Epitope Specificities of Murine Monoclonal and Rabbit PolyclonalAntibodies against Enterobacterial Lipopolysaccharides
of the Re ChemotypeANTONI ROZALSKI,1t LORE BRADE,1 PAUL KOSMA,2 BEN J. APPELMELK,3 CHRISTOF KROGMANN,4
AND HELMUT BRADE1*Division ofBiochemical Microbiology, Institut fur Experimentelle Biologie und Medizin, Forschungsinstitut Borstel,
Parkallee 22, D-2061 Borstel, Federal Republic of Germany'; Institut fur Chemie der Universitat fur Bodenkultur, A-i 180Vienna, Austria2; Research Group on Commensal Infections, School of Medicine, Vrije Universiteit, 1081 BT Amsterdam,The Netherlands3; and Institut fur Organische Chemie der Universitat, D-2000 Hamburg, Federal Republic of Germany4
Received 6 February 1989/Accepted 24 May 1989
Murine monoclonal and rabbit polyclonal antibodies raised against the lipopolysaccharides (LPS) of Remutants of Salmonella minnesota, Proteus mirabilis, and Escherichia coli were serologically characterized.Using natural Re LPS and natural and synthetic partial structures thereof, representing the 3-deoxy-D-manno-2-octulosonic acid (KDO) or lipid A region or both, the epitope specificities of four monoclonalantibodies were defined. Clones 20 (immunoglobulin M [IgM]) and 25 (IgG3) recognize a terminal a-
pyranosidically linked KDO monosaccharide residue and the a-2,4-linked KDO disaccharide, respectively, as
the immunodominant group. Therefore, these two antibodies are core antibodies which do not require thepresence of lipid A constituents for binding. The minimal structure enabling binding of clone 17 (IgG2b) is apseudotetrasaccharide of the sequence a-KDO-(2--4)-a-KDO-(2-->6)-p-glucosamine-(1--*6)-glucosaminitolwith two amide-linked 3-hydroxytetradecanoic acid residues. The smallest structure with which clone 22 (IgG3)reacted was de-O-acylated Re LPS. Therefore, clones 17 and 22 are LPS antibodies requiring both the lipid Aand the KDO region for binding. Phosphoryl residues of the lipid A moiety in Re LPS are dispensable for thereaction with clone 17, whereas they are necessary for that with clone 22. These four different antibody typeswere also detected in polyclonal rabbit antisera and could be distinguished from each other by absorptionexperiments. It was found that type 20 and 25 antibodies either were not present or were present only in smallamounts and that the majority of the antibodies were of types 17 and 22. From these data, we conclude thatthe immunodominant structures of Re LPS comprise both the KDO and lipid A domains.
Lipopolysaccharides (LPS) are common constituents ofthe outer membrane of gram-negative bacteria. They func-tion as the major surface antigens of gram-negative organ-isms and exert numerous physiological and pathophysiolog-ical activities, as a result of which LPS are also calledendotoxins (2).LPS are chemically composed of a lipid part, lipid A,
which is responsible for the endotoxic activities of LPS (8),and a heteropolysaccharide which generally consists of anoligosaccharide, termed the core region, and, in some bac-terial families, of the so-called 0 chain, composed of repeat-ing oligosaccharide units. Each of the three regions, lipid A,core, and 0 chain, exhibit distinct immunoreactive proper-ties which are used for serotyping gram-negative bacteria ingeneral and for clinical microbiology. In addition, antibodiesagainst common structural elements, i.e., the evolutionarilywell conserved core and lipid A region, have been consid-ered to exhibit in vitro cross-reactivity and in vivo cross-protectivity against the deleterious effects of endotoxins.Whereas the immunoreactivity of the 0 chain and the lipid Amoiety are well understood on the molecular level, that ofthe core region remained obscure until recently when we
determined its chemical structure in LPS of bacterial species
* Corresponding author.t Present address: Institute of Microbiology, University of Lodz,
PL-90-237 Lodz, Poland.
