Use of synthetic antigens to determine the epitope specificities of monoclonal antibodies against the 3-deoxy-D-manno-octulosonate region of bacterial lipopolysaccharide.

Mouse monoclonal antibodies were raised against heat-killed bacteria of the Re mutant R595 of Salmonella minnesota and characterized by the passive hemolysis and passive hemolysis inhibition tests and by double immunodiffusion experiments using lipopolysaccharide (LPS) from different rough mutants of S. minnesota and synthetic antigens. The latter were copolymerization products of acrylamide with the alpha- and beta-allylglycosides of 3-deoxy-D-manno-octulosonic acid (KDO) and the alpha-2,4-linked KDO disaccharide [poly-alpha-KDO, poly-beta-KDO, and poly-(alpha-KDO)2, respectively], and sodium (3-deoxy-D-manno-octulopyranosyl)onate-(2----6)-(2-deoxy-2-[ (R)-3- hydroxytetradecanoylamino]- beta-D-glucopyranosyl)-(1----6)-(2-deoxy-2-[(R)-3-hydroxytetradecanoy lam ino]-D-glucose) [alpha-KDO-(GlcNhm)2], representing a part structure of Re LPS. One antibody (clone 20, immunoglobulin M) was found to recognize a terminal alpha-linked KDO residue, since it reacted in the passive hemolysis assay with alpha-KDO-(GlcNhm)2 and all LPS tested, it was inhibited by all synthetic antigens containing alpha-linked KDO residues, and it gave a reaction of identity with poly-alpha-KDO and poly-(alpha-KDO)2 in double immunodiffusion experiments. A second antibody (clone 25, immunoglobulin G3) was identified as specific for an alpha-2,4-linked KDO disaccharide, since it reacted in immunodiffusion exclusively with synthetic poly-(alpha-KDO)2 and not with the monosaccharide derivatives in either anomeric configuration, and it was inhibited only with poly-(alpha-KDO)2 and with LPS from S. minnesota R595 (Re) and R345 (Rb2). The reaction of this antibody with R345 LPS is attributed to the quantitative substitution with KDO disaccharide present as a side chain, which is not present in stoichiometric amounts in the other LPS.     

Characterization of murine monoclonal and murine, rabbit, and human polyclonal antibodies against chlamydial lipopolysaccharide

Murine monoclonal and rabbit, murine, and human polyclonal antibodies against chlamydial lipopolysaccharide (LPS) were characterized by the passive hemolysis and passive hemolysis inhibition assays and by absorption experiments with LPSs of Chlamydia psittaci, Chlamydia trachomatis, and a recombinant strain of Salmonella minnesota Re (r595-207) expressing the chlamydia-specific LPS epitope, as well as natural and synthetic partial structures of chlamydial LPS. Eleven monoclonal antibodies of the immunoglobulin M and G classes were characterized as chlamydia-specific by their failure to react with Re-type LPS, binding to a similar epitope for which the trisaccharide alpha-3-deoxy-D-manno-2-octulosonic acid (KDO)-(2-8)-alpha-KDO-(2-4)-alpha-KDO was 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. A similar antibody specificity was detected in lapine and murine hyperimmune sera after immunization with chlamydia, in addition to those recognizing more complex (e.g., those requiring the presence of phosphoryl residues) and less complex epitopes. Among the latter were those cross-reacting with Re-type LPS, which could be 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 immunization protocol. The preferential formation of chlamydia-specific antibodies was observed after immunization with liposome-incorporated immunogens. Human sera from patients with suspected genital chlamydial infections were also found to contain chlamydia-specific and cross-reactive antibodies, the latter of which could be removed by absorption with Re-type LPS.     

Anti-idiotypic immunization provides protection against lethal endotoxaemia in BALB/c mice.

