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INFECTION AND IMMUNITY, Jan. 1990, p. 205-213 Vol. 58, No. 10019-9567/90/010205-09$02.00/0Copyright © 1990, American Society for Microbiology

Characterization of Murine Monoclonal and Murine, Rabbit,and Human Polyclonal Antibodies against

Chlamydial LipopolysaccharideLORE BRADE,' OTTO HOLST,1 PAUL KOSMA,2 YOU-XUN ZHANG,3t HANS PAULSEN,4 REA KRAUSSE,5

AND HELMUT BRADE1*Division ofBiochemical Microbiology, Institut fur Experimentelle Biologie und Medizin, Forschungsinstitut Borstel,

Parkallee 22, D-2061 Borstel, Federal Republic of Germany1; Institut fur Chemie der Universitat fur Bodenkultur, A-i 180Vienna, Austria2; Laboratory of Microbial Structure and Function, Rocky Mountain Laboratory, Hamilton, Montana

598403; Institut fur Organische Chemie der Universitat, D-2000 Hamburg, Federal Republic of Germany 4; and Abteilungfur Medizinische Mikrobiologie der Universitat, D-2100 Kiel, Federal Republic of Germany5

Received 10 July 1989/Accepted 19 September 1989

Murine monoclonal and rabbit, murine, and human polyclonal antibodies against chlamydial lipopolysac-charide (LPS) were characterized by the passive hemolysis and passive hemolysis inhibition assays and byabsorption experiments with LPSs of Chlamydia psittaci, Chlamydia trachomatis, and a recombinant strain ofSalmonella minnesota Re (r595-207) expressing the chlamydia-specific LPS epitope, as well as natural andsynthetic partial structures of chlamydial LPS. Eleven monoclonal antibodies of the immunoglobulin M and Gclasses were characterized as chlamydia-specific by their failure to react with Re-type LPS, binding to a similarepitope for which the trisaccharide a-3-deoxy-D-manno-2-octulosonic acid (KDO)-(2-8)-a-KDO-(2-4)-CL-KDOwas an absolute prerequisite. For optimal binding, parts of the lipid A moiety were also involved; however,phosphoryl and ester-linked acyl groups and the reducing glucosamine residue of lipid A were dispensable. Asimilar antibody specificity was detected in lapine and murine hyperimmune sera after immunization withchlamydia, in addition to those recognizing more complex (e.g., those requiring the presence of phosphorylresidues) and less complex epitopes. Among the latter were those cross-reacting with Re-type LPS, which couldbe removed by absorption. The titers of different antibody specificities, in particular the ratio of chlamydia-specific to cross-reactive antibodies, present in murine polyclonal antisera depended on the immunizationprotocol. The preferential formation of chlamydia-specific antibodies was observed after immunization withliposome-incorporated immunogens. Human sera from patients with suspected genital chlamydial infectionswere also found to contain chlamydia-specific and cross-reactive antibodies, the latter of which could beremoved by absorption with Re-type LPS.

Chlamydiae are pathogenic, obligatory intracellular para-sites causing a variety of diseases in animals and humans.Little is known about molecular mechanisms of pathogenic-ity of these unique microorganisms and the host defensemechanisms against them. Surface structures of chlamydiaeare involved in the early steps during infection (adhesion andpenetration), and these at the same time are surface antigensagainst which antibodies are raised during infection.

It is known that chlamydiae possess a surface glycolipidantigen which harbors a genus-specific epitope containing animmunodominant sugar chemically related to 3-deoxy-D-manno-2-octulosonic acid (KDO) (11, 12). Chemical studieson this antigen have shown that it contains typical chemo-taxonomical markers for lipopolysaccharide (LPS), such asKDO, D-glucosamine, phosphorus, and long-chain 3-hy-droxy fatty acids (9, 19). This composition is similar to thatof LPS from enterobacterial Re mutants (8) and thus pro-vides a basis to understand cross-reactions between chla-mydiae and other bacterial species on the molecular level.Studies on monoclonal antibodies against the Re-type LPS,containing an a-2,4-linked KDO disaccharide in its saccha-ride portion, have shown that antibodies which recognize theKDO (5) or lipid A region (3) or both (22) can be selected.

* Corresponding author.t Permanent address: Beijing Institute of Ophthalmology, Beijing

100005, People's Republic of China.

Such antibodies also react with chlamydial LPS (1) and areobviously present in polyclonal anti-chlamydia antisera, thuscausing their cross-reaction with Re-type LPS. In addition,monoclonal and polyclonal antibodies which recognize thechlamydia-specific LPS epitope and do not cross-react withRe-type LPS have been described (7, 9, 10, 20).We found that chlamydial LPS contains a linear KDO

trisaccharide of the sequence (x-KDO-(2-8)-a-KDO-(2-4)-KDO, in which the oa-2,8-linked disaccharide portion wasassumed to represent the immunodominant region of thegenus-specific epitope (1, 16). In the meantime, we haveprepared a variety of synthetic compounds and chemicallydefined partial structures of LPS which can be used asantigens to characterize chlamydial LPS antibodies. Herewe report on our results obtained with murine monoclonalantibodies and polyclonal antisera from mice, rabbits, andhumans.

MATERIALS AND METHODS

Bacteria and bacterial LPS. The Re mutant of Salmonellaminnesota (strain R595) was transformed with plasmidpFEN207 (17) and propagated as described previously (6).LPS was extracted by the phenol-chloroform-petroleumether method (14) and purified by repeated ultracentrifuga-tion, followed by conversion to the uniform triethylammo-nium salt after electrodialysis (13). This LPS has been shownto contain the genus-specific epitope of chlamydial LPS (1);

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206 BRADE ET AL.