of the families Enterobacteriaceae, Neisseriaceae, Vibrio-naceae, and Chlamydiaceae and of Haemophilus influenzae(for literature, see reference 2).The initial concept of the immunogenic and antigenic
properties of rough mutant bacteria of enterobacterial generais based on the work of Luderitz and co-workers (13, 14).They proposed that antibodies against rough LPS are di-rected against the core oligosaccharide, the terminal sugarbeing immunodominant. We (5) have reported on monoclo-nal antibodies against the LPS of the Re mutant R595 ofSalmonella minnesota which were directed against the ter-minal 3-deoxy-D-manno-2-octulosonic acid (KDO) and thea-2,4-linked KDO disaccharide portion of LPS, thus sup-porting the above hypothesis. In this study, we character-ized additional monoclonal antibodies which, by use ofsynthetic and chemically defined partial structures of naturalLPS of the Re chemotype from S. minnesota, Proteusmirabilis, and Escherichia coli, allowed us to dissect anddefine the antibody specificities present in polyclonal anti-sera. It was shown that the majority of antibodies in poly-clonal rabbit antisera are directed against complex epitopescomprising parts of both the lipid A and the inner coreregion.
MATERIALS AND METHODS
Bacterial LPS. LPS was extracted by the phenol-chloro-form-petroleum ether method (9) from S. minnesota rough
a KDO, 3-Deoxy-a-D-manno-2-octulopyranosylonic acid; PA, polyacrylamide.b GlcNhm, 2-Deoxy-2-([R]-3-hydroxytetradecanamido)-D-glucopyranose.
mutants of chemotypes Ra (strain R60), Rb2 (strain R345),Rc (strain R5), Rd1 (strain R7), Rd2 (strain R4), and Re(strain R595), from P. mirabilis chemotype Re (strain R45),and from E. coli chemotype Re (strain F515). In the follow-ing text the strain numbers are used. LPS was purified byrepeated ultracentrifugation followed by conversion to theuniform triethylammonium salt after electrodialysis (7). Al-kali-treated LPS (LPS-OH) was prepared with sodium hy-droxide (0.25 M at 56°C for 2 h) followed by precipitationwith ethanol. De-O-acylated and dephosphorylated LPS(LPS-HF) was prepared in the following way. LPS (100 mg)was dried overnight in a desiccator over phosphorus pentox-ide, dissolved in anhydrous hydrazine (5 ml), and stirred for30 min at 37°C. After the addition of chilled (-20°C) acetone,the precipitate was collected by centrifugation, washedextensively with acetone, dissolved in water (5 ml), precip-itated with acetone, dried, dissolved in water (5 ml), andfinally lyophilized, leading to the formation of de-O-acylatedLPS. The dry sample was suspended in 48% aqueous hydro-gen fluoride (5 ml) in a polypropylene tube and stirred at 4°Cfor 2 days. After centrifugation, the sediment was washedseveral times with ethanol, dissolved in water (5 ml), andlyophilized; the yield was 40 to 60% LPS. LPS-HF (20 mg)was dissolved in water (2 ml) followed by the addition ofsodium borohydride (20 mg), and the mixture was stirredwith a magnet at room temperature for 1 h. The reactionmixture was acidified with acetic acid and evaporated sev-eral times to dryness after the addition of methanol, fol-lowed by dialysis against water and lyophilization, yieldingde-O-acylated, dephosphorylated, and reduced LPS (LPS-HFr).
