Neutralizing and cross-reactive antibodies against enterobacterial lipopolysaccharide

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International Journal of Medical Microbiology 297 (2007) 321–340 www.elsevier.de/ijmm

SHORT SURVEY

Neutralizing and cross-reactive antibodies against enterobacterial lipopolysaccharide Sven Mu¨ller-Loennies, Lore Brade, Helmut Brade Division of Medical and Biochemical Microbiology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany

Abstract Lipopolysaccharide (LPS, endotoxin) elicits an immune reaction which is responsible for many of the harmful effects seen in septic shock patients. The eradication of bacteria by antibiotics is insufficient to resolve the pathology due to the lack of LPS neutralization. LPS-neutralizing antibodies have been described; however, these were specific for the serotype of the infecting bacteria and thus not useful for the treatment of septic shock patients. Structural analyses revealed that the LPS structures of Escherichia coli and Salmonella are structurally conserved in the inner core region. Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides, which we have obtained from all E. coli core types (K-12, R1, R2, R3 and R4), Salmonella enterica, and the mutant strain E. coli J-5, we have identified an epitope which is bound with high affinity by the monoclonal antibody WN1 222-5, which has been shown previously shown to be cross-reactive against a large collection of blood, fecal, and urinary isolates of E. coli, S. enterica, some Citrobacter, independently of the serotype [Di Padova, F.E., Brade, H., Barclay, G.R., Poxton, I.R., Liehl, E., Schuetze, E., Kocher, H.P., Ramsay, G., Schreier, M.H., McClelland, D.B., Rietschel, E.T., 1993. A broadly cross-protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharides. Infect. Immun. 61, 3863–3872]. Importantly, WN1 222-5 was protective in various models of endotoxic shock. The minimal structural element necessary for high-affinity binding consists of R1-a-D-Glcp-(1-3)-[L-a-D-Hepp-(1-7)]-L-a-D-Hepp 4P-(1-3)-R2 (R1, R2 ¼ additional sugars of LPS) in which the side-chain heptose and the 4-phosphate on the branched heptose are the main determinants of the epitope. Additional sugars of the outer core (R1) enhance the affinity, whereas loss of an intact Kdo region and/or lipid A (R2) prevent binding. The identification of the epitope provides the structural basis for the rational development of a potential vaccine against E. coli LPS. r 2007 Elsevier GmbH. All rights reserved. Keywords: Lipopolysaccharide; Sepsis; Antibody; Therapy; Epitope; Core

Introduction The cell walls of Gram-negative and Gram-positive bacteria differ fundamentally (Fig. 1) and only the former contain an outer membrane. The outer leaflet of this outer membrane is made from a phosphoglycolipid Corresponding author. Tel.: +49 45 3718 8774; fax: +49 45 3718 8419. E-mail address: [email protected] (S. Mu¨ller-Loennies).

1438-4221/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2007.04.002

called lipopolysaccharide (LPS, endotoxin), while the inner leaflet is made from phospholipids (Funahara and Nikaido, 1980; Nikaido, 2003). Gram-negative bacteria of the Enterobacteriaceae such as the genera Escherichia, Salmonella, Klebsiella, Proteus, Yersinia, and Shigella comprise harmless or even vitally important members of the commensal flora of mammals, but the same genera also contain important human pathogens. After invasion, an infection may be established during which bacteria reach

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Fig. 1. Schematic view of the cell envelope of Gram-positive and Gram-negative bacteria. Gram-negative bacteria contain an outer membrane (OM) which is absent in Gram-positive bacteria. It creates an additional compartment, the periplasmic space. The outer layer of the OM is composed of lipopolysaccharides (LPS) whereas the inner layer is composed of phospholipids (Funahara and Nikaido, 1980; Nikaido, 2003).

subepithelial tissues and the bloodstream. This may also occur after traumatic stress, surgery and severe burns which compromise the protective function of the physical barriers. This potentially life-threatening event leads, as a first line of defense, to the activation of innate immune responses, which are mediated by dendritic cells, neutrophils, endothelial cells, and monocytes and macrophages. The latter appear to play a prominent role since the transfer of LPS-responsive macrophages into LPS non-responder mice was sufficient to confer responsiveness (Freudenberg et al., 1986). These cells have developed mechanisms to recognize molecules of microbial origin to eliminate the potentially life-threatening microbes from the infected site and continuously monitor the body for the presence of LPS and other microbial products which are commonly referred to as pathogen-associated molecular patterns (PAMP). The toxic effects of heat-stable components of the Gram-negative bacterium Vibrio cholerae were already described in 1892 by Richard Pfeiffer, and the term endotoxin was introduced to distinguish this class of toxins from actively secreted heat-labile exotoxins. Subsequently, chemical analysis revealed that endotoxins are LPS and both terms are used synonymously since. By orders of magnitude the most potent stimulus of all PAMP is LPS from S. enterica and E. coli (described below) due to a characteristic chemical structure (Rietschel et al., 1994). The interested reader is referred to reviews on the history of endotoxin which have been published recently (Rietschel and Westphal, 1999; Beutler and Rietschel, 2003). Various receptors of immune cells are involved in the innate immune recognition (pattern recognition recep-

tors), among which Toll-like receptors (TLR) have been shown to play an important role (Alexander and Rietschel, 2001; Akira and Takeda, 2004). Apart from the innate defense system, the adaptive immune response is also activated. The processes which lead to the activation of the immune system following an LPS stimulus are not yet completely understood at the molecular level, but significant progress has been made in the past decade. According to the current view, the serum component LPS-binding protein (LBP) complexes LPS and catalyses its transfer to CD14 (membrane bound or soluble), which then associates with Toll-like receptor (TLR) 4 and forms together with the protein myeloid differentiation-2 (MD-2), a signaling complex within the membrane (Beutler et al., 2003). Additional molecules such as moesin (Tohme et al., 1999; Amar et al., 2001; Iontcheva et al., 2004), the MaxiK channel (Seydel et al., 2001) and membranebound LBP (Mu¨ller et al., 2003) may be part of the signaling complex within the membrane. Downstream events include the activation of components of the interleukin-1 (IL-1) receptor pathway employing several adaptor molecules (Fitzgerald et al., 2003) leading to the activation of transcription factors of the nuclear factor kB family. The activation of the immune system may lead to the eradication of bacteria at the infected site; however, upon dissemination an overwhelming hyper-inflammatory response (systemic inflammatory response syndrome, SIRS) may occur, which according to the current concept of sepsis development (for discussion see Opal, 2007) is counter regulated, leading to the suppression of the immune system and the subsequent

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failure to manage accurately the infection (Riedemann et al., 2003). Symptoms of infection and sepsis such as fever, leukopenia, tachycardia, and vasoconstriction, which are later followed by vasodilation, are due to an increased production and release of a large set of diverse mediators which act synergistically or antagonistically (e.g. pro- and anti-inflammatory; Fig. 2). A central event is the activation of macrophages and blood monocytes to release tumor necrosis factor-a (TNF-a) and IL-1 (Freudenberg et al., 1986; Vogel, 1990). Further mediators comprise lipid mediators such as leukotrienes, prostaglandins and platelet-activating factor, as well as further interleukins (IL-6, IL-8, IL-12, IL-23), nitric oxide, and interferon g. Furthermore, complement is activated, and the activation of the coagulation cascade under simultaneous inhibition of fibrinolysis may lead to disseminated intravascular coagulation. All these events contribute to organ damage or even organ failure, which potentially result in the death of patients. This devastating state, known as ‘‘sepsis’’ or ‘‘septic shock’’, can be elicited apart from Gram-negative bacteria also by other pathogens such as Gram-positive bacteria, viruses, and fungi. Septic episodes, with an estimated incidence of 4750,000 cases each year in the United States of America, are associated with a high mortality rate among patients who develop septic shock, ranging

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from 30% to 70% (Cohen, 2002). Frequent Gramnegative isolates of blood cultures are E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa (Ziegler et al., 1982). Also Haemophilus influenzae and Neisseria meningitidis are frequently identified as the cause of fulminant sepsis. Despite the diverse nature of these pathogens, they elicit similar innate immunological host responses. Biological activities of LPS have been well established in vitro and in vivo, but some uncertainty remained with regard to the role as a virulence factor during infections. Knowledge of the biosynthesis of LPS (Raetz and Whitfield, 2002) together with established structure–activity relationships (Rietschel et al., 1994) allowed the construction of mutant bacteria containing an LPS, which was functional as an outer-membrane component but lost its endotoxic properties (Khan et al., 1998). After infection of mice such a mutant was unable to cause disease, despite an in vivo growth rate comparable to the wild-type strain, which indicated that LPS can be regarded as an important virulence factor during infections with Gram-negative bacteria. Accordingly, it can be expected that the neutralization of endotoxic activities is beneficial for the outcome of sepsis and may be able to prevent its development.

Fig. 2. Immunological events in the development of septic shock. Immunocompetent cells, such as neutrophils, endothelial cells, monocytes, and macrophages, are activated by endotoxins to produce large amounts of cytokines and other bioactive mediators as an innate defense mechanism. Additionally, complement and the coagulation cascade are activated. These events lead to symptoms such as fever, coagulopathy, vasodilation, and capillary leakage, and as a hallmark of septic shock to organ damage.

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Current strategies for the treatment of septic patients rely mainly on the eradication of bacteria by antibiotics and additional intensive care measurements. Antibiotic treatment, however, does not prevent the release of LPS into the bloodstream, and in fact an increased release of LPS has been observed after treatment with certain antibiotics (Rodriguez et al., 2005). This may be one explanation why, despite the efficient killing of the bacteria, shock caused by Gram-negative pathogens could not be significantly reduced (David, 2001). Attempts of treatment by the removal of mediators from the circulation by hemoadsorption or by directly targeting proinflammatory cytokines such as TNF-a and IL-1 have been considered (Baumgartner et al., 1999) but proved unsuccessful in clinical studies. Since not a single factor can be held responsible for the pleiotropic effects exerted by LPS, but instead a large number of mediators derived from different cell types, which form a complex network of interactions (Fig. 2), it is certainly advantageous to inhibit the activation cascades at a very early stage by either blocking at the receptor site using, e.g., LPS antagonists (Pollack and Ohl, 1996) or by targeting LPS directly, e.g. by removing it from the circulation in a biologically inactive form. This has been attempted with bactericidal/permeability increasing protein (BPI), polymyxin B, other antibacterial polycationic peptides, LBP, adsorption technologies, and antibodies. However, as discussed by Opal (2007), such attempts were unsuccessful in clinical studies, so far, although several promising results were obtained in in vitro and also in vivo models. This surprising fact may be explained by individual differences among patients and the complex immunological events during septic episodes, which makes the treatment of septic patients difficult, and until today the mortality of sepsis remains unacceptably high (Beutler and Rietschel, 2003; Riedemann et al., 2003). The development of novel treatment strategies requires a deeper understanding of endotoxin activity and the establishment of structure–function-relationships. For the latter a detailed knowledge of the chemical structures of LPS is a prerequisite. Such studies are hampered by the amphiphilic properties of LPS which result in the formation of aggregates in solution, and therefore pure preparations are difficult to obtain. The development of extraction procedures for the preparation of pure LPS (Westphal and Jann, 1965; Galanos et al., 1969) provided the first basis for biological meaningful experiments. Degradation by chemical means showed that upon treatment with mild aqueous acid, a precipitate could be obtained which was termed lipid A and represents the lipid anchor of the LPS molecule in the outer membrane (Westphal and Lu¨deritz, 1954; Imoto et al., 1985), and which was later on identified as the endotoxic principle of LPS (Lu¨deritz

et al., 1984; Galanos et al., 1985; Kotani et al., 1985). A detailed investigation of structure–activity relationships (Kotani et al., 1984; Takada et al., 1988; Rietschel et al., 1994), including a biophysical characterization of biologically active and inactive lipid A revealed structural parameters which influence the biological activities of LPS (Rietschel et al., 1990; Seydel et al., 1999).

The chemical structure of LPS In general, LPS are phosphorylated glycolipids that possess complex chemical structures. The chemical structures of LPS and their biosynthesis from many bacteria have been analyzed (Holst, 1999, 2002; Za¨hringer et al., 1999; Jansson, 1999; Gronow and Brade, 2001; Raetz and Whitfield, 2002; Trent, 2004; Frirdich and Whitfield, 2005). From these studies it became evident that a tremendous variability of chemical structures of LPS is synthesized by bacteria, reflecting that LPS have evolved to support bacterial growth in different environments. Individual LPS molecules may differ in their degree of acylation, phosphorylation, glycosylation, and further less common structural modifications. In order to adapt to different environmental conditions, bacteria have developed the ability to sense environmental changes in pH, salt concentration, and temperature by two-component regulatory systems such as PhