Vol. 60, No. 6

INFECTION AND IMMUNITY, June 1992, p. 2201-2210 0019-9567/92/062201-10$02.00/0 Copyright ) 1992, American Society for Microbiology

Characterization of the Epitope Specificity of Murine Monoclonal Antibodies Directed against Lipid A HELLA-MONIKA KUHN,' LORE BRADE,1 BEN J. APPELMELK,2 SHOICHI KUSUMOTO,3 ERNST T. RIETSCHEL,1 AND HELMUT BRADE1* Division of Biochemical Microbiology, Forschungsinstitut Borstel, Institut fiirEperimentelle Biologie und Medizin, D-2061 Borstel, Germanyl; Department of Medical Microbiology, Vrije Universiteit, 1007 MC Amsterdam, The Netherlands2; and Department of Chemistry, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan3 Received 1 November 1991/Accepted 23 March 1992

A series of monoclonal antibodies directed against lipid A was characterized by using synthetic lipid A analogs and partial structures. These compounds vary in phosphate substitution, acylation pattern (type, number, and distribution of fatty acids), and, in the case of monosaccharides, in their backbone glycosyl residue. The monoclonal antibodies tested could be subdivided into five groups according to their reactivity patterns. One group reacted exclusively with 1,4'-bisphosphoryl lipid A, and a second also reacted with 4'-monophosphoryl lipid A. Two further groups recognized either 4-phosphoryl or 1-phosphoryl monosaccharide partial structures of lipid A. The fifth group reacted with 4-phosphoryl monosaccharide structures and with phosphate-free compounds. Antibodies reactive with monosaccharide structures also recognized their epitopes in corresponding phosphorylated disaccharide compounds. Both groups of monosaccharide and monophosphoryl lipid A-recognizing antibodies have access to their epitopes in bisphosphoryl compounds as well. Because of this unidirectional reactivity with more complex structures, the various specificities cannot be distinguished by using bisphosphoryl lipid A (e.g., Escherichia coli lipid A) as a test antigen. The epitopes recognized by the various monoclonal antibodies all reside in the hydrophilic backbone of lipid A, and there was no indication that fatty acids were part of the epitopes recognized. Nevertheless, the reactivities of compounds in the different test systems are strongly influenced by their acylation patterns; i.e., acyl groups may modulate the exposure of lipid A epitopes.

ical assay system used. Thus, in the present study, passive immunohemolysis and enzyme immunoassay (EIA) were compared. We will show that the specificities characterized in polyclonal sera are indeed also expressed by MAbs, as estimated in all serological assay systems employed.

Lipopolysaccharides (LPSs) (endotoxin) are amphipathic macromolecules present as common constituents in the cell wall of gram-negative bacteria. All LPSs, regardless of their origin, share a general architecture: they contain a lipid component (termed lipid A) and a heteropolysaccharide. The heteropolysaccharide consists of a core oligosaccharide (core region) and, in the case of members of the family Enterobacteriaceae, of the 0-specific chain. Each of the three LPS regions exhibits distinct immunoreactive properties (4). The numerous pathophysiological effects (8) of endotoxin are caused by the lipid A portion and can be elicited by synthetic lipid A as well (18). In addition, lipid A is immunogenic and antigenic and lipid A antibodies crossreact with lipid A from various bacterial species (19). Since lipid A represents the toxic principle of LPS, antibodies to lipid A may offer a tool for neutralizing the harmful effects of endotoxin. In recent studies, we have identified five distinct lipid A antibody specificities in rabbit sera (6, 7). All specificities were dependent on the presence of phosphate. One of these specificities comprised the 1,4'-bisphosphoryl hexosamine disaccharide of lipid A, two further specificities comprised either the 1- or the 4'-monophosphorylated disaccharide, and two comprised 1- or 4-phosphoryl monosaccharide partial structures of lipid A. Since these studies were performed with polyclonal rabbit sera, we asked the question whether similar specificities may be obtained with monoclonal antibodies (MAbs). Since the antigenicity of lipid A is greatly influenced by physicochemical conditions, we also studied how far antibody reactivities depend on the serolog*

MATERIALS AND METHODS Bacterial lipid A. Bisphosphoryl lipid A (lipA-Ac) and 4'-monophosphoryl lipid A (lipA-HCl) were obtained from Escherichia coli Re mutant F515 by treating phenol-chloroform-petroleum ether-extracted LPS with either acetate buffer or HCl (5). Synthetic lipid A and partial structures. The structures of synthetic disaccharide antigens and the various substituents are shown in Fig. 1 and Table 1. Compounds LA-15-PP, LA-15-PH, LA-15-HP, and LA-15-HH represent synthetic hexaacyl E. coli lipid A and its 4'-phosphoryl, 1-phosphoryl, and 1,4'-dephosphoryl partial structures, respectively (23, 29). Preparations LA-14-PP, LA-14-PH, LA-14-HP, and LA-14-HH (24) correspond to tetraacyl precursor Ia (22) and its dephosphoryl derivatives. Compounds LA-19-PP, LA-19PH, and LA-19-HP represent synthetic counterparts of alkali-treated (de-O-acylated) lipid A and its monophosphoryl derivatives. LA-16-PP corresponds to the heptaacyl species of Salmonella minnesota lipid A (17). LA-20-PP represents synthetic pentaacyl precursor lb (22, 40), and LA-20-PH represents its 4'-monophosphoryl partial struc-

ture. LA-21-PP and LA-21-PH are isomers of precursor lb carrying the hexadecanoic residue at the nonreducing glucosamine (40). LA-22-PP is a synthetic analog of Chromobacterium violaceum lipid A (39). The structures and substi-

Corresponding author. 2201





R FIG. 1. Chemical structure of disaccharide lipid A antigens. See also Table 1.

FIG. 2. Chemical structure of monosaccharide partial structures of lipid A. See also Table 2.

tuents of synthetic monosaccharides are given in Fig. 2 and Table 2. Compounds LA-11-HP and LA-11-PH represent lipid X and its 4-phosphoryl isomer (28). The other synthetic monosaccharide lipid A analogs (30) vary with respect to acylation and phosphorylation. Additionally, in some compounds, D-glucosamine (GlcN) is replaced by either D-glucose (Glc), 3-amino-3-deoxy-D-glucose (Glc3N), or 2,3-diamino-2,3-dideoxy-D-glucose (GlcN3N). The latter glycosyl residue is present in the backbone of the lipid A of a variety of phototrophic and nonphototrophic bacteria (33, 35, 36). Liposome membrane-embedded antigens. The preparation of antigens embedded in liposome membranes has been described previously (6). All lipids were obtained from Sigma Chemical Co. (Munich, Germany). Immunization and cell fusion. For immunization with 4'monophosphoryl lipid A, HCI-treated bacteria of the E. coli Re mutant F515 were coated with free lipA-HCl (19) at a ratio of 10:1 (bacteria/lipid A [wt/wt]). The immunization time schedule of Girard and Chaby (20) was modified: mice (BALB/c, 10 animals) were initially injected intraperitoneally with 10 ,ug of lipA-HCI-coated bacteria, followed by five booster injections (50 ,ug each, intraperitoneally) on days 60, 110, 157, 192, and 280. Synthetic compound LA-15-HP was incorporated into liposomal membranes as 1-phosphoryl lipid A immunogen. Four BALB/c mice were immunized as described by Mashimo et al. (31), with some modifications.

Per mouse, 20 ,ug of immunogen was injected on days 0 and 7, 60 p.g was injected on day 14, and 120 ,ug was injected on day 21; two booster injections were then administered on days 70 and 132 (50 ,ug each). Ten days after each injection, blood samples were tested with the passive immunohemolysis system (see below). Only after the fourth injection did 4 of 10 mice respond to lipA-HCI-coated bacteria with a low titer. Similarly, mice immunized with LA-15-HP liposomes responded only after the last two injections (three of four mice), and the titers were even lower than those of lipAHCI-immunized mice. In contrast, all 10 control mice immunized with lipA-Ac-coated bacteria already had significant titers to lipA-Ac after the first booster. Obviously, the complete structure of lipid A is a better immunogen for mice than monophosphoryl derivatives. Four days after the last injection, spleen cells from one mouse of each group (except the control mice) were fused with mouse myeloma subclone X63Ag at a ratio of 1:1. Fusion was performed by means of 50% (wt/vol) polyethylene glycol (PEG 1500; Boehringer Mannheim, Germany) according to standard procedures. As soon as hybridoma growth was macroscopically visible, supernatants were screened for antibody production by EIA with natural E. coli lipid A. Cloning of positive cultures was carried out by limiting dilution as described by Brandt et al. (9). MAbs H348 and D7188 (see below and Table 3) were raised with HCI-treated S. minne-

TABLE 1. Synthetic disaccharide antigensa

TABLE 2. Synthetic monosaccharide antigensa

R, X, and Y components'

R4 Y3X


LA-16-PP LA-15-PP LA-15-HP LA-15-PH LA-15-HH LA-22-PP LA-22-PH LA-20-PP LA-20-PH LA-21-PP LA-21-PH LA-14-PP LA-14-HP LA-14-PH LA-14-HH

LA-19-PP LA-19-HP LA-19-PH

C14-O-C16 C14-OH C14-OH C14-OH C14-OH

C14-OH C14-OH C14-OH C14-OH C14-OH

C14-O-C12 C14-O-C12 C14-O-C12 C14-O-C12 C14-O-C12

C14-O-C14 P

C14-O-C14 C14-O-C14 C14-O-C14 C14-O-C14



C14-O-C14 C14-OH C14-O-C14 C14-OH



C14-O-C14 C14-OH C14-O-C14 C14-OH C14-OH C14-O-C16 C14-OH C14-OH





C14-OH C14-O-C16 C14-OH C14-OH C14-OH C14-OH C14-O-C16 C14-OH C14-OH C14-OH C14-O-C16 C14-OH

C14-OH C14-OH C14-OH C14-OH C14-OH C14-OH


C14-OH C14-OH C14-OH C14-OH H H H

C14-OH C14-OH C14-OH C14-OH

C14-OH C14-OH

C14-OH C14-OH

C14-OH C14-OH C14-OH



a See structure in Fig. 1. " P, phosphoryl group; C14-O-C14, (R)-3-(tetradecanoyloxy)tetradecanoic acid; C14-OH, (R)-3-hydroxytetradecanoic acid.


880.503 880.200 LA-11-HP 89.695 89.397 880.201 880.244 880.124 880.421 880.319 880.125 LA-li-PH 880.475 89.575 89.589

R' NH-Ac

NH-C14-OH NH-C14-OH O-C14-OH NH-C14-OH NH-C14-OH NH-C14 O-C14-OH NH-C14-OH

NH-C14-O-C14 O-C14-O-C14 NH-C14-OH NH-C14-OH NH-C14-OH


R, X, and Y componentsb ~ R2


0-C14-OH O-C14-OH NH-C14-OH 0-C14


NH-C14-OH O-C14-O-C14 O-C14-OH O-C14-O-C14 O-C14-OH NH-C14-OH

NH-C14-OH NH-C14-O-C14





P P P P P H H Me Me

a See structure in Fig. 2. and 0 indicate amide- and ester-bound fatty acids, respectively, depending on the nature of the backbone sugar. Compounds LA-li-PH and 880.475 represent mixtures of the a and , anomers; compounds 89.575 and 89.589 are ,-methyl glycosides. Me, methyl group. See also footnote b to Table 1.



VOL. 60, 1992


TABLE 3. Antibodies used and their reactivities with lipid A Serum or


Reactivity with free lipid A in":




K64 (RS) K81 (RS) K46 (RS) K77 (RS)

Si (S) S11-7 (S) A23 (A) A24 (A) A6 (A) Al (A) A43 (A) lC3f (A) 8Ai (A) 5E4 (A) H348 (A) D7188 (A)

IgG2b IgM IgG2a IgG2b IgG2b IgG2b IgM IgG2a IgGi

IgGi IgG2b IgGi

LA-15-PH (L)' LA-15-HP (L) LA-15-PP (L) LA-15-HH (L) E. coli lipA-HCld LA-15-HP (L) S. minnesota R595 cells S. minnesota R595 cells E. coli J5 cells E. coli J5 cells S. minnesota R595 cells E. coli J5 cells E. coli J5 cells E. coli J5 cells S. minnesota R595 lipA8 S. typhimurium G30/C21 lipAg

4 4

4 8 This study This This This 1 1 This 3 3 3 This This

study study study study

+++ +++ +++ + _e ++ ++ +++ +++ +++ ++ ++


study study

+ ++


++ ++ ++

++ ++

+++ +++ +++ +++

+++ ++ ++ ++ ++ +++

a RS, rabbit serum; S, MAb supernatant; A, MAb ascites. b Reactivity with E. coli lipA-Ac; in EIA, 100 ng per well was used, and in PIH, 100 ,ug per 4 ml of a 5% suspension of SRBCs was used. + ++, strong; + +, medium; +, weak; - negative. (L), incorporated into liposomal membranes. d E. coli Re mutant bacteria were treated with 0.1 M HCI (100°C for 1 h) and additionally coated with 4-monophosphoryl lipid A. e This antibody does not react with antigen-coated SRBCs in PIH but does react in PHA. f MAb 1C3 is an isotype switch of MAb 8A1. g 5. minnesota R595 or S. typhimurinum G30/C21 bacteria were treated with 0.1 M HCI (100°C for 1 h) and additionally coated with lipid A obtained from the respective LPSs by hydrolysis (1% acetic acid; 100°C for 2 h).

sota R595 and Salmonella typhimunum G30/C21 bacteria additionally coated with free lipid A (19) liberated from the respective LPSs by 1% acetic acid (100°C for 2 h) at a ratio of 10:1 (bacteria/lipid A [wt/wt]). For MAb H348, BALB/c mice were injected subcutaneously with 100 ,ug of lipid A-coated bacteria on day 0 and with 50 ,ug on day 14; they were then injected intravenously (25 ,ug) on day 21. For MAb D7188, mice were injected subcutaneously twice with 100 ,ug of immunogen (days 0 and 21) and with 50 ,ug of immunogen on day 35; 25 ,ug was then injected intravenously on day 45. Spleen cells were fused with myeloma line SP2/0 4 days after the last injection. Rabbit antisera and murine MAbs. Sera and MAbs used are listed in Table 3. Polyclonal rabbit sera were described previously (6, 7). MAbs S1 and S11-7 were derived from immunizations described above. MAbs A23, A24, and A43 were obtained from the same fusion as were Al and A6 (1). MAbs 1C3, 8A1, and 5E4 were kindly provided by R. Coughlin, Malvern, Pa., and H348 and D7188 were kindly provided by T. Ulrich, Hamilton, Mont. Passive immunohemolysis (PIH) and hemolysis inhibition assay (19). Antigens were added to aliquots of sheep erythrocytes (SRBCs, 5% suspension in phosphate-buffered saline [PBS]), incubated at 37°C for 30 min, and washed three times. Resuspended SRBCs (50 VIl; 0.5% in Veronal-buffered saline) were added to twofold serially diluted antibodies (50 ,ul). The addition of 25 ,u1 of guinea pig serum as a complement source (in excess) was followed by incubation at 37°C for 1 h. The antigen doses needed to obtain the highest antibody titer were determined in advance (200 ,ug/4 ml of 5% SRBC suspension was never exceeded). For passive hemolysis inhibition, sensitized SRBCs (50 ,ul) were added to a mixture of inhibitor (in twofold serial dilutions) and antibody (25 ,ul each) which was preincubated at 37°C for 15 min. The addition of complement was followed by incubation at 37°C for 1 h. The antibody dilution resulting in 2 to 3

hemolytic units was determined in advance; one hemolytic unit was defined as the antibody dilution yielding 50% lysis of SRBCs. Absorption of antibodies. Glutardialdehyde-fixed SRBCs were coated with antigen as described above. After several washings, packed cells were suspended in serum or cell culture supernatant (50 p.1 of packed SRBCs per ml of antibody solution) and incubated at 37°C for 30 min. After centrifugation, the supernatant was tested in passive hemolysis or EIA. Passive hemagglutination. SRBCs were sensitized as described for PIH; however, twice the amount of antigen was used. SRBCs were suspended in PBS with 0.1% bovine serum albumin (BSA) to yield a 0.3% suspension. SRBCs (50 ,ul) were added to 50 p.1 of antibody, twofold serially diluted (in BSA-supplemented PBS), and incubated at 37°C for 1 h. EIA and EIA inhibition. Based on the method described by Peters et al. (38), an EIA for amphipathic antigens was developed. Polyvinyl plates (Falcon 3911; Becton Dickinson, Paramus, N.J.) were coated overnight at 4°C or for 1 h at 37°C with various amounts of antigen (50 p.l) per well diluted in- buffer A (0.01 M phosphate buffer [pH 7.3] with 0.9% saline). With water-insoluble compounds LA-15-HH and LA-14-HH, coating was performed as described previously (14). Each further incubation step was performed at 37°C under gentle agitation, followed by four washes in an automatic washer. Coated plates were washed with buffer A and blocked for 1 h with milk buffer (10% skim milk in buffer A). Incubation (1 h) with diluted antibody (25 p.1 in milk buffer) and washing (0.2% gelatin in buffer A) were followed by a 1-h incubation with 25 p.1 of enzyme-conjugated secondary antibody (1:1,000 diluted in milk buffer). After a washing with gelatin, two washes were performed with 0.1 M citrate buffer (pH 4.5). Substrate [25 p.1 of citrate buffer containing 2 mg of 2,2'-azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS) per ml and 25 p.1 of 0.1% H202 per ml]



KUHN ET AL. TABLE 4. Hemolytic antibody titers of MAbs and rabbit sera with synthetic compounds of various phosphorylation and acylation patterns

Hemolytic titer with rabbit serum or MAbb:

Antigen (compound)a

No. of Noyo

hcyl chains



LA-19-PH LA-19-HP

2 2


Characterization of the epitope specificity of murine monoclonal antibodies directed against lipid A.

A series of monoclonal antibodies directed against lipid A was characterized by using synthetic lipid A analogs and partial structures. These compound...
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