Synthetic antigens. Sodium (allyl 3-deoxy-a-D-manno-2-octulopyranosyl)onate and disodium [3-deoxy-a-D-manno-2- octulopyranosylonate - ( 2-- 4)- allyl 3-deoxy-a -D -manno-2-octulopyranosyl)]onate were synthesized as describedpreviously (11). Trisodium [3-deoxy-a-D-manno-2-octulo-pyranosylonate - (2 -*4) -3 - deoxy - a - D - manno-2 - octulo-pyranosyl)]onate-(2->4)-(allyl 3-deoxy-a-D-manno-2-octu-lopyranosyl)]onate (Table 1) was synthesized as will bereported elsewhere (P. Kosma, G. Schulz, F. M. Unger, andH. Brade, Carbohydr. Res., in press). The allylglycosideswere copolymerized with acrylamide as described previ-ously (6, 11), yielding the high-molecular-weight antigenslisted in Table 1. In the formulas of Table 1, n is 100 to 150,corresponding to molecular sizes between 60 and 100kilodaltons. Sodium 3 - deoxy -c - D -manno -2- octulopyrano-sylonate - ( 2 -> 6) - 2 - deoxy - 2 - [(R) - 3 - hydroxytetradecan-amido]-,B-D-glucopyranosyl(1--6)-2-deoxy-2-[(R)-3-hydro-xytetradecanamido]-D-glucose was synthesized as reportedpreviously (16). Disodium [3-deoxy-a-D-manno-2-octulopy-
ranosylonate - (2 -*4) -3 - deoxy - aL- D - manno-2 - octulopyr-anosylonate]-( 2-6 ) -2 - deoxy -2 - [(R) - 3-hydroxytetrade-canamido] - 3- D -glucopyranosyl] - (1-- 6) -2-deoxy - 2 - [(R)-3 -hydroxytetradecanamido]-D-glucose and trisodium [3-deoxy-x-D - manno- 2 - octulopyranosylonate- (2--> 4) - 3-deoxy - a - D -manno- 2 - octulopyranosylonate - (2 -4 ) -3 - deoxy - a - D -
manno- 2 - octulopyranosyl]onate - (2-- 6) - 2-deoxy - 2 - [(R) - 3 -hydroxytetradecanamido ] - p - D - glucopyranosyl - ( 1-> 6) - 2 -deoxy-2- [(R)- 3 -hydroxytetradecanamido] -D-glucose weresynthesized as will be reported elsewhere (H. Paulsen and C.Krogmann, unpublished data). The synthetic antigens usedin this study are abbreviated in the text according to Table 1.Synthetic lipid A of E. coli (compound 506 [10]) and its 1-and 4'-dephospho derivatives (compounds 505 and 504 [12],respectively) were kindly provided by S. Kusumoto andDaichi Co., Osaka, Japan.
Antibodies and serological assays. The protocol for thepreparation of murine monoclonal antibodies has been de-scribed in detail previously (1). Briefly, BALB/c mice wereimmunized with killed S. minnesota R595 bacteria by fourintravenous injections over 42 days. Fusions were per-formed 3 days after the last injection. Screening was per-formed by an enzyme immunoassay with S. minnesota R595LPS, and subcloning was done by conventional methods, aswas the production of ascites fluid and the determination ofimmunoglobulin isotypes and subgroups. Monoclonal anti-bodies of the immunoglobulin (IgG) isotype were purified byaffinity chromatography with protein A-Sepharose 4B (Phar-macia/LKB, Freiburg, Federal Republic of Germany).Monoclonal antibody clones 20 and 25 were also purified byan immunosorbent with immobilized KDO ligands as re-ported elsewhere (A. Rozalski, L. Brade, P. Kosma, B. J.Appelmelk, H. Paulsen, and H. Brade, Carbohydr. Res., inpress). Polyclonal antisera were prepared by injecting rab-bits with 50, 100, 100, and 200 jig of heat-killed bacteria ondays 0, 4, 7, and 11, respectively, and exsanguinating therabbits on day 16 (IgM-rich antisera). Some animals receivedbooster injections (100 ,ug intramuscularly) over a 1-yearperiod every 2 months, leading to IgG-rich antisera. Serawere absorbed with sheep erythrocytes (SRBC) and storedin aliquots at -20°C.The passive hemolysis and passive hemolysis inhibition
tests were performed as described previously (4). Some ofthe hemolysis assays were run in parallel on crude ascitesand on purified antibodies. No differences in specificity wereobserved; however, the purified immunoglobulins tended toaggregate during storage. Therefore, all inhibition experi-ments (in which the antibody dilution is critical) wereperformed with crude ascites. Absorption was carried out at4°C for 1 h on 1-ml aliquots of prediluted antisera (1:10 in
aSynthetic E. coli lipid A (compound 506) and its 4'- and 1-dephospho partial structures (compounds 505 and 504, respectively) were used as previouslydescribed (4).
phosphate-buffered saline) with 100 ,ul of packed SRBCcoated with the respective antigen.