CONH2 CONH2

I IH-[(CH2-CH)x-CH2-CH-(CH2-CH)y]n-H

CH20 - R

Compound Nature of substituent Amt (nmol) of ligand/

(abbreviated) R = mg of copolymerisate

KDO-PA KDO-(2 -e

2.4-KDO2-PA KD0-(2-.4)-KD0-(2--e

2.8-KDO2-PA KDO-(2- 8)-KDO-(2--"2.8.4-KD03-PA KDO-(2--_8)-KDO-(2-e4)-KDO-(2-.,

323

338

295

106

FIG. 1. Synthetic copolymerization products used as antigens in this study. KDO-PA, KDO-polyacrylamide. Values were determined bythe thiobarbiturate assay.

it will be referred to as r595-207 LPS. LPS was prepared ina similar way from an Escherichia coli Re mutant (strainF515). Chlamydial LPS of Chlamydia psittaci PK 5082 (23)and Chlamydia trachomatis serotype L2 were prepared asreported previously (9, 19).

Preparation of LPS partial structures. Alkali-treated LPS(LPS-OH); de-O-acylated and dephosphorylated LPS (LPS-HF); and de-O-acylated, dephosphorylated, and reducedLPS (LPS-HFr) were prepared as reported previously (22).

Synthetic antigens. The copolymerization products listedin Fig. 1 were synthesized as described previously (15, 16).Sodium 3-deoxy-at-D-manno-2-octulopyranosylonate-(2---6)-2 -deoxy -2 - [(R) - 3 -hydroxytetradecanamido] - , -D-glucopyr-anosyl-(1---6)-2-deoxy-2-[(R)-3-hydroxytetradecanamido]-D-glucose and disodium [3-deoxy-ot-D-manno-2-octulopyranos-ylonate - (2-*4) - 3 - deoxy - a - D - manno - 2 - octulopyranosyl-onate] -(2-6) -2 -deoxy - 2 - [(R) - 3 -hydroxytetradecanamido] -,1- D-glucopyranoanosyl] -(1-+6)-2-deoxy-2- [(R) -3 -hydroxy-tetradecanamido]-D-glucose were synthesized as previouslydescribed (C. Krogmann, Ph.D. thesis, University of Ham-burg, Hamburg, Federal Republic of Germany, 1989; 21).These compounds are partial structures of Re-type LPScontaining the glucosamine backbone of lipid A with twoamide-linked 3-hydroxymyristic acid residues and one ortwo KDO residues and will be abbreviated as KDO-GlcNhm2 and KDO2-GlcNhm2, respectively.

Preparation of immunogens. Liposome-incorporated im-munogens were prepared as previously described (3). Heat-killed bacteria were prepared by boiling an overnight cultureat 100°C for 1 h. Sheep erythrocyte (SRBC)-coated immu-nogens were prepared as described below by using 200 ,ug ofantigen per 200 ,ul of SRBCs.Animal antisera. Rabbit antisera against heat-killed bacte-

ria were prepared as described previously (6). Mouse anti-sera from female, 8- to 10-week-old BALB/c mice in groupsof four were used. Antisera against heat-killed bacteria wereprepared as described for rabbits; however, injections weredone intraperitoneally. Sera against SRBC-coated and lipo-some-incorporated immunogens were obtained after fiveintraperitoneal injections of increasing amounts (20 to 50 ,ug)of antigen in a total volume of 200 ,ul of SRBCs or liposomesuspension, respectively, over a 2-month period. The ani-mals were then tested for the presence of antibodies againstchlamydial LPS. The one with the highest titer was used forthe preparation of monoclonal antibodies, whereas the oth-

ers were given a booster 3 months later and were exsan-guinated 1 week after the last injection.

Serology. (i) Passive hemolysis and passive hemolysis inhi-bition assays. The hemolysis test was carried out in 96-wellmicrodilution plates. SRBCs were washed three times inphosphate-buffered saline. Packed cells (0.2 ml) in 5 ml ofphosphate-buffered saline were mixed with graded amounts(1 to 200 ,ug) of the respective antigen and incubated at 37°Cfor 30 min with occasional shaking. The antigen-coated cellswere washed three times in phosphate-buffered saline andfinally suspended in 40 ml of Veronal-buffered saline (VBS)to give a 0.5% suspension. Serial twofold dilutions of anti-body in VBS (50 pI) were mixed with 50 pul of antigen-coatedSRBCs and 25 pul of guinea pig serum (prechecked for theabsence of chlamydial LPS antibodies and diluted 1:20 inVBS) as a source of complement, followed by incubation at37°C for 1 h. After centrifugation, 50% endpoint titers weredetermined. One hemolytic unit is defined as the amount ofantibody causing 50% of hemolysis under these test condi-tions. The passive hemolysis inhibition test was performedby preincubating 3 to 4 hemolytic units of antibody in 25 pIof VBS with serial twofold dilutions of inhibitor in 25 ,ul ofVBS at 37°C for 15 min. After the addition of antigen-coatedSRBCs (50 pul) and 25 pu1 of complement, the plates wereincubated at 37°C for 1 h. Inhibition values are expressed asamount of inhibitor causing 50% inhibition of hemolysis.

(ii) Enzyme immunosorbent assay. Polyvinyl plates (96-well, type 3911; Falcon Plastics) were coated with 50 1.l of asolution of LPS (4 p,g/ml) in phosphate-buffered saline (PBS)at 37°C overnight, followed by blocking in PBS containing10% defatted dry milk at 37°C for 1 h. The following stepswere done according to standard procedures.

(iii) Absorption. Absorption was carried out at 4°C for 1 hon 500-pA samples of prediluted antisera (1:10 in PBS) with50 pI of packed SRBCs coated with the respective antigen.Absorption with uncoated SRBCs served as a control.Monoclonal antibodies. The following clones were ob-

tained after immunization with purified elementary bodies ofC. psittaci PK 5082 (23) (clones S5-10 and S10-3) and C.trachomatis serotype L2 (clone L21-6), serotype E (cloneEVI-Hl), serotype G (clones GIII-C3 and GII-B3), andserotype F (clones FVI-A4 and FI-A6). Clones S15-1, S15-2,and S15-6 were obtained after immunization with recombi-nant r595-207 LPS-OH, and clone S19-3 was obtained afterimmunization with recombinant r595-207 LPS-HF, both

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ANTIBODIES AGAINST CHLAMYDIAL LPS 207

TABLE 1. Reactivity of monoclonal antibodies with chlamydial and recombinant LPSs and with partial structures from them

Hemolytic titer against SRBCs coated with:

Antibody Isotype S. minnesota r595-207 C. psittaci C. trachomatis

LPS (150)" LPS-OH (150) LPS-HFr (50) LPS (200) (100) LPS-OH (200) LPS (200)

S10-3 IgM <4 64 64 <4 64 <4 <4S15-1 IgM <4 128 512 NDC <4 <4 <4S15-2 IgM 32 512 512 ND 512 64 64S15-6 IgM <4 1,024 1,024 ND 128 <4 64S19-3 IgM 64 1,024 1,024 ND 1,024 512 128S5-10 IgG3 64 64 32 <4 64 64 128L21-6 IgG3 64 64 64 <4 64 64 64E VI-Hl IgG2a 12,800 6,400 12,800 12,800 6,400 6,400 12,800G III-C3 IgG3 1,600 3,200 3,200 400 3,200 1,600 800G II-B3 IgG2b 400 400 400 100 400 400 200F VI-A4 IgG3 1,600 1,600 1,600 1,600 1,600 800 800F I-A6 IgG2b 1,600 800 400 400 800 400 1,600

a Amount (micrograms) of antigen per 0.2 ml of packed SRBCs.b De-0-acylated with sodium methylate.c ND, Not determined.

incorporated into liposomes. Screening was performed withRe-type LPS of S. minnesota R595 and E. coli F515 and withLPS and LPS-HFr of S. minnesota r595-207. Subcloning anddetermination of immunoglobulin isotypes and subgroupswere carried out by conventional methods. A monoclonalantibody recognizing a single oa-pyranosidically linked KDO(5) was provided by B. J. Appelmelk, Amsterdam, TheNetherlands.Human antisera. Sera which were sent to our routine-

serology laboratory were collected from male and femalepatients with clinical symptoms of genital infections sus-pected to be caused by chlamydiae.

RESULTS

Characterization of murine monoclonal antibodies. Anti-bodies were screened for chlamydia specificity and for theirability to fix complement. Eleven antibodies were character-ized further by using recombinant and chlamydial LPSs, aswell as synthetic and natural partial structures from them.Table 1 shows the results. Clones EVI-Hl and FVI-A4exhibited similar titers with all antigens tested, with differ-ences not exceeding one dilution step. Clones S5-10 andL2I-6 gave similar results; however, they were negative withSRBCs coated with native LPS of C. psittaci. All immuno-globulin M (IgM) antibodies had significantly lower titerswith native LPS compared with those against LPS-OH andLPS-HFr. Since the epitope density of a given antigen on theerythrocyte surface is influenced by its physicochemicalproperties (3), some antibodies were titrated against SRBCscoated with graded amounts of antigen. The results areshown in Table 2. Clones S5-10 and L21-6 yielded themaximal titers of 128 and 64, respectively, with all threeantigens. However, the amount of antigen causing a suffi-cient epitope density varied over a wide range. Whereas 128,Ig per 0.2 ml of SRBCs was required with native LPS toobtain the optimal reactivity, 16 ,ug was enough with LPS-OH, and even smaller amounts were sufficient with LPS-HFr (8 and 4 ,ug for clones S5-10 and L2I-6, respectively).With clones S15-1 and S15-2, a similar pattern was observed,although at a higher level. When clone S15-1 was tested withnative LPS, no activity was seen even at the highest dose of200 jig per 0.2 ml of SRBCs. Larger amounts were nottested, since nonspecific lysis then occurred. These data

suggested that the negative results shown in Table 1 did notreflect major differences in antibody specificity but, rather,affinity. Thus, the hemolytic activity of an antibody dependson the epitope density on the erythrocyte membrane, whichin turn depends on the physicochemical properties of thesensitizing antigen. Therefore, we tested all antibodiesagainst SRBCs coated with graded amounts of various LPSpreparations of C. psittaci, C. trachomatis, and the recom-binant strain r595-207. The results are shown in Table 3,where the amounts of antigen yielding half-maximal titersare listed. The IgM antibodies required larger amounts ofantigen than did those of the IgG type, and native LPS had

TABLE 2. Hemolytic titers of monoclonal antibodies againstSRBCs coated with graded amounts of recombinant LPS and

partial structures thereof

Amt Hemolytic titer obtained with

Antigen ........(,ug)/0.2monoclonal antibody:ml of S5-10 L21-6 S15-1 S15-2SRBCs (IgG3) (IgG3) (IgM) (IgM)

S. minnesota 8 <4 <4 <4 <4r595-207 16 <4 <4 <4 <4LPS 32 <4 4 <4 <4

64 16 32 <4 <4128 128 64 <4 32200 128 64 <4 64

S. minnesota 4 <4 <4 <4 <4r595-207 8 32 32 <4 <4LPS-OH 16 64 64 <4 <4

32 128 64 <4 <464 128 64 <4 64128 128 64 16 256200 128 64 128 512

S. minnesota 1 4 16 <4 <4r595-207 2 32 32 <4 <4LPS-HFr 4 64 64 <4 <4

8 128 64 <4 <416 128 64 8 12832 128 64 256 51264 128 64 512 512128 128 64 512 512200 128 64 512 512

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208 BRADE ET AL.

TABLE 3. Coating efficiency of recombinant and chlamydial LPSs and partial structures from them for SRBCs

Amt (,ug) per 0.2 ml of packed SRBCs yielding optimal titersa

Antibody Isotype S. minnesota r595-207 C. psittaci C. trachomatis

LPS LPS-OH LPS-HFr LPS LPS-OH LPS-OCH3 LPS

EVI-Hl IgG2a 64 8 2 32 8 16 32GIII-C3 IgG3 128 16 8 >200 16 16 128GII-B3 IgG2b 128 16 8 >200 64 16 >200FVI-A4 IgG3 128 32 8 200 64 32 128FI-A6 IgG2b 128 16 8 >200 128 32 128L2I-6 IgG3 64 8 2 >200 8 32 128S5-10 IgG3 128 16 2 >200 16 32 128S10-3 IgM >200 64 16 >200 >200 32 >200S15-1 IgM >200 200 32 NDb ND ND NDS15-2 IgM >200 128 32 ND ND ND NDS15-6 IgM >200 128 64 ND ND ND NDS19-3 IgM >200 64 16 ND ND ND ND

a Amount of antigen yielding half-maximal titers.b ND, Not determined.

a lower coating efficiency than did LPS-OH, LPS-OCH3 orLPS-HFr. LPS of C. psittaci was even less active than C.trachomatis LPS. Since antibodies of the IgM isotype aremore efficient in lysing antigen-coated erythrocytes in com-plement-dependent assays, the results suggested a low af-finity of the selected IgM monoclonal antibodies.