Against lipid A (the conserved moiety of lipopolysaccharides from Gram-negative bacteria) neutralizing IgM monoclonal antibodies (mAb) 8-2 and 26-20 anti-idiotypic (Ab2) mAb were produced: Ab2 mAb KM-04 (IgG1) against mAb 8-2, and Ab2 mAb PW-1 (IgG2a) and PW-2 (IgG1) against mAb 26-20. The binding of Ab2 mAb KM-04 to 8-2 (Ab1) was strongly inhibited by a lipopolysaccharide (LPS) extract from either Salmonella minnesota R595 (Re LPS) or Escherichia coli J5 (Rc LPS), whereas the binding of Ab2 mAb PW-1 and PW-2 to 26-20 (Ab1) was only marginally inhibited by both Re LPS and Rc LPS. The results indicated that Ab2 mAb KM-04 recognizes a lipid A-binding site related idiotope on mAb 8-2 and therefore KM-04 might bear the internal image of a neutralization determining epitope of lipid A. Consequently Ab2 KM-04 might induce antibodies to lipid A. Indeed anti-idiotypic immunization of syngeneic BALB/c mice with Ab2 mAb KM-04 resulted in development of lipid A-binding anti-anti-idiotypic (Ab3) antibodies in serum. Similar immunizations with Ab2 mAb PW-1 and PW-2 were unsuccessful. However, induction of lipid A-binding Ab3 by mAb KM-04 proved to be genetically restricted to BALB/c mice. DBA/2 mice, Swiss mice and rabbits did not develop lipid A-binding antibodies upon immunization with mAb KM-04. In protection experiments, it was shown that BALB/c mice vaccinated with mAb KM-04 showed significantly enhanced survival from challenge with either rough (Re) LPS from Salmonella minnesota or smooth LPS from E. coli 0111:B4 when compared to BALB/c mice immunized with a non-relevant Ab2 mAb. The results suggest that mAb KM-04 constitutes a non-internal image vaccine to the lethal effect of lipid A in BALB/c mice. Furthermore an Ab3 mAb was prepared against Ab2 mAb KM-04 that showed reactivity with Re LPS. This Ab3 mAb, designated LE-21 (IgG2a) protected mice against an otherwise lethal challenge of Re LPS.     

Discrimination by rabbit anti-idiotypic antibodies of two murine IgM monoclonal antibodies directed against lipid A.

Two murine IgM monoclonal antibodies (MAs) directed against the lipid A portion of bacterial lipopolysaccharide (LPS) were compared in their binding to Re LPS and lipid A and their idiotypic make-up with rabbit anti-idiotypic sera. Horseradish peroxidase (HRPO)-labelled MAs 8-2 and 26-20 bound equally well to Re LPS. The binding of HRPO-labelled MA 8-2 to synthetic lipid A was low compared to the relatively strong binding of labelled 26-20. The MAs proved to be competitive in a competition binding assay (CBA) with Re LPS as coating antigen. Rabbit immune sera were raised against individual MAs. Anti-idiotypic antibodies (anti-id Abs) were detected with two sensitive enzyme immunoassays (EIA): a solid-phase EIA and an inhibition EIA. The rabbit antisera proved to be idiotype specific, indicating that both MAs recognize separate epitopes. We expect that anti-id Abs will prove to be of value for the differentiation of panels of LPS specific MAs.     

Cloning and expression in Escherichia coli of a Haemophilus influenzae type b lipooligosaccharide synthesis gene(s) that encodes a 2-keto-3-deoxyoctulosonic acid epitope.

The composition of lipooligosaccharide (LOS) can modify the virulence of Haemophilus influenzae type b (Hib). A genomic library of Hib strain A2 was constructed in the lambda bacteriophage EMBL3. Twenty-six phage clones expressed a Hib LOS oligosaccharide epitope in Escherichia coli that was detected by the monoclonal antibody (MAb) 6E4. None of the clones bound a polyclonal sera specific for Hib A2 LOS or an anti-H. influenzae lipid A MAb. One clone, designated EMBLOS-1, assembled an oligosaccharide with an apparent molecular weight of 1,400 (the 1.4K oligosaccharide) on a 4.1K lipopolysaccharide (LPS) species in E. coli LE392 and produced a novel 5.5K LPS that bound 6E4. Binding of 6E4 to the 5.5K EMBLOS-1 LPS band was abolished by treatment with sodium metaperiodate but was not affected by digestion with proteinase K, confirming the carbohydrate nature of the epitope. The EMBLOS-1 Haemophilus insert hybridized to similar restriction fragments in type b and nontypeable strains regardless of whether they expressed the 6E4 epitope. The 6E4 epitope did not undergo phase variation in Hib strain A2 at a frequency of greater than 10(-3). The oligosaccharide of the Salmonella minnesota Re mutant and 2-keto-3-deoxyoctulosonic acid (KDO) inhibited binding of 6E4 to Hib A2 LOS. We conclude that a gene(s) encoding an enzyme(s) that assembles a stable Hib LOS epitope containing KDO is conserved in H. influenzae and that the cloned Hib LOS synthesis gene products assemble a Hib LOS epitope on an E. coli K-12 LPS core.     

A synthetic glycoconjugate representing the genus-specific epitope of chlamydial lipopolysaccharide exhibits the same specificity as its natural counterpart.

The tetrasaccharide 3-deoxy-alpha-D-manno-2-octulosonic acid (alpha-KDO) (2----8)-alpha-KDO(2----4)-alpha-KDO(2----6)-beta GlcNAc, a partial structure of chlamydial lipopolysaccharide (LPS) representing a genus-specific epitope, was synthesized and covalently linked to bovine serum albumin, resulting in an artificial glycoconjugate antigen. Mice were immunized with the glycoconjugate to prepare chlamydia-specific monoclonal antibodies. They were selected with chlamydia-specific LPS antigens and the structurally and antigenically related Re-type LPS of a Salmonella minnesota rough mutant. Characterization of the selected antibodies was by (i) hemagglutination of sheep erythrocytes coated with recombinant chlamydia-specific LPS, (ii) inhibition by synthetic polyacrylamide derivatives containing the genus-specific epitope or partial structures thereof, (iii) enzyme immunoassay with recombinant LPS and synthetic bovine serum albumin glycoconjugates as solid-phase antigens, (iv) immunofluorescence of L929 monolayers infected with Chlamydia psittaci or C. trachomatis, and (v) Western immunoblots with glycoconjugates and LPS as the antigen. Two groups of monoclonal antibodies were obtained; the monoclonal antibodies in one group cross-reacted with chlamydial and Re-type LPS, but those of the other group were chlamydia specific. Among the latter, KDO trisaccharide-specific antibodies that had the same epitope specificity as antibodies obtained after immunization with chlamydial elementary bodies were identified; however, they exhibited a more than 100-fold higher affinity. In addition, antibodies that bound preferentially to the 2.8-linked KDO disaccharide were detected, although with lower affinity. The data show that the artificial glycoconjugate antigen is similar to its natural counterpart.     

Monoclonal anti-human IgG antibodies for quantitation of allergen-specific IgG in human sera

Eight monoclonal anti-human IgG antibodies were fully characterized and evaluated as possible reagents in solid phase radioimmunoassay for quantitating allergen-specific IgG antibody. Four monoclonal antibodies (HG24D, HG2-14, HG2-18, and HG2-25) recognize CH2 domain of human IgG and bind to human IgG fixed to microtiter plate with high affinities. These monoclonal antibodies were more suitable than polyclonal rabbit anti-human IgG antibody in Phadebas RAST for honey bee venom-specific IgG antibody. Nonspecific binding was much lower, and the slopes of standard curves were much steeper. In contrast to polyclonal antibody, the standard curve was hardly influenced by human serum IgG in sample diluent. These advantages of monoclonal antibodies that recognize CH2 domain of human IgG made it possible to quantitate egg white- and Dermatophagoides pteronyssinus-specific IgG antibodies with use of allergen disks prepared for IgE RAST. This property allows a single system to be used for measurement of IgG and IgE antibodies against clinically relevant allergens.     