RESULTS
Murine monoclonal antibodies were obtained after immu-nization with whole bacteria of the Re chemotype of S.minnesota. The monoclonal antibodies so prepared were
tested in the passive hemolysis assay to select complement-fixing antibodies. The antigen screening panel included allrough chemotypes of S. minnesota and synthetic E. coli lipidA, as well as partial structures of the latter. Of thoseantibodies reacting with at least the R595 LPS-OH, four(clones 20, 25, 17, and 22) were selected for further charac-terization. The results obtained in the passive hemolysisassay are shown in Table 2. None of the antibodies reactedwith synthetic E. coli lipid A or its 1- and 4'-dephosphopartial structures. Clone 20 (IgM) reacted with all LPS to a
similar extent, whereas clone 25 (IgG3) reacted exclusivelywith R595 and R345 LPS. These two antibodies have beencharacterized previously (5; Rozalski et al., in press); theyreact with a terminal a-pyranosidically linked KDOmonosaccharide residue (clone 20) and an a-2,4-linked KDOdisaccharide (clone 25), respectively. Clones 17 and 22reacted exclusively with LPS of the Re chemotype, and theirfurther characterization is described below.
Characterization of monoclonal antibody clones 17 (IgG2b)and 22 (IgG3). The antibodies were tested with SRBC coatedwith various antigens including native and alkali-treated ReLPS from S. minnesota, E. coli, and P. mirabilis. Since thereactivity of monoclonal antibodies may depend on theisotype on the one hand and on the epitope density on thecell surface on the other hand, graded amounts of antigenwere used for sensitization of SRBC. The results are shownin Table 3. Clones 17 and 22 had titers of 3,200 to 6,400 and25,600 to 51,200, respectively, against Re LPS from S.minnesota, E. coli, and P. mirabilis. With LPS-OH, titerswere obtained similar to those obtained with LPS; however,lesser amounts of antigen were required for optimal sensiti-zation. For P. mirabilis, only LPS-OH could be tested sincethe native LPS did not sensitize SRBC properly. The optimalreactivity of native LPS was obtained with a sensitizationdose of 64 to 128 p.g/0.2 ml of SRBC, whereas 8 to 16 p.g/0.2ml of SRBC was required with LPS-OH. The antibodieswere then tested against de-O-acylated and dephosphory-lated Re LPS of E. coli (F515 LPS-HF) and against the sameantigen after reduction (F515 LPS-HFr). Clone 17 was stillactive with both antigens, whereas clone 22 did not reactwith either of them even at high epitope densities (Table 3).Since natural LPS is heterogeneous even after extensivepurification (15), which may lead to misinterpretation of the
TABLE 3. Hemolytic titers of monoclonal antibody clones 17 and 22 against SRBC coated with graded amounts of Re mutant LPSand natural and synthetic partial structures thereof
Hemolytic titer against SRBC coated with:
Clone Amnt (p.g) of S. minnesotano. antigen/0.2 ml R595 P. mirabilis R45 E. coli F515 Synthetic structure
" Two to three hemolytic units of antibody and R595 LPS-OH-coated SRBCwere used.
b Data from reference 5.
results, the phosphate- and O-acyl-less structures KDO-2-deoxy - 2 - ([R] - 3 - hydroxytetradecanamido) - D - glucopyr-anose (GlcNhm2), KDO2-GlcNhm2, and KDO3-GlcNhm2were synthesized. Clone 17 reacted with KDO2-GlcNhm2and KDO3-GlcNhm2 to a similar extent but did not reactwith KDO-GlcNhm2, whereas clone 22 was unreactive withall three synthetic antigens (Table 3). These results showthat clones 17 and 22 differ in their specificities depending onthe presence of phosphoryl groups in the antigens.To elucidate the role of the KDO region in the specificity
of clones 17 and 22, we performed inhibition experimentsusing synthetic KDO antigens (copolymerization products ofvarious KDO allylglycosides with acrylamide). The resultsare shown in Table 4. None of the antigens used inhibited thetwo monoclonal antibodies (inhibition value of >5,000 ng).LPS and LPS-OH were active inhibitors for both antibodies,whereas LPS-HF inhibited only clone 17.