Next, inhibition experiments were performed (Table 4).LPS and LPS-OH were good inhibitors for all antibodiesexcept clone S19-3, which could not be inhibited withLPS-OH in amounts of up to 1 jig. LPS-HFr was ofcomparable activity, when compared with LPS-OH (thedifference not exceeding one dilution step), with clonesS10-3, L21-6, and S5-10; it was less active (up to two dilutionsteps difference) with clones EVI-Hl, GIII-C3, GII-B3, andFVI-A4. It was not active (more than two dilution stepsdifference) with clones S15-2, S19-3, and FI-A6. We thenused synthetic antigens containing the complete KDO regionof chlamydial LPS, i.e., the ot-KDO-(2---8)-x-KDO-(2->4)-a-KDO trisaccharide and mono- and disaccharide partialstructures thereof for the inhibition of monoclonal antibod-ies. A monoclonal antibody (clone A20) recognizing a singleoa-pyranosidically linked KDO (5) was included as a positivecontrol. None of the chlamydial antibodies could be inhib-ited with KDO mono- or disaccharide structures in amountsof up to 25 ,ug. The KDO trisaccharide inhibited all clonesexcept S15-2, S19-3, GIII-C3, and FI-A6 with amounts

TABLE 4. Inhibition of monoclonal antibodies with recombinantLPS and partial structures thereof

Inhibition value (ng) obtained withAntibody' S. minnesota r595-207

LPS LPS-OH LPS-HFr

S10-3 4 32 32S15-2 16 63 >1,000S19-3 63 >1,000 >1,000L21-6 32 32 63S5-10 63 32 63EVI-Hl 63 32 125GIII-C3 63 32 250GII-B3 63 32 250GVI-A4 63 63 250FI-A6 125 63 1,000

a Antibodies were diluted to give 3 to 4 hemolytic units with SRBCs coatedwith S. minnesota r595-207 LPS-OH.

between 2.5 and 10 ,ug. However, the inhibition of the KDOmonosaccharide-specific clone A20 was more effective by 1order of magnitude (data not shown). These results show (i)that in LPS, ester-linked acyl and phosphoryl groups and thereducing glucosamine of lipid A do not participate in theconstitution of the epitope recognized and (ii) that thecomplete KDO trisaccharide is required for binding.

Characterization of rabbit polyclonal antisera against chia-mydial LPS. IgM- and IgG-rich antisera obtained after im-munization with chlamydia were titrated in the passivehemolysis assay by using chlamydial and recombinant LPSsand natural and synthetic partial structures from them. Theresults are shown in Table 5. All animals had high titersagainst the antigens tested, those with r595-207 LPS, LPS-OH, and LPS-HFr being comparable, never differing bymore than one dilution step. In addition, antibodies againstRe LPS of E. coli and against the synthetic partial structuresKDO-GlcNhm2 and KDO2-GlcNhm2 were observed, withsignificant titers. To determine whether these were due tothe presence of cross-reactive or distinct antibodies, absorp-tion experiments were performed (Table 6). Absorptionswere not carried out successively, but separately. Theactivities of the respective absorbed samples against thehomologous antigen could be completely abolished; how-ever, in a few cases, two absorptions were required. Afterabsorption with Re LPS-OH, all antisera still reacted withrecombinant r595-207 LPS-OH and LPS-HFr with hightiters, indicating that Re-reactive and chlamydia-reactiveantibodies were present and separated from each other byabsorption. Whereas the latter were specific for recombinantLPS, the former cross-reacted with recombinant LPS, bywhich they could be absorbed. The Re-reactive antibodiescould be further divided into phosphate dependent andphosphate independent, as shown by absorption with thephosphateless synthetic partial structure KDO2-GlcNhm2,which did not result in the removal of all antibodies reactingwith phosphate-containing Re LPS-OH. Phosphate depen-dency was also observed with chlamydia-specific antibodies.Whereas absorption with r595-207 LPS-OH abolished thereactivity towards the homologous antigen and against thedephosphorylated LPS-HFr, absorption with LPS-HFr re-moved only the homologous antibody and not that againstLPS-OH. As expected, absorption with LPS-HFr was alsounable to abolish the reactivity of phosphate-dependentRe-reactive antibodies.

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TABLE 5. Hemolytic titers of polyclonal rabbit antisera raised against chlamydial LPS

Hemolytic titer against SRBCs coated with:

Natural structures Synthetic structuresAntiseruma Immunogenb

S. minnesota r595-207 E. coli

LPS LPS-OH LPS-HFr Re LPS-OH KDO-GlcNhm2 KDO2-GlcNhm2K49/16 C. psittaci 2,560 5,120 5,120 2,560 2,560 1,280K175/16 C. psittaci 1,280 1,280 2,560 1,280 2,560 640K54/16 C. trachomatis 10,240 10,240 10,240 2,560 10,240 1,280K55/16 C. trachomatis 5,120 10,240 10,240 5,120 10,240 2,560K174/16 C. trachomatis 2,560 5,120 2,560 2,560 2,560 1,280K137/60 C. trachomatis 1,280 1,280 2,560 1,280 2,560 1,280K138/16 C. trachomatis 10,240 10,240 10,240 2,560 5,120 1,280K138/60 C. trachomatis 2,560 2,560 1,280 640 640 320

a Sera were collected on day 16 (IgM rich) or on day 60 (IgG rich).b Purified elementary bodies (C. psittaci) and purified outer membranes (C. trachomatis) were used for immunization.

Re-reactive antibodies not requiring phosphate could stillbe represented by a variety of antibody specificities, such asthose recognizing a single terminal KDO monosaccharide, aKDO disaccharide (5), or a KDO disaccharide together withparts of the glucosamine backbone of lipid A (22). Therefore,absorption was performed with synthetic KDO-GlcNhm2and KDO2-GlcNhm2. The results are shown in Table 7.Absorption with KDO-GlcNhm2 resulted in the removal ofthe homologous reactivity and, in some cases, in the reduc-tion or complete absorption of the reactivity directed againstKDO2-GlcNhm2, indicating that KDO monosaccharide- ordisaccharide-reactive antibodies were present in these anti-sera, with different titers. Most surprisingly, absorption withKDO2-GlcNhm2 was unable to abolish the reactivitytowards KDO-GlcNhm2, although the latter represents apartial structure of the former: Therefore, these antibodiescould not be specific for a KDO monosaccharide but wereKDO-GlcNhm2 specific and did not cross-react with themore complex structure KDO2-GlcNhm2. Accordingly,these antibodies could not be inhibited with KDO-polyacryl-amide but were inhibited with KDO-GlcNhm2 (data notshown). Such antibodies have been also detected in serafrom rabbits immunized with Re bacteria (22).

Characterization of murine polyclonal antisera against chia-mydial LPS. Hyperimmune sera were prepared in mice byimmunization with heat-killed bacteria and liposome-incor-porated immunogens. The titers obtained in the passivehemolysis assay are listed in Table 8. All animals hadantibodies against recombinant r595-207 LPS, LPS-OH, andLPS-HFr; however, the titers varied depending on theimmunogen. Highest titers were observed after immuniza-tion with LPS-OH and LPS-HF, both used as liposome-incorporated immunogens. Also, the type of the predomi-nant antibody specificity depended on the immunizationprotocol. Whereas the reactivities with r595-207 LPS, LPS-OH, and LPS-HFr were comparable in sera from animalsimmunized with heat-killed bacteria, the others varied con-siderably. Most prominent were the differences in seraobtained from animals immunized with liposome-incorpo-rated r595-207 LPS-HF. In this case, the titer against LPSwas up to four dilution steps lower than that against LPS-OHor LPS-HFr. In addition, antibodies against Re LPS and thesynthetic partial structure KDO2-GlcNhm2 were detected.None of the animals had titers against the synthetic antigenKDO-GlcNhm2. Again, the titers of such cross-reactiveantibodies were dependent on the immunization schedule.Whereas immunization with liposome-incorporated LPS and

LPS-OH yielded high titers of Re-reactive antibodies, theother immunogens did not stimulate their production. Theseresults showed that the immune response to chlamydial LPSdepends on the primary structure of the antigen on the onehand and on the physicochemical environment on the other.

Characterization of human patient antisera. Sera frompatients with suspected genital chlamydial infection weretested in the passive hemolysis assay against SRBCs coatedwith S. minnesota r595-207 LPS-OH, r595-207 LPS-HFr, E.coli Re LPS-OH, and synthetic KDO-GlcNhm2. The corre-lation of titers against the two recombinant LPS was deter-mined for 54 sera (Table 9); the linear correlation coefficientwas 0.883, indicating that antibodies against the native andthe dephosphorylated LPSs were present in comparabletiters. From 54 sera, 26 did not react with E. coli Re LPS-OHand synthetic KDO-GlcNhm2. Those being positive with oneof these antigens were absorbed with E. coli Re LPS-OH andretested. A representative selection of sera is shown in Table10. In all cases, the antibodies reacting with E. coli ReLPS-OH and synthetic KDO-GlcNhm2 were absorbed. Insome sera, e.g., serum no. 36, 40, and 49, the titer againstr595-207 LPS-OH was not significantly reduced, whereas inothers (no. 42, 45, 46, and 56) a significant reduction by morethan two dilution steps was observed. The reactivitiesagainst the dephosphorylated r595-207 LPS HFr were re-duced to a comparable extent, except for serum no. 49 and50.

DISCUSSIONThe LPS and the major outer membrane protein are

integral components of the cell wall of chlamydial elemen-tary bodies and, at the same time, represent their majorsurface antigens. Whereas the major outer membrane pro-tein harbors genus-, species-, and subspecies-specificepitopes, little is known about the chemical and antigenicstructure of chlamydial LPS, although the latter is used as animmunochemical marker to detect chlamydiae in clinicalspecimens and as an antigen to detect chlamydial antibodiesin patient body fluids. Chemical (1, 9, 19) and serological (1,7, 9, 18, 21) investigations have shown that chlamydial LPSis structured in a manner similar to enterobacterial Re LPSand shares antigenic determinants with this type of LPS. Inaddition, it harbors a chlamydia-specific epitope which isgenus specific (10) and based on the unique chemical struc-ture of the chlamydial KDO region, i.e., a KDO trisaccha-ride of the sequence a-KDO-(2-8)-a-KDO-(2-4)-KDO (1, 16).Although some of the immunochemical properties of chla-

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210 BRADE ET AL.

TABLE 6. Determination by absorption of different antibody specificities present in rabbit antiseraafter immunization with chlamydial LPS

Antigen for Hemolytic titer against SRBCs coated withb:Antiserum absorptiona E. coli Re LPS-OH KDO2-GIcNhm2 r595-207 LPS-OH r595-207 LPS-HFr

1;r At% OBInYl t,- <KNcC<t Cf 1 II c 1 IMSKBCEc Re LPS-OHKDO2-GlcNhm2r595-207 LPS-OHr595-207 LPS-HFr