Production of potent polyclonal antibodies to bacterial membrane amphiphiles

Lipid A (LA), ketodeoxyoctonate (KDO) and lipoteichoic acids (LTA) were used to produce homologous polyclonal antibodies. These haptens were administered to rabbits in differing immunogenic forms, using multiple intradermal and intraperitoneal injections with complete Freund adjuvant. Booster injections were either made intradermally with incomplete Freund adjuvant or intravenously in saline. The immune-response was monitored regularly with an enzyme-immunoassay. Lipid A and KDO covalently linked to bovine serum albumin (BSA), with hapten densities per BSA molecule of 17 and 9, respectively, produced nondetectable immune-response. Acid-hydrolysed and intact cells of Salmonella minnesota Re 595 used as LA and KDO immunogens, respectively, produced significant immune-response when administered intradermally or intraperitoneally. Good immune-response was obtained with LTA covalently linked to BAS. However, a better result was obtained with crude LTA, containing 21.5% proteins. Generally, the lengthy immunization schedules used produced IgG antibodies to the antigens and the highest reciprocal titres attained were 75,000, 55,000 and 150,000 for LA, KDO and LTA, respectively. Meaningful expression of antisera titres by enzyme-immunoassay is discussed. We defined titre as the reciprocal antiserum dilution of the intercept of the mid-point on the linear section ending at 0.2 absorbance on the antiserum dilution curve.     

Cross-reactive polyclonal antibodies to the inner core of lipopolysaccharide from Neisseria meningitidis

Sera from mice immunized with native or detergent-extracted outer membrane vesicles derived from lipopolysaccharide (LPS) mutant 44/76(Mu-4) of Neisseria meningitidis were analyzed for antibodies to LPS. The carbohydrate portion of 44/76(Mu-4) LPS consists of the complete inner core, Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->5KDO[4-->2 alpha KDO]. Immunoblot analysis revealed that some sera contained antibodies to wild-type LPS which has a fully extended carbohydrate chain of immunotype L3,7, as well as to the homologous LPS. Sera reacted only weakly to LPS from 44/76(Mu-3), which lacks the terminal glucose of the inner core. No binding to more truncated LPS was observed. Consequently, the cross-reactive epitopes are expressed mainly by the complete inner core. Dephosphorylation of wild-type LPS abolished antibody binding to LPS in all but one serum. Thus, at least two specificities of cross-reactive antibodies exist: one is dependent on phosphoethanolamine groups in LPS, and one is not. Detection of these cross-reactive antibodies strongly supports the notion that epitopes expressed by meningococcal LPS inner core are also accessible to antibodies when the carbohydrate chain is fully extended. Also, these inner core epitopes are sufficiently immunogenic to induce antibody levels detectable in polyclonal antibody responses. Meningococci can escape being killed by antibodies to LPS that bind only to a specific LPS variant, by altering the carbohydrate chain length. Cross-reactive antibodies may prevent such escape. Therefore, inner core LPS structures may be important antigens in future vaccines against meningococcal disease.     

Preparation of anti-idiotypic antibody against avian influenza virus subtype H9

To generate monoclonal anti-idiotypic antibodies(mAb2)against avian influenza virus subtype H9(H9 AⅣ),BALB/c mice were immunized with purified chicken anti-H9-AⅣ IgG and the splenocytes of immunized mice werefused with myeloma cells NS-1.Hybridoma cells were screened by indirect enzyme-linked immunosorbent assayswith both chicken and rabbit anti-H9-AⅣ IgG as coating antigens.One hybridoma cell clone secreting monoclonalantibody against idiotypes shared by both chicken and rabbit anti-H9-AⅣ IgG was established.Experimentsdemonstrated the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AⅣ IgG and to inducechickens to generate hemagglutination inhibition antibodies,indicating this anti-species-sharing-idiotypic antibodybore the internal image of hemagglutinin on avian influenza virus.Cellular & Molecular Immunology.2005;2(2):155-157.     