Characterization of polyclonal antisera. Polyclonal antiserawere prepared by immunizing rabbits with whole bacteria ofthe Re chemotype of S. minnesota, E. coli, and P. mirabilis.A short-term immunization protocol with bleeding on day 16was used to obtain IgM-rich antisera. In addition, IgG-richantisera were prepared by booster injecting animals over a1-year period. The antisera were tested with Re LPS from S.minnesota, E. coli, and P. mirabilis in the passive hemolysisassay (Table 5). All animals had high antibody levels againstthe LPS of the immunizing strain with titers ranging from1,280 to 20,480 (except animal K205, with a homologous titerof only 160). The titers obtained with the heterologous ReLPS were comparable, with the differences never exceedingone dilution step. In addition, all antisera reacted either withrough LPS other than Re (R7 and R345) or with natural (F515LPS-HFr) and synthetic (KDO-GlcNhm2 and KDO2-
GlcNhm2) partial structures of Re LPS or with both. Thereactivity pattern and the titers obtained with heterologousantigens varied from less than 40 to 5,120.To dissect the different antibody specificities present in
polyclonal antisera, we performed absorption experiments.Individual samples of antisera were absorbed with differentantigens and retested. Absorption with SRBC was used as acontrol to compensate for the dilution during the experi-ment. The results with four representative antisera areshown in Table 6. In all cases, the reactivity toward theantigen used for absorption could be reduced by more than95%, defining the efficacy of the absorption. After absorptionwith F515 LPS-OH, the reactivity against the heterologousantigens was abolished except with antiserum K50, forwhich even a second absorption left a titer of 160 againstF515 LPS-HFr and the corresponding synthetic structureKDO2-GlcNhm2. Some of the reactivities in Table 6 couldarise from different antibody specificities, e.g., the reactivitywith KDO2-GlcNhm2 could be caused by antibodies such asclones 20 and 25 recognizing partial structures of KDO2-GlcNhm2, i.e., the cx-pyranosidically linked KDO monosac-charide residue and the x-2,4-linked KDO disaccharide,respectively, or by antibodies similar in specificity to clone17. Therefore, inhibition experiments were designed inwhich hemolytic antigen-antibody systems of KDO-GlcNhm2- or R345 LPS-OH-coated SRBC and differentantisera (adjusted to two to three hemolytic units with therespective antigen) were used. R345 LPS-OH was used sinceit contains a KDO disaccharide as a side chain in stoichio-metric amounts (3) which allows interaction with KDOdisaccharide-specific antibodies such as clone 25 (5). Thesesystems were then inhibited with synthetic antigens. Repre-sentative results are shown in Table 7. The antiserum K4was inhibited equally by KDO monosaccharide- and disac-charide-containing antigens independent of whether KDO-GlcNhm2- or R345 LPS-OH-coated SRBC were used asindicator cells. The reactivity of K50 with KDO-GlcNhm2-coated SRBC could not be inhibited with KDO alone but wasinhibited equally with KDO-GlcNhm2 and KDO2-GlcNhm2.However, the reactivity of the same antiserum with R345LPS-OH-coated SRBC was inhibited with the KDO disac-charide-containing antigens KDO2-polyacrylamide (PA) andKDO2-GlcNhm2. Similar results were obtained with theIgG-rich antiserum K203.
DISCUSSIONLPS represent the major surface antigens of gram-negative
bacteria. The three chemically well-defined regions of LPS,i.e., the 0 chain, the core oligosaccharide, and the lipid A
TABLE 5. Hemolytic antibody titers of polyclonal antisera from rabbits immunized with heat-killed Re mutant bacteria
Antibody titer against SRBC coated with:Antiserum" Immunogen
" All animals were immunized by a short-time immunization protocol with bleeding on day 16 (IgM rich), except K203 and K205, which were booster injectedover a 1-year period (IgG rich).