SRBCEc Re LPS-OHKDO2-GIcNhm2r595-207 LPS-OHr595-207 LPS-HFr

SRBCEc Re LPS-OHKDO2-GlcNhm2r595-207 LPS-OHr595-207 LPS-HFr





SRBCEc Re LPS-OHKDO2-GlCNhm2r595-207 LPS-OHr595-207 LPS-HFr

<20640<20320

1,280<20160<20160

2,560<20

2,560<20640

5,120<20640<20640

2,560<20320<20640

1,280<20640<20320

2,560<20

1,280<20

1,280

640<20320<20160

2,)6u<20<20<20<20

640<20<20<20<20

1,280<20<20<20<20

2,560<20<20<20<20

1,280<20<20<20<20

1,280<20<20<20<20

1,280<20<20<20<20

320<20<20<20<20

J), I U2,5602,560<20320

D),IL2,5602,560<20<20

2,560640640<20<20

1,280640640<20<20

10,2405,1205,120<20

1,280

10,2405,1202,560<20

2,560

5,120640

1,280<20640

1,2801,2801,280<20640

10,2402,5605,120<20

5,120

2,5601,2802,560<20320

10,2405,1202,560<20<20

10,2405,1202,560<20<20

2,560640320<20<20

2,560640640<20<20

10,2405,1205,120<20<20

1,280640640<20<20

a Antisera (500 ,ul, prediluted 1:20 in PBS) were absorbed at 4°C with 50 pd of antigen-coated SRBCs. Ec, E. coli.b Homologous titers in italics.

mydial LPS were described already many years ago in aphenomenological manner (11, 12), a detailed analysis of thiscomplex antigen on the molecular level only became ame-nable by the work of Nano and Caldwell (17), who cloned agene which coded for an enzyme (presumably a glycosyl-transferase) which, when introduced into Re mutant bacteria(6), resulted in the expression of a chlamydia-specific LPSepitope as defined by monoclonal antibodies. The availabil-ity of unlimited amounts of such recombinant LPS allowedus to determine the chemical structure of the KDO region asao-KDO-(2-8)-ot-KDO-(2-4)-KDO (1, 16). Based on thisknowledge, we started an immunochemical investigation onthe specificity of mono- and polyclonal antibodies, theresults of which are reported here.

By using different immunogens (purified elementary bod-ies, cell walls of C. psittaci and C. trachomatis, and isolatedchlamydial and recombinant LPSs), we obtained 11 mono-clonal antibodies of the IgG and IgM types. The resultsobtained in the passive hemolysis assay in which differentchlamydial and recombinant LPS preparations, as well asnatural and synthetic partial structures from these sources,were used showed that none of the antibodies required thepresence of ester-linked fatty acids or phosphate, since theyreacted similarly with LPS-OH and LPS-HFr of S. minne-sota r595-207. This also indicated that the reducing glu-cosamine residue was not a major component of the epitoperecognized by these antibodies. However, some antibodiesexhibited low reactivities with some LPS preparations, e.g.,

K49

K175

K54

K55

K174

K137/60

K138/16

K138/60

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TABLE 7. Hemolytic titers of polyclonal rabbit antisera raisedagainst chlamydial LPS with synthetic partial structures

of Re-type LPS

Hemolytic titer against SRBCsAntiserum Antigen for coated withb:absorptiona

KDO-GlcNhm2 KDO2-GIcNhm2

K49 SRBC 2,560 1,280KDO-GlcNhm2 <20 160KDO2-GlcNhm2 640 <20

K175 SRBC 2,560 640KDO-GlcNhm2 <20 <20KDO2-GlcNhm2 640 <20

K54 SRBC 10,240 1,280KCO-GIcNhm2 <20 <20KDO2-GlcNhm2 2,560 <20

K55 SRBC 10,240 2,560KDO-GlcNhm2 <20 <20KDO2-GlcNhm2 2,560 <20

K174 SRBC 2,560 1,280KDO-GlcNhm2 <20 80KDO2-GlcNhm2 320 <20

K137/60 SRBC 2,560 1,280KDO-GlcNhm2 <20 640KDO2-GlcNhm2 640 <20

K138/16 SRBC 5,120 1,280KDO-GlcNhm2 <20 640KDO2-GlcNhm2 2,560 <20

K138/60 SRBC 640 320KDO-GlcNhm2 <20 80KDO2-GlcNhm2 320 <20

a Antisera (500 ,ul, prediluted 1:20 in PBS) were absorbed at 4°C with 50 pdof antigen-coated SRBCs.

b Homologous titers in italics.

TABLE 9. Linear regression of human antibody titers againstr595-207 LPS and dephosphorylated LPS-HFr

No. of reactants with indicated titer against r595-207Titer against LPS-HFrr595-207 LPS

<16 16 32 64 128 256 512 1,024

<16 12 0 0 0 0 0 0 016 9 11 0 0 0 0 0 032 3 4 6 0 0 0 0 064 0 0 1 2 0 0 0 0128 0 0 0 2 1 0 0 0256 0 0 0 0 0 1 0 0512 0 0 0 0 1 0 0 0

1,024 0 0 0 0 0 0 0 1

native recombinant and chlamydial LPSs. This observationsuggested that these antigens had a low coating efficacy forSRBCs, resulting in a low epitope density on the erythrocytesurface which sufficed for lysis by high-affinity but not bylow-affinity antibodies. That indeed the coating efficacy ofthe antigens varied considerably was shown by coatingSRBCs with graded amounts of antigen. Since the affinity ofLPS for erythrocyte membranes depends mainly on the ratioof hydrophilicity to hydrophobicity (3), it is well understoodthat the removal of ester-linked fatty acids influences thesensitization of SRBCs. This also explains why chlamydialLPS, in particular, has a very low coating efficacy comparedwith that of recombinant LPS, since the former contains inits lipid A portion fatty acids with up to 22 carbon atoms (9,19), whereas the latter contains fatty acids with 12 to 16carbon atoms (2). Therefore, the results of the hemolysisassay are understood more as a difference of these antibod-ies in affinity rather than in specificity. This assumption wasfurther supported by the results of inhibition experiments, inwhich LPS and LPS-OH yielded similar inhibition valueswith all antibodies except one (clone S19-3). LPS-HFr

TABLE 8. Appearance of different LPS antibody specificities in mouse sera obtained by different immunization protocolsHemolytic titer against SRBCs coated with:

Immunogen (S. minnesota Animal Natural structures Synthetic structuresr595-207) no. S. minnesota r595-207 E l

LPS LPS-OH LPS-HFr Re LPS-OH KDO-GcNhm2 KDO2-GlcNhm2

Heat-killed bacteria 1 1,280 1,280 640 80 <40 <402 320 320 160 <40 <40 <403 1,280 1,280 640 <40 <40 <40

Liposome-incorporated LPS 1 320 320 320 160 <40 602 320 1,280 1,280 320 <40 804 640 1,280 1,280 640 <40 <40

Liposome-incorporated LPS-OH 1 6,400 51,200 25,600 320 <40 3203 6,400 51,200 25,600 640 <40 640

Liposome-incorporated LPS-de-O-aCa 1 160 320 1,280 <40 <40 <403 320 640 1,280 <40 <40 <404 80 160 320 <40 <40 <40

Liposome-incorporated LPS-HF 1 12,800 102,400 102,400 <40 <40 <403 6,400 102,400 102,400 <40 <40 <404 6,400 51,200 51,200 <40 <40 <40

LPS-HF-coated SRBCs 2 800 1,600 1,600 <40 <40 <403 800 3,200 3,200 <40 <40 <404 800 1,600 1,600 80 <40 <40

a De-O-acylated LPS after treatment with hydrazine at 37°C.

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212 BRADE ET AL.

TABLE 10. Determination by absorption of different antibody specificities against chlamydial LPS present in human sera

Hemolytic titer against SRBCs coated with:Serum r595-207 LPS-OHa r595-207 LPS HFr E. coli Re LPS-OH Synthetic KDO-GlcNhm2no. E.________________________Synthetic____KDO___G__cNhM2_

Before absorption After absorption Before absorption After absorption (before absorption)b (before absorption)b36 512 256 128 128 32 12840 32 32 32 32 32 3242 32 <16 <16 <16 32 3245 16 <16 16 <16 16 <1646 32 <16 16 <16 16 1649 128 128 128 32 64 1650 256 32 256 <16 256 6456 16 <16 16 <16 16 16

a Antisera (250 ,ul, prediluted 1:16 in PBS) were absorbed at 4°C with 25 p.l of SRBCs coated with E. coli Re LPS-OH.bAll sera had titers of <16 after absorption.

yielded, with most of the antibodies, comparable inhibitionvalues; however, with some antibodies, no or low inhibitionwas observed. Most striking were the results obtained withantibody S19-3, which could not be inhibited with LPS-OHand LPS-HFr, although it reacted with both antigens in thehemolysis assay. Since this antibody required a high epitopedensity and since in the inhibition assay the inhibitor com-petes with the SRBC-coated antigen, this behavior is attrib-uted to a very low affinity.By using the synthetic trisaccharide ot-KDO-(2-8)-ot-KDO-

(2-4)-a-KDO, the disaccharide partial structures ot-KDO-(2-8)-a-KDO and a-KDO-(2-4)-a-KDO, and KDO monosac-charide, all prepared as multivalent polyacrylamidecopolymerization products, none of the antibodies could beinhibited with any of the partial structures. The KDOtrisaccharide inhibited 6 out of 11 antibodies; however, thevalues, compared with those of a KDO monosaccharide-specific antibody, were 1 order of magnitude lower. Atpresent it is difficult to decide whether these data should betaken as an indication that the KDO trisaccharide alone isnot the complete epitope recognized by these antibodies orwhether the epitope is not accessible to the antibody due toconformational changes in the copolymerization product.Our current investigations on the conformation of the KDOtrisaccharide linked to glucosamine (as in LPS) or to thepolyacrylamide chain indicate that the ligand may not befreely accessible to antibodies in the latter. However, theKDO monosaccharide-specific antibody can react with theterminal KDO residue, being separated from the acrylamidechain by two KDO residues. In summary, the data show thatall antibodies tested recognize closely related epitopes re-quiring the presence of three KDO residues in a definedlinkage (since they do not react with Re and other LPS) andthat they do not require the presence of phosphoryl orester-linked acyl groups or the reducing glucosamine residueof lipid A. The role of amide-linked fatty acids and the KDOproximal glucosamine remains to be elucidated. At least forsome antibodies, amide-linked fatty acids are also dispens-able, since they bound to completely deacylated LPS cou-pled to Sepharose (data not shown).On the basis of these results and those reported on

monoclonal and polyclonal antibodies against Re LPS (22),we tried to dissect different antibody specificities present inpolyclonal antisera of rabbits, mice, and humans. In this partof the study, we showed that rabbit antisera against chla-mydiae contained chlamydia-specific antibodies and thosecross-reacting with Re LPSs, which could be separated fromeach other by absorption with defined antigens. (Note: theterm cross-reaction is used in the following in a descriptive

way rather than as an immunochemical term. We do notknow at present whether Re-reactive antibodies react withthe Re portion of chlamydial LPS or whether chlamydialLPS is heterogeneous and contains a population of LPSswith only two KDO residues. The answer to this questionwill be given when chemically homogeneous LPSs or partialstructures thereof have been synthesized or prepared fromnatural sources). By using natural and synthetic partialstructures of recombinant and Re LPSs, it was found thatamong both, the chlamydia-specific and the Re-reactiveantibodies, those requiring phosphate groups and those forwhich the presence of phosphate was dispensable werepresent. The results also showed that the Re-reactive onescould be absorbed without affecting the chlamydia-specificantibodies. Whereas chlamydia-specific and Re-reactive an-tibodies were present in comparable titers in rabbit antisera,mouse antisera contained predominantly chlamydia-specificantibodies. Here, the immunization protocol had a stronginfluence on the titer and the ratio of these two types ofantibodies. Particularly high titers were obtained after im-munization with dephosphorylated LPS incorporated intoliposomes.The cross-reactivity of chlamydial antisera with Re LPS is

one of the disadvantages from which routine chlamydialserology with patient sera suffers, since a positive reactionwith chlamydial LPS is obtained with chlamydia-specific andRe-reactive antibodies. Since the latter may arise fromcontact with chlamydial, Re-type LPS, or any other LPS,the results are difficult to interpret. Different serologicalassays do not overcome this general problem as long as theantigen is LPS. Therefore, we also looked for antibodyspecificities present in human sera from patients suspectedto have a genital chlamydial infection. Although we havestudied only a small number of sera (n = 54), it is evidentthat chlamydia-specific and Re-reactive antibodies occuralso in human sera and, as shown for rabbit antisera, aredependent or not dependent on the presence of phosphategroups. Absorption experiments with some sera showed thatthe ratio of the different specificities to each other variedconsiderably. At present, we are carrying out studies on serafrom clinically defined chlamydial infections which mayallow us in the future to assign a certain type or stage ofchlamydial disease to distinct reactivity patterns, with aselection of antigens to be defined.

ACKNOWLEDGMENTSWe thank H. D. Caldwell for his advice and for providing

monoclonal antibody L21-6, B. J. Appelmelk for providing mono-clonal antibody A20, and F. E. Nano for plasmid pFEN207. The

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expert technical assistance of S. Werner, U. Albert, and V. Susottis gratefully acknowledged. We thank M. Grunefeld and E. Brandtfor their help in establishing the hybridoma technology.

This work was supported by the Bundesministerium fur Fors-chung und Technologie (grant 01 ZR 8604 to H.B.).

LITERATURE CITED

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2. Brade, H., L. Brade, and E. T. Rietschel. 1988. Structure-activity relationships of bacterial lipopolysaccharides (endotox-ins). Zentralbl. Bakteriol. Mikrobiol. Hyg. Ser. A 268:151-179.

3. Brade, L., K. Brandenburg, H.-M. Kuhn, S. Kusumoto, I.Macher, E. T. Rietschel, and H. Brade. 1987. The immunoge-nicity and antigenicity of lipid A are influenced by its physico-chemical state and environment. Infect. Immun. 55:2636-2644.

4. Brade, H., C. Galanos, and 0. Luderitz. 1983. Differentialdetermination of the 3-deoxy-D-mannooctulosonic acid residuesin lipopolysaccharides of Salmonella minnesota rough mutants.Eur. J. Biochem. 131:195-200.

5. Brade, L., P. Kosma, B. J. Appelmelk, H. Paulsen, and H.Brade. 1987. Use of synthetic antigens to determine the epitopespecificities of monoclonal antibodies against the 3-deoxy-D-manno-2-octulosonate region of bacterial lipopolysaccharide.Infect. Immun. 55:462-466.

6. Brade, L., F. E. Nano, S. Schlecht, and H. Brade. 1987.Antigenic and immunogenic properties of recombinants fromSalmonella typhimurium and Salmonella minnesota rough mu-tants expressing in their lipopolysaccharide a genus-specificepitope of chlamydiae. Infect. Immun. 55:482-486.

7. Brade, L., M. Nurminen, P. H. Maikela, and H. Brade. 1985.Antigenic properties of Chlamydia trachomatis lipopolysaccha-ride. Infect. Immun. 48:569-572.

8. Brade, H., and E. T. Rietschel. 1984. a-2.4-Interlinked 2-keto-3-deoxy-D-mannooctulosonic acid disaccharide: a common con-stituent of enterobacterial lipopolysaccharides. Eur. J. Bio-chem. 145:231-236.

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11. Dhir, S. P., S. Hakomori, G. E. Kenny, and J. T. Grayston. 1972.Immunochemical studies on chlamydial group antigen (presenceof a 2-keto-3-deoxycarbohydrate as immunodominant group). J.Immunol. 109:116-122.

12. Dhir, S. P., G. E. Kenny, and J. T. Grayston. 1971. Character-ization of the group antigen of Chlamydia trachomatis. Infect.Immun. 4:725-730.

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15. Kosma, P., J. Gass, G. Schulz, R. Christian, and F. M. Unger.1987. Artificial antigens. Synthesis of polyacrylamide copoly-mers containing 3-deoxy-D-manno-2-octulopyranosylonic acid(KDO) residues. Carbohydr. Res. 167:39-54.

16. Kosma, P., G. Schulz, and H. Brade. 1988. Synthesis of atrisaccharide of 3-deoxy-D-manno-2-octulopyranosylonic acid(KDO) residues related to the genus-specific lipopolysaccharideepitope of Chlamydia. Carbohydr. Res. 183:183-199.

17. Nano, F. E., and H. D. Caldwell. 1985. Expression of thechlamydial genus-specific lipopolysaccharide epitope in Esche-richia coli. Science 228:742-744.

18. Nurminen, M., M. Leinonen, P. Saikku, and P. H. Maikela. 1983.The genus-specific antigen of Chlamydia: resemblance to thelipopolysaccharide of enteric bacteria. Science 220:1279-1281.

19. Nurminen, M., E. T. Rietschel, and H. Brade. 1985. Chemicalcharacterization of Chlamydia trachomatis lipopolysaccharide.Infect. Immun. 48:573-575.

20. Nurminen, M., E. Wahlstrom, M. Kleemola, M. Leinonen, P.Saikku, and P. H. Mfikela. 1984. Immunologically related ke-todeoxyoctonate-containing structures in Chlamydia trachoma-tis, Re mutants of Salmonella species, and Acinetobacter cal-coaceticus var. anitratus. Infect. Immun. 44:609-613.

21. Paulsen, H., and M. Schuller. 1987. Bausteine von oligosaccha-riden. LXXVIII. Synthese von KDO-haltigen lipoid-A-analoga.Justus Liebigs Ann. Chem. 1987:249-258.

22. Rozalski, A., L. Brade, P. Kosma, B. J. Appelmelk, C. Krog-mann, and H. Brade. 1989. Epitope specificities of murinemonoclonal and rabbit polyclonal antibodies against enterobac-terial lipopolysaccharides of the Re chemotype. Infect. Immun.57:2645-2652.

23. Sadecky, E., M. Travnicek, J. Balascak, R. Brezina, J. Kazar, J.Urvolgyi, I. Flesar, and J. Cizmar. 1978. Enzootic abortion ofewes in Roznava district. Vet. Med. (Prague) 23:25-28.

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