Chemical, biological, and immunochemical properties of the Chlamydia psittaci lipopolysaccharide.

The lipopolysaccharide (LPS) of Chlamydia psittaci was extracted from yolk sac-grown elementary bodies, purified, and characterized chemically, immunochemically, and biologically. The LPS contained D-galactosamine, D-glucosamine, phosphorus, long-chain fatty acids, and 3-deoxy-D-manno-2-octulosonic acid in the molar ratio of approximately 1:2:2:6:5. The antigenic properties of the isolated LPS were compared with those of the LPS from Chlamydia trachomatis and Salmonella minnesota Re by the passive hemolysis and passive hemolysis inhibition tests, absorption, hydrolysis kinetics, and Western blot analysis with rabbit polyclonal antisera against chlamydiae and with a mouse monoclonal antibody recognizing a genus-specific epitope of chlamydial LPS. Two antigenic determinants were identified, one of which was chlamydia specific and the other of which was cross-reactive with Re LPS. Both determinants were destroyed during acid hydrolysis, whereby a third antigen specificity was exposed which was indistinguishable from the lipid A antigenicity. In rabbit polyclonal antisera prepared against Formalin-killed elementary bodies or detergent-solubilized membranes, two antibody specificities were differentiated. One of these was chlamydia specific, and the other was cross-reactive with Re LPS. The LPS of C. psittaci was inactive within typical endotoxin parameters (lethal toxicity, pyrogenicity, local Shwartzman reactivity); it was, however, active in some in vitro assays, such as those testing for mouse B-cell mitogenicity and the induction of prostaglandin E2 in mouse peritoneal macrophages.     

Inhibition by lipid A-specific monoclonal antibodies of priming of human polymorphonuclear leucocytes by endotoxin

Exposure to lipopolysaccharide (LPS) primes polymorphonuclear leucocytes (PMNL) for enhanced release of oxygen metabolites after subsequent stimulation. The metabolic response of human PMNL primed with LPS and stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP) was measured by chemiluminescence (CL) as a parameter for endotoxic activity. Polymyxin B (PMB) and monoclonal antibodies (MAbs) with specificity for lipid A were tested for inhibition of the priming effect of Re LPS of Salmonella minnesota R595, Rc LPS of Escherichia coli J5 and smooth LPS of E. coli O111. The CL response of PMNL primed with Re LPS or Rc LPS was higher than that of PMNL primed with smooth LPS. Pre-incubation of rough or smooth LPS with PMB caused dose-dependent inhibition of priming of PMNL. Two IgM MAbs, 8-2 and 26-20, which recognise different epitopes on the hydrophobic part of lipid A, also completely prevented priming of PMNL by either rough or smooth LPS. The dose-dependent inhibitory effect of both MAbs was similar to the inhibition by PMB. These results indicate that the binding of MAbs to the hydrophobic part of lipid A is important in blocking lipid A-mediated effects.     

Immunological Detection of Lipopolysaccharide Antigens of Thermophilic Campylobacters Captured on Polymyxin-Coated Polyester Cloth

Cholate-extracted lipopolysaccharide (LPS) antigens from thermophilic campylobacters were captured on polymyxin-coated polyester cloth. The captured antigens were detected by sequential reactions with rabbit anli-Campylobacter antibody, anti-rabbit IgG peroxidase conjugate and chromogenic peroxidase substrate. A polyclonal rabbit antibody elicited against a single Campylobacter jejuni strain detected the reference strains of the twenty most frequently isolated thermophilic campylobacters in the Lior serotyping scheme. Moreover, LPS antigens of six C. jejuni Penner serotypes fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting were recognized by four antisera prepared against homologous and heterologous Penner serotypes. The results suggest the potential application of polymyxin-cloth enzyme immunoassay for rapid detection of thermophilic campylobacters where monoclonal antibodies can be raised to possible common LPS epitopes.     