a For immunizing bacterial strains, see Table 5.b Antisera (1 ml, 1:10 diluted in phosphate-buffered saline) were absorbed at 4°C with SRBC (100 .1l of packed cells) sensitized with the indicated antigens.' This activity could not be absorbed by a second absorption.
component, exhibit distinct immunoreactive properties.Those of the core and lipid A are of particular biomedicalinterest since they have similar structures and thus mayinduce broadly cross-reactive and potentially cross-protec-tive antibodies. There is an ongoing discussion whether ornot the concept of cross-protectivity of anticore and anti-lipid A antibodies against LPS effects is correct (for litera-ture, see B. J. Appelmelk, Ph.D. thesis, Free University ofAmsterdam, Amsterdam, The Netherlands, 1987). To pro-vide a molecular basis for understanding how antibodies andother proteins act on these unique carbohydrate structures,we started some years ago to investigate the chemicalstructure of lipid A and the adjacent core region in relation totheir immunogenic and antigenic properties.
Recently, we have reported (5) on monoclonal antibodiesagainst Re mutant LPS of members of the family Enterobac-teriaceae which are made up of lipid A and KDO only.Among the monoclonal antibodies obtained, two have been
characterized in detail, namely, clones 20 and 25, which arespecific for an ot-pyranosidically linked KDO monosaccha-ride residue and an a-2,4-linked KDO disaccharide, respec-tively (5; Rozalski et al., in press). In the present study, weanalyzed two additional monoclonal antibodies which re-acted exclusively with Re LPS and did not react with roughLPS other than Re, nor with free lipid A, nor with partialstructures of the KDO region. Therefore, these antibodiesrequire for binding parts of both lipid A and the KDO region.Since the Re LPS of different enterobacterial genera sharecommon structural elements such as the 1,4'-bisphosphory-lated ,B-1,6-linked D-glucosamine disaccharide carrying 4mol equivalents of 3-hydroxymyristic acid and the a-2,4-linked KDO disaccharide but differ in the type, number, anddistribution of nonhydroxylated fatty acids and in the sub-stitution with 4-amino-4-deoxy-L-arabinopyranose, ethanol-amine, and phosphate, we first wanted to know whetherthese variations led to differences in the recognition by
TABLE 7. Inhibition of distinct antibody specificities present in polyclonal rabbit antisera against Re mutant bacteria
Inhibition value (ng) obtained in the hemolytic antigen-antibody system ofa:Inhibitor K4- K4-R345 K50- K50-R345 K203- K203-R345
aKDO, 3-deoxy-cx-D-manno-2-octulopyranosylonic acid; GlcN, 2-amino-2-deoxy-D-glucopyranose; GlcNhm, 2-deoxy-2-([R]-3-hydroxytetradecanamido)-D-glucopyranose; GlcNhm-ol, 2-deoxy-2-([RI-3-hydroxytetradecanamido)-D-glucitol; P, phosphate; PA, polyacrylamide. The substitution of the Re LPS (1) andthose other than Re (2) with fatty acids is not shown. R, Core oligosaccharide.
b Positive and negative reactions may occur depending on the degree of substitution with the third KDO residue.
monoclonal antibody clones 17 and 22. We tested theseantibodies with native and alkali-treated Re LPS from S.minnesota, P. mirabilis, and E. coli and found their reactiv-ities to be similar with all antigens. Therefore, the epitopesrecognized by clones 17 and 22 are located primarily in thestructure common to these three LPS. The role of the KDOregion was investigated by inhibition experiments with syn-thetic KDO-containing antigens, which it was shown thatboth antibodies required the presence of the KDO disaccha-ride. Up to this stage of the study, the two antibodiesappeared to be very similar in specificity. However, whende-O-acylated and dephosphorylated LPS (F515 LPS-HF) orLPS-HF after additional reduction of the reducing glu-cosamine residue of lipid A (F515 LPS-HFr) was tested,major differences became evident. Whereas clone 22 wasinactive with both antigens in the passive hemolysis andpassive hemolysis inhibition assays, clone 17 retained itsreactivity with both antigens. This result was confirmed byusing the synthetic structures KDO-GlcNhm2, KDO2-GlcNhm2, and KDO3-GlcNhm2. From these data the follow-ing conclusions can be drawn. Phosphoryl groups of the ReLPS are essential for the binding of clone 22, whereas theyare dispensable for the binding of clone 17. Moreover, theepitope recognized by clone 17 does not require the reducingglucosamine of lipid A. In addition, the unchanged reactivityof clone 17 with KDO3-GlcNhm2 indicates that binding ofthis monoclonal antibody to its epitope is not hindered by thepresence of an additional KDO residue in position 4 of theterminal KDO.We were not able to decide how important fatty acids are
for the binding of clone 17. When the synthetic tetrasaccha-ride KDO2-GlcN2 and the completely deacylated and par-tially dephosphorylated carbohydrate backbone of Re LPSwere coupled to bovine serum albumin and tested in anenzyme immunoassay system, the latter reacted stronglywith clone 17, whereas the former exhibited only weakactivity (data not shown). This may be regarded as anindication that fatty acids as such are dispensable; however,with the concomitant loss of phosphoryl groups and coupling
to a protein carrier, the conformation of the KDO-glu-cosamine region could be disturbed and made unable to bindclone 17.
Also, further details on the specificity of clone 22, e.g., thequestion of whether both the ester-linked and glycosidicallylinked phosphoryl groups in Re LPS are necessary and againthe role of fatty acids, could not be investigated now sincethe antigens required for such studies are not yet available.Such compounds will become available in the future bychemical synthesis.
Nevertheless, we now can define the specificities of fourmonoclonal antibodies against Re LPS, which can be brieflycharacterized as follows. Clones 20 (IgM) and 25 (IgG3)recognize in Re LPS the terminal o-pyranosidically linkedKDO monosaccharide residue and the ot-2,4-linked KDOdisaccharide, respectively (5). Therefore, these two antibod-ies are core antibodies in the classical sense, since they reactwith the core oligosaccharide of LPS, with which the termi-nal carbohydrate residue contributes most to the specificityof the epitope in terms of immunodominance (13, 14). Forclones 17 (IgG2b) and 22 (IgG3), neither the free lipid A northe KDO disaccharide alone fulfills the structural require-ments for binding. Therefore, clones 17 and 22 are anti-LPSantibodies. Clone 17 binds mainly to the carbohydrate back-bone of Re LPS; phosphoryl groups and ester-linked fattyacids are not essential in constituting the correspondingepitope. Clone 22 exhibits the most complex specificity,which require for binding, in addition to the KDO disaccha-ride, certainly the phosphorylated and questionably theacylated lipid A moiety. It is obvious that a hierarchy interms of structural requirement for these four monoclonalantibodies can be defined. So far, the minimal structurerecognized is a single KDO residue for clone 20, a KDOdisaccharide for clone 25, and a trisaccharide of two KDOand one glucosamine residues for clone 17. Clone 22 requiresfor binding additional phosphoryl groups and possibly asecond glucosamine residue together with fatty acids. Inanalogy to the observations made on polyclonal lipid Aantibodies (4), the Re monoclonal antibodies directed against
partial structures of LPS also recognize these structures inmore complex molecules. A summary of the possible reac-tivity patterns with a representative selection of natural andsynthetic antigens is given in Table 8.We then asked whether the specificities observed with
