-if test of Wang (16) was used to determine the serological specificity of rabbit antisera raised against purified MOMPs. Antibodies were measured with fluorescein isothiocyanate-conjugated goat antiserum specific

1026 CALDWELL AND SCHACHTER 94K 88K 43K MOMP' 30K L.2 B D G H MW 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 21K ~ 14.3K VW' FIG. 1. SDS-PAGE protein pattems found at different steps in the isolation of MOMPs from several C. trachomatis serotypes by sequential treatment of intact EBs with Sarkosyl and SDS. Lanes 1, whole cell EB lysates prepared by treating purified EBs with Laemmli solubilizing buffer; lanes 2, supernatant material recovered after Sarkosyl treatment of EB; lanes 3, soluble fraction obtained after treating the Sarkosylinsoluble outer membrane fraction from lanes 2 with 2%6 SDS buffer. Protein-molecular-weight standards are designated by MW. Note that the MOMP is quantitatively recovered in nearly a homogenous form for each serotype by treating isolated outer membranes with SDS (lanes 3). The protein proffes observed for the C. psittaci Mn strain are not shown but gave very similar results to those shown above for C. trachomatis serotypes. against rabbit immunoglobulins (Cappel Laboratories, Cochranville, Penn.). C. trachomatis serotypes A, B, Ba, C, D, E, F, G, H, I, J, K, LI, L2, and L3 were tested individually. The C. psittaci strains tested were guinea pig inclusion conjunctivitis, feline pneumonitis, Mn strain Cal-10, and 6BC. RESULTS Isolation of outer membranes and MOMPs. In a previous study, intact chlamydial outer membranes were prepared by simply treating intact L2 EBs with 2% Sarkosyl buffer (2). The chlamydial outer membrane was present in the Sarkosyl-insoluble pellet recovered after centrifugation. It was also shown in those studies that the 39.5K L2 MOMP was quantitatively extracted in nearly a homogenous form by treating these Sarkosyl-isolated outer membranes with 2% SDS. Since a major objective of this study was to purify and immunochemically characterize the MOMP from different chlamydial strains, experiments were designed to determine if this extremely simple procedure could be applied to non-lgv C. trachomatis serotypes for the preparatory isolation of their MOMPs. The SDS-polyacrylamide gel protein patterns of several C. trachomatis serotypes obtained during different steps in the MOMP isolation procedure are shown in Fig. 1. The protein patterns of whole EBs for each serotype studied are shown in lanes 1 and represent the total SDS-soluble protein composition of the organisms. The Sarkosyl-soluble EB proteins are shown in lanes 2. It is evident that the Sarkosylsoluble protein profiles are very similar to those obtained from whole EBs with SDS solubilization, with the exception of the MOMP. The MOMPs of each of the C. trachomatis serotypes were only poorly extracted from intact EBs with Sarkosyl. For each serotype studied, this protein remained associated with the Sarkosyl-insoluble outer membrane pellet and was quantitatively solubilized by treating the isolated membranes with SDS (Fig. 1, lanes 3). Similar 94 K 68K 1 2 3 MW L2 B e. do _: 4 5 6 7 D G H Mn 43K 1141111'iWaIW: ei: 30 K _ 21 K3 14.3 K B INFECT. IMMUN. FIG. 2. SDS-PAGE showing protein homogeneity and variability in apparent subunit MW of chlamydial MOMPs purified by preparatory SDS-PAGE. Lane 1, MW markers; lane 2, L2 serotype 39.5K MOMP; lane 3, B serotype 39.5K MOMP; lane 4, D serotype 40.5K MOMP; lane 5, G serotype 38K MOMP; lane 6, H serotype 44.5K MOMP; lane 7, C. psittaci Mn (Cal-10) strain 40K MOMP. Approximately 15,ug of protein from each MOMP preparation was electrophoresed.

VOL. 35, 1982 results were also obtained with the C. psittaci Mn strain (results not shown). These results clearly show that the procedures described previously for isolating the MOMP of the L2 serotype are not uniquely applicable to serotype or to species but appear suitable for studies on all Chlamydia spp. In addition to the above findings, Fig. 1 also shows that the total protein profiles of each of the serotypes tested were very similar. Distinct differences were evident, however, with respect to the apparent subunit MWs of the MOMPs. The MOMPs of the L2 and B serotypes had identical MWs of 39.5K. In contrast, the MOMPs of the D, G, and H serotypes had apparent MWs of 40.5K, 38K, and 44.5K, respectively. The purity of the MOMPs isolated from six different chlamydial serotypes by preparatory SDS-PAGE is shown in Fig. 2. Each MOMP was homogenous and consisted of a single band by SDS-PAGE. Yields of MOMPs obtainable by SDS extraction of outer membranes were estimated to be greater than 60%o. The isolated proteins had identical subunit MWs to those observed before preparatory SDS-PAGE and electrophoretic concentration. MOMPs purified by this procedure were immunogenic, and the antisera obtained after immunization were specific for the MOMPs. This is shown in Fig. 3 in which SDS-polyacrylamide gel-separated proteins of L2 EB whole cell lysates were electrophoresed into antiserum raised against the purified 39.5K MOMP; a single immunoprecipitin line was formed with the 39.5K major polypeptides. Similar results were obtained for each of the isolated MOMPs except for the C. psittaci Mn 40K protein. Antiserum raised against the Mn 40K polypeptide was not reactive by this procedure. The reason(s) for this failure is unknown since the concentration of the Mn 40K protein used for immunization was very similar to those of the immunogenic C. trachomatis MOMPs. Antibody against the Mn 40K protein was present in hyperimmunized rabbit sera, although at a much reduced concentration, since the sera reacted when tested against whole Mn EBs by micro-if (see below). Serological specificity of antisera raised against purified MOMPs. The specificity of anti-momp sera was evaluated by both rocket immunoelectrophoresis and micro-if. The results of rocket immunoelectrophoresis are shown in Fig. 4. Immunological cross-reactivity was observed among each of the C. trachomatis MOMPs. In contrast, none of the C. trachomatis anti- MOMP sera reacted with the 40K MOMP of the C. psittaci Mn strain. These results indicate that the MOMP of C. trachomatis is a speciesspecific antigen. The possibility of these results CHLAMYDIAL MAJOR OUTER MEMBRANE PROTEIN 1027 being non-immunological reactions due to SDSserum protein interactions (13) is very unlikely since antibodies raised against ovalbumin, that was also prepared by preparatory SDS-PAGE for immunization, reacted only with the homologous ovalbumin antigen. No precipitin reactions were observed when anti-ovalbumin was electrophoresed against any of the chlamydial MOMPs (Fig. 4f). Although immunological cross-reactivity was evident among the C. trachomatis MOMPs, distinct qualitative and quantitative differences among immunoprecipitation reactions were observed. The homologous MOMP antibody-antigen reactions were always the most intensely precipitated, and the immunoprecipitate peak heights of these reactions tended to be less (with the exception of the H serotype) than the precipitin peaks obtained with heterologous MOMP antigen. These results suggest that more antibody specific for the homologous MOMP antigen is present in these sera since the height or area of the immunoprecipitin peak in rocket immunoelectrophoresis is directly proportional to the antibody-antigen ratio ' CD II 4 cs ~~~~S SDS- FIG. 3. Crossed imunoelectrophoresis of SDSpolyacrylamide gel-electrophoresed protein complexes demonstrating the specificity of antisera raised against preparatory SDS-PAGE-purified MOMP. The arrow illustrates the single immunoprecipitate obtained after electrophoresing SDS-protein complexes from a whole cell lysate of the L2 serotype against antiserum raised to purified 39.5K L2 MOMP. Approximately 25 Fg of protein from a L2 whole cell EB lysate was electrophoresed on an SDS-polyacrylamide gel. The lane containing the electrophoresed proteins was removed from the slab gel and electrophoresed against agarose containing antiserum as described in the text. A duplicate lane containing an identical L2 whole cell lysate mixture was excised and stained with CBB. This stained gel was used in the figure to illustrate the position of the proteins. Similar results were obtained for each of the antisera prepared against C. trachomatis MOMPs. Ab, antibody.,*i-- N.-4-ambodopow-ft

1028 CALDWELL AND SCHACHTER (17). It is also evident that some sera form much less intense immunoprecipitates with heterologous MOMP antigens, suggesting that antibodies to fewer determinants are present. The serological reactivity of anti-momp sera measured by micro-if is shown in Table 1. Serial twofold dilutions of each antiserum were tested against EBs from each of the 15 C. trachomatis serotypes and from 4 C. psittaci strains. The data show several interesting findings. (i) Anti-MOMP sera were species specific. No interspecies reactions were observed with any of the antisera tested. These findings are in agreement with those observed by rocket immunoelectrophoresis. (ii) Serological reactions among C. trachomatis serotypes were somewhat complex, but distinct serological patterns were evident. For example, antibody titers were consistently greater for the homologous anti- MOMP serum EB reaction with the exception of anti-b-momp serum, which reacted equally well with the K, Li, and L2 serotypes. C. trachomatis serotypes could be separated into INFECT. IMMUN. two distinct serological groups according to their reactivity with the various anti-momp sera. The composition of these two groups was identical to the two major serotype complexes that have been identified by micro-if and have been called, by Wang (16), the B and C complexes. B complex serotypes (B, Ba, D, E, F, G, K, Li, L2, and L3) cross-reacted strongly with B, D, G, and L2 anti-momp sera. These same antisera, however, reacted poorly when tested against the C complex serotypes (A, C, H, I, and J). In contrast to the above findings, anti-h MOMP serum was considerably more specific. This antiserum reacted at a 1:4,096 dilution with its homologous EB antigen but only at titers of 1:512 or less with each of the heterologous C. trachomatis serotypes. The serological specificity of rabbit hyperimmune antiserum raised against purified viable L2 EB was very similar to that observed with anti-l2 MOMP serum. This antiserum was species specific and also differentiated C. trachomatis serotypes into the same B and C complexes. (Ab-L2) (39. 5K( A (A b-b ) B (Ab-D \ \405K5 C1 AA!'A B 12 D G (Ab-G3 ( 38K / H MnOva - B 12 D D (Ab-H \44.5K) N S:.A G H MnOva BL2 D G H MnOva E (Ab-Ovo) F AA At B L2 D0i H MnOvo B 12 D G H MnOvc B L2 D G H M -0v FIG. 4. Rocket immunoelectrophoresis of isolated MOMPs demonstrating the serological relatedness of the SDS-soluble proteins among different chlamydial strains. (A) anti-l2 39.5K MOMP; (B) anti-b 39.5K MOMP; (C) anti-d 40.5K MOMP; (D) anti-g 38K MOMP; (E) anti-h 44.5K MOMP; and (F) anti-ovalbumin control serum. All electropherograms contain the same antigens. Antigens are preparatory SDS-PAGE-purified polypeptide proteins shown in Fig. 2. Ten microliters of each MOMP preparation (approximately 5 to 7,ug of protein) was electrophoresed against a 15% antibody-containing agarose gel. Conditions for electrophoreses are described in the text. Note that none of the anti-momp sera react with the C. psittaci Mn (Cal 10) MOMP and that cross-reactions among all C. trachomatis MOMPs are apparent. The control serum (anti-ovalbumin) only precipitated ovalbumin antigen. No reaction occurred when ovalbumin was electrophoresed against each anti- MOMP sera or when the isolated MOMPs were electrophoresed against hyperimmune anti-ovalbumin serum raised against ovalbumin which was isolated by preparatory SDS-PAGE.

VOL. 35, 1982 TABLE 1. CHLAMYDIAL MAJOR OUTER MEMBRANE PROTEIN 1029 Micro-IF titers of rabbit antisera prepared against isolated chlamydial MOMPs or whole EBs Organismsa Antiserum titers (C. trachomatis MOMP (serotype/mw) Whole chlamydial serotype/strain) Woeclmda B(39.5K) D(40.5K) G(38K) L2(39.5K) H(44.5K) Mn(40K) EB (L2 serotype) B complex serotype B/TW-5 2,048b 1,024 256 4,096 64 -C 8,192 Ba/Ap-2 512 1,024 256 2,048 256-8,192 D/UW-3 1,024 4,096 2,048 512 256-8,192 E/UW-5 1,024 1,024 512 2,048 256-8,192 F/UW-6 512 256 2,048 2,048 128-8,192 G/UW-57 1,024 512 4,096 4,096 256-8,192 K/UW-31 2,048 1,024 2,048 4,096 512-8,192 L1/440 2,048 2,048 1,024 2,048 256-8,192 L2/434 2,048 1,024 1,024 8,192 512-16,384 L3/404 2,048 2,048 2,048 4,096 512-8,192 C complex serotype A/G-17 64 64 32 32 64 _b 2,048 C/TW-3 16 8 128 32 - - 1,024 H/UW-4 64 64 64 256 4,096-1,024 I/UW-12 16-16 64 128-1,024 J/UW-36 16 32 64 256 128-1,024 C. psittaci strain GPIC - - - - - 16 - FN - - - - - - - 6BC - - - - - 32 - Mn - - - - - 128 - a Intact Chlamydia spp. used as test antigen. b Homologous titers. C -, Negative at a 1:8 dilution. DISCUSSION The results of this study demonstrate that the MOMPs of C. trachomatis form an antigenically complex family and that species, subspecies, and perhaps type-specific antigenic determinants are present on the SDS-denatured forms of these proteins. It is also suggested from these studies that this protein is a serodominant surface antigen and that antigenic heterogeneity in the portions of the protein that are exposed on the surfaces of the organisms differentiates the species into two distinct serogroups (Table 1). The B complex is composed of serotypes B, Ba, D, E, F, G, K, Ll, L2, and L3. These serotypes all cross-reacted strongly by micro-if with anti- MOMP sera serotypes B, D, G, and L2. In contrast, these anti-momp sera reacted only weakly with the C complex isolates A, C, H, I, and J. Antiserum specific against the MOMP of the H serotype was highly reactive with its homologous EB antigen but failed to cross-react significantly with any of the other serotypes. These serological results suggest that B complex organisms have similar, but not identical, antigenic sites of their MOMPs exposed on their surfaces, whereas the exposed antigenic determinants'of the MOMPs of C complex organisms appear to be more unique immunologically. These results are quite similar to the patterns observed by the micro-if test with antisera raised against viable C. trachomatis organisms. In that system, some of the C complex strains such as H and I are far less likely to cross-react with other C. trachomatis serotypes, and B complex strains are often markedly cross-reactive. Obviously, one cannot directly compare micro-if groupings with those obtained with antisera against MOMPs until all serotype-specific MOMPs have been tested. However, with the results at hand, it seems clear that the antigens responsible for the serotype-specific responses are, at least in part, on the portions of the MOMP exposed on the surfaces of the organisms. It is clear that the MOMP antigen is distinct from other protein antigens described for C. trachomatis organisms. The C. trachomatis species-specific 155K polypeptide antigen described by Caldwell et al. (3, 5) differs greatly in subunit MW compared with the 38 to 44.5K polypeptides described in this study. Although portions of the 155K protein are exposed on the cell surface (15), quantitatively this protein is only a minor constituent of the organism (3). The MOMP antigen is also apparently unrelated to

1030 CALDWELL AND SCHACHTER the type-specific surface protein of approximately 30K described by Sacks and MacDonald (14) and Hourihan et al. (9). Besides the obvious discrepancy in size, the above investigators have clearly shown that antisera against the 30K polypeptide reacts only with this protein in Triton X-100 extracts from homologous EB preparations. The antigenic unrelatedness between MOMPs of strains between the two chlamydial species is not surprising. Members of the two species are known to differ considerably antigenically (4) and share only 5 to 10% DNA homology (10). The findings reported here provide further evidence supporting the genetic differences between the two chlamydial species. The ability of antibodies raised against SDSdenatured polypeptides to react with acetonefixed whole Chlamydia indicates that at least some of the antibodies are specific for the more native form of the MOMP. These findings indicate that the antigenic sites being detected are nonconfirmational since antibody raised against SDS-solubilized MOMP probably recognizes only primary and some secondary protein structures. These nonconfirmational antigenic determinants are clearly important, since they appear to be the antigen(s) to which a considerable amount of antibody is made after immunization with intact viable organisms. This conclusion is supported by the close similarity in serospecificity observed by micro-if with anti-l2 MOMP (39.5K) and antiserum raised against intact L2 organisms (Table 1). The differences in reactivity of anti-momp sera observed by rocket immunoelectrophoresis and micro-if indicate that these two techniques are measuring different antigenic determinants present on the protein. For example, anti-h MOMP serum intensely precipitated each of the heterologous C. trachomatis-soluble MOMP antigens by rocket immunoelectrophoresis. In contrast, this same antiserum was only weakly cross-reactive with the same C. trachomatis serotypes by micro-if. These dissimilar observations may simply reflect the availability of certain antigenic determinants of the protein to react with antibody in each of the two assays. For example, the soluble form of the protein used in rocket immunoelectrophoresis probably has numerous antigenic determinants that are accessible to react with antibody. In comparison, the micro-if test would measure those determinants on the portion of the protein exposed on the surface of the outer membrane of the organism. The findings of this study are perhaps also relevant with respect to the diagnosis of C. trachomatis infection and deserve discussion. It seems reasonable, considering the properties of INFECT. IMMUN. this protein, that it would be a logical candidate antigen to detect in clinical specimens suspected of containing C. trachomatis organisms. The properties that make this protein desirable in the development of such an assay are: (i) it is the most abundant protein constituent of the organism, comprising as much as 60% of the total outer membrane protein (2) and approximately 10 to 12% of the total protein of the whole organisms (unpublished data). (ii) The protein is shared by both the developmental forms of Chlamydia spp. (1) and therefore would constitute a considerable antigenic mass since a substantial proportion of a given chlamydial population within intracellular inclusions consists of noninfectious reticulate bodies. It should also be considered, although there is no supporting data for this possibility, that the protein may exist within cytoplasmic inclusions in a non-cell-associated form. (iii) In its soluble form, the protein is C. trachomatis species specific with respect to its antigenic properties. This property is particularly attractive considering the objective, since only a single antiserum with antibodies specific for the common antigenic determinant(s) of the protein(s) would be required, making the assay system independent of the infecting C. trachomatis strain. This characteristic is important since there are at least 15 distinct serotypes of C. trachomatis (7). The use of monoclonal antibodies or polyclonal antibodies prepared by immunoadsorption would be particularly useful in the development of such an assay. These studies are currently under way in this laboratory. ACKNOWLEDGMENTS We thank Jim Simmons and Linda Perry for their expert technical experience, Susan Smaus for secretarial assistance in preparing this manuscript, and the professional staff of the Department of Microbial Structure and Function for their critical evaluation of the manuscript. LITERATURE CITED 1. Caldwell, H. D. 1980. Relation of surface membrane proteins to virulence of the chlamydial agents, p. 47-54. In G. R. O'Conner (ed.), Immunologic diseases of the mucous membranes. Masson Publishing USA, Inc., New York. 2. Caldwell, H. D., J. Kromhout, and J. S. Schachter. 1981. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 31:1161-1176. 3. Caldwell, H. D., and C. C. Kno. 1977. Purification of a Chlamydia trachomatis-specific antigen by immunoadsorption with monospecific antibody. J. Immunol. 118:437-441. 4. Caldwell, H. D., C. C. Kuo, and G. E. Kenny. 1975. Antigenic analysis of chlamydiae by two-dimensional immunoelectrophoresis. I. Antigenic heterogeneity between C. trachomatis and C. psittaci. J. Immunol. 115:963-968. 5. Caldwell, H. D., C. C. Kuo, and G. E. Kenny. 1975. Antigenic analysis of chlamydiae by two-dimensional immunoelectrophoresis. II. A trachoma-lgv-specific antigen. J. Immunol. 115:969-975.

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