Prevention of lethal endotoxemia in actinomycin D-sensitized mice by incubation of Salmonella minnesota R595 lipopolysaccharide with monoclonal antibodies to R595

Murine monoclonal antibodies reacting with lipopolysaccharide (LPS) of Salmonella minnesota strain R595 (Re chemotype) were prepared, and tested for their ability to protect actinomycin D-sensitized mice against lethal endotoxemia. Protection was found with some antibodies up to a 90-fold increase in LD50, whereas others exhibited no protection. The various protective antibodies did not all bind to the same epitope. The same applied for non-protective clones. Protective and non-protective clones could not be discriminated by ELISA. One protective monoclonal antibody (clone 20) was specific for ketodeoxyoctonate, a structural element common to various LPS. These findings show that the involvement of lipid A in the binding site of monoclonal antibodies is no prerequisite for protection.     

Immunopharmacological activities of 2-keto-3-deoxyoctonic acid-(alpha 2----6)-linked 4-O-phosphono-D-glucosamine derivatives carrying N- and 3-O-acyl substituents.

The immunopharmacological activities of 2-keto-3-deoxyoctonic acid (KDO)-(alpha 2----6)-linked lipid A-subunit analogs, 4-O-phosphono-D-glucosamine derivatives carrying N- and 3-O-acyl substituents, were compared with those of the corresponding analogs without KDO, GLA-27, GLA-47, and GLA-60. Among the analogs tested, GLA-60, a 4-O-phosphono-D-glucosamine carrying N-3-hydroxytetradecanoyl and 3-O-3-tetradecanoyloxytetradecanoyl groups, exhibited the most intensive activities in terms of mitogenicity, adjuvanticity, and mediator (tumor necrosis factor and colony-stimulating factor) induction. Binding (alpha 2----6) of KDO to GLA-60 failed to enhance the activities. Similarly, the activities of GLA-27 and GLA-47 were also decreased by introduction of KDO to the O-6 of the analogs. This indicates that the strengths of the activities of the subunit analogs depend on the kinds of N- and 3-O-linked acyl substituents and not on the presence of the KDO linked to the O-6.     

Effective Inhibition of Cardiolipin-Binding Antibodies in Gram-Negative Infections by Bacterial Lipopolysaccharide

Anticardiolipin antibodies (ACA) were detected by solid-phase enzyme immunoassay in the majority of sera from patients with Gram-positive and Gram-negative bacterial infections. The response involved all the major immunoglobulin classes IgG, IgM, and IgA. The specificity of the ACA was studied in competitive inhibition experiments with three putative antigens: cardiolipin, lipopolysaccharide (LPS) isolated from Salmonella minnesota, strain Re 595, and synthetic Escherichia coli lipid A. The binding of IgG class ACA from the sera of five patients with Gram-negative infections was effectively inhibited by LPS, whereas 100-fold more cardiolipin was required for comparable inhibition. Pure lipid A was a less effective inhibitor of anticardiolipin activity than LPS. This pattern of reactivity was not seen in sera from patients with Gram-positive infections, syphilis, or systemic lupus erythematosus. Our findings suggest that cardiolipin may not be the inducing antigen for the cardiolipin-binding antibodies that develop in Gram-negative infections.     

A novel trait of naturally occurring anti-DNA antibodies: dissociation from immune complexes in neutral 0.3-0.5 M NaCl

Monoclonal and polyclonal anti-DNA antibodies from autoimmune mice, and experimentally induced rabbit anti-nucleic acid polyclonal antibodies were tested for stability of binding to nucleic acids in the presence of various concentrations of NaCl by an enzyme-linked immunosorbent assay (ELISA). Murine monoclonal antibodies 2C10 (IgG2b) and 1A2 (IgG2a), which are known to react specifically with double-stranded (ds) DNA, dissociated completely from their complexes with DNA when washed with a neutral 0.5 M NaCl solution. Another monoclonal antibody (MoAb) (IgM,kappa), polyreactive with single-stranded (ss) DNA, cardiolipin, and trinitrophenylhapten (TNP), was also dissociated from its complexes with ss DNA, but not from its complexes with TNP, by 0.3-0.5 M NaCl. Similar differences were observed in the binding stability of serum antibodies from autoimmune mice to DNA and TNP. In contrast, anti-nucleic acid polyclonal antibodies induced in rabbits by immunization with poly(I), poly(dT) or poly(ADP-ribose) were not significantly dissociated from their immune complexes with relevant antigens or DNA by 0.5 M NaCl. The finding that nucleic acid antigens were not detached from a solid phase by washing with 0.5 M NaCl solution indicated that the reduction of binding of anti-DNA antibodies in both MoAbs and naturally occurring antibodies was really due to dissociation of the antibodies from immune complexes. This is the first demonstration that DNA epitopes recognized by naturally occurring antibodies in both SLE and its mouse models are sensitive to neutral NaCl concentrations. This novel trait of naturally occurring antibodies will be very useful in studies on the nature of immune complexes in sera and kidneys of cases of systemic lupus erythematosus (SLE).     

Monoclonal antibodies to salmonella lipopolysaccharide: functional analysis of anti-lipid A antibodies.

We have produced monoclonal antibodies (MoAb) to the Rb core and lipid A regions of Salmonella lipopolysaccharide (LPS) and have assessed their ability to inhibit LPS-mediated mitogenic responses in vitro, and to protect against LPS toxicity and lethal Salmonella infection in vivo. Monoclonal antibodies RC-8 and RC-16 were specific for LPS Rb core determinants, and MoAb LA-1, LA-2, LA-3, LA-4 and LA-5 were specific for lipid A. Anti-lipid A MoAb LA-2, LA-3 and LA-5 were found to abrogate mitogenic responses of C3H/HeN spleen cells to smooth S. typhimurium LPS (S LPS) and to rough S. minnesota R595 LPS (Re LPS). Monoclonal antibody LA-5 was effective in extending the median length of survival of C3H/HeN mice challenged with a lethal dose of either S LPS or Re LPS. Antibody LA-2 could extend the median length of survival of C3H/HeJ mice challenged with Re LPS but not with S LPS, and failed to extend significantly the length of survival of S LPS-challenged C3H/HeN and DBA/2 mice. Neither 20 micrograms of anti-Rb core or anti-lipid A MoAb nor 200 micrograms of anti-lipid A MoAb were able to protect C3H/HeN or BALB/c mice, respectively, against lethal infection with S. typhimurium SR-11. These results suggest that the importance of anti-lipid A antibodies in host defence may lie more in their ability to neutralize pathological effects of LPS, than in their ability to protect against bacterial infection.     

Minimal oligosaccharide structures required for induction of immune responses against meningococcal immunotype L1, L2, and L3,7,9 lipopolysaccharides determined by using synthetic oligosaccharide-protein conjugates.