monoclonal antibodies occur as well in polyclonal rabbitantisera. With the aid of the above monoclonal antibodiesand of chemically and serologically defined antigens, wewere able to dissect the antibody specificities present inpolyclonal antisera obtained after immunization of rabbitswith heat-killed Re bacteria of the enterobacterial species E.coli, S. minnesota, and P. mirabilis. To facilitate the follow-ing discussion, the monoclonal antibody specificities de-scribed are regarded as prototypes and will be assigned toreactivities present in polyclonal antisera. By using succes-sively more defective partial structures of Re LPS as anti-gens to sensitize SRBC, the hemolytic titers so obtainedshould roughly reflect the distribution of the different spec-ificities. As an example, we discuss the data obtained withantiserum K203 in Table 6. The titer of 1,280 againsthomologous Re LPS could arise from all four antibodytypes, clones 20, 25, 17, and 22. However, the lower titer of320 with KDO2-GlcNhm2 indicates that the reactivity againstLPS is that of type 22, since other antibody types could stillreact with the phosphate-free antigens F515 LPS-HFr andKDO2-GlcNhm2. At the same time, the data show that thereactivities against F515-HFr and KDO2-GlcNhm2 corre-spond to a type 17 antibody since they resisted absorptionwith KDO-GlcNhm2 and R345 LPS-OH, which would re-move antibodies of type 20 and 25. The titer of 160 againstR345 LPS-OH could reflect the reactivity of a type 20 or type25 antibody since this LPS is known to contain an (x-2,4-linked KDO disaccharide as a side chain in stoichiomet-ric amounts (3). The absorption data in Table 6 show that thereactivity against R345 LPS-OH could not be absorbed withthe KDO monosaccharide-containing antigen KDO-GlcNhm2 and thus represents a KDO disaccharide-specificantibody of type 25. The titer of 160 of this antiserum againstKDO-GlcNhm2 could be due to a type 20 antibody. How-ever, this reactivity could not be absorbed with R345 LPS-OH, revealing that it represents a new type of antibodywhich is specific for KDO-GlcNhm2 and is not yet known ina monoclonal form. This interpretation was confirmed byinhibition experiments (Table 7). When the antiserum dilu-tion of K203 was adjusted to two to three hemolytic unitswith KDO-GlcNhm2-coated SRBC (an antigen-antibody sys-tem in which type 20 and KDO-GlcNhm2-specific antibodieswould be expected to react), the inhibitors KDO-PA andKDO2-PA were inactive, whereas KDO2-GlcNhm2 and,even better, KDO-GlcNhm2 were good inhibitors. When,however, the antiserum dilution was adjusted with R345LPS-OH-coated SRBC (an antigen-antibody system inwhich type 20 and 25 antibodies would be expected to react),only those inhibitors containing a KDO disaccharide such asKDO2-PA or KDO2-GlcNhm2 were active. Therefore, in thisinhibition system, the disaccharide-specific antibody type 25is the limiting specificity of the antiserum. The titer of theKDO-GlcNhm2-specific antibody was particularly high inK50 and K51, which were obtained after immunization withheat-killed Re bacteria of P. mirabilis, but was not detectedor was detected only in low titers in animals immunized withE. coli and S. minnesota (Table 5). An explanation for thisobservation could be the substitution of the KDO and lipid Aregions in P. mirabilis LPS with 4-amino-4-deoxy-L-arab-inopyranose (17), which is completely lacking in E. coli andpresent as a nonstoichiometric substitute of the lipid A
region in S. minnesota. The presence of this positivelycharged amino sugar in the negatively charged KDO regionmay alter the immunogenic properties of this LPS.
In summary, the data show that the majority of antibodiespresent in polyclonal rabbit antisera against Re-type bacteriaare of types 22 and 17, antibodies which bind to an epitopecomposed of the core constituent KDO and lipid A. There-fore, the concept of the immunogenicity and antigenicity ofrough LPS established in the early 1970s has to be refined.Anticore antibodies in the classical sense are only those oftypes 20 and 25, for which indeed core constituents (KDOand KDO disaccharide for Re LPS) are immunodominant.Their reactivity does not depend on the presence of lipid Acomponents. However, these antibodies constitute only aminor fraction in polyclonal antisera. Our results show thatthe major specificities in an Re antiserum are represented bytypes 17 and 22. They are neither pure anti-KDO noranti-lipid A antibodies but require both these LPS regions forbinding. Thus, type 17 and 22 antibodies are true anti-LPSantibodies which react with a complex epitope composed ofthe entire KDO-lipid A domain.
ACKNOWLEDGMENTS
We thank E. T. Rietschel and H. Paulsen for fruitful discussionsand C. Bielfeldt, S. Werner, U. Albert, C. Reinfeldt, and V. Susottfor expert technical assistance.
This work was supported by grants from the Deutsche Forschungs-gemeinschaft (Br731/7-1) and the Bundesministerium fur Forschungund Technologie (01 ZR 8604) to H.B.
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