The 12 types of meningococcal lipopolysaccharide (LPS) (immunotypes) contain immunotype-specific and cross-reactive epitopes situated on the oligosaccharide part of the LPS molecules. To identify useful cross-reactive epitopes and to determine minimal oligosaccharide structures required for the induction of an immune response against the most prevalent immunotypes, L1, L2, and L3,7,9, synthetic as well as native LPS-derived oligosaccharides were conjugated with tetanus toxoid. L3,7,9 phosphoethanolamine (PEA) group-containing oligosaccharide-tetanus toxoid conjugates evoked high immunoglobulin G (IgG) antibody levels in rabbits which were detected by an L2-, L3,7,9-, and, depending on the antiserum, L1-specific enzyme-linked immunosorbent assay (ELISA). Inhibition studies revealed that an identical antibody population was detected by L1 and L3,7,9 ELISA, indicating a similar tertiary structure of the inner core oligosaccharide of these two immunotypes. These antibodies recognize PEA group-containing epitopes present on the L1 and L3,7,9 LPS. An L2 PEA group-containing oligosaccharide-tetanus toxoid conjugate elicited L2- and L3,7,9-specific IgG antibodies, but in contrast with the L3,7,9 conjugates, no L1-specific IgG antibodies were evoked. These results indicate that L1 and L2 LPS do not contain cross-reactive epitopes, whereas both L2 and L3,7,9 LPS and L1 and L3,7,9 LPS possess common determinants. Three linear oligosaccharides and one branched oligosaccharide, representing partial structures of the inner core oligosacchardes of meningococcal LPS, were synthesized. Only the branched synthetic oligosaccharide-containing conjugate was able to induce and L1- and L3,7,9-specific immune response, whereas the linear oligosaccharide-protein conjugates evoked L2-specific immune responses. The branched oligosaccharide (beta-D-Glcp(1----4)-[L-alpha-D-Hepp(1----3)]-L-alpha-D-Hepp ) is therefore considered a minimal structure required for the induction of an immune response against L1 and L3,7,9 LPS and part of a cross-reactive epitope between these two immunotypes. For L2-specific immune responses, oligosaccharide structures terminating in beta-D-Glcp(1----4), alpha-D-GlcNAcp(1----2), or L-alpha-D-Hepp(1----5) are needed. The results suggest that it is possible to prepare an oligosaccharide structure with the ability to evoke an immune response against L1, L2, and L3,7,9 LPS. A feasible structure for such a "hybrid" oligosaccharide is discussed.     

Lactoferrin-Lipid A-Lipopolysaccharide Interaction: Inhibition by Anti-Human Lactoferrin Monoclonal Antibody AGM 10.14

Lactoferrin (LF) is a glycoprotein that exerts both bacteriostatic and bactericidal activities. The interaction of LF with lipopolysaccharide (LPS) of gram-negative bacteria seems to play a crucial role in the bactericidal effect. In this study, we evaluated, by means of an enzyme-linked immunosorbent assay, the binding of biotinylated LF to the S (smooth) and R (rough) (Ra, Rb, Rc, Rd1, Rd2, and Re) forms of LPS and different lipid A preparations. In addition, the effects of two monoclonal antibodies (AGM 10.14, an immunoglobulin G1 [IgG1] antibody, and AGM 2.29, an IgG2b antibody), directed against spatially distant epitopes of human LF, on the LF-lipid A or LF-LPS interaction were evaluated. The results showed that biotinylated LF specifically binds to solid-phase lipid A, as this interaction was prevented in a dose-dependent fashion by either soluble uncoupled LF or lipid A. The binding of LF to S-form LPS was markedly weaker than that to lipid A. Moreover, the rate of LF binding to R-form LPS was inversely related to core length. The results suggest that the polysaccharide O chain as well as oligosaccharide core structures may interfere with the LF-lipid A interaction. In addition, we found that soluble lipid A also inhibited LF binding to immobilized LPS, demonstrating that, in the whole LPS structure, the lipid A region contains the major determinant recognized by LF. AGM 10.14 inhibited LF binding to lipid A and LPS in a dose-dependent fashion, indicating that this monoclonal antibody recognizes an epitope involved in the binding of LF to lipid A or some epitope in its close vicinity. In contrast, AGM 2.29, even in a molar excess, did not prevent the binding of LF to lipid A or LPS. Therefore, AGM 10.14 may represent a useful tool for neutralizing selectively the binding of LF to lipid A. In addition, the use of such a monoclonal antibody could allow better elucidation of the consequences of the LF-lipid A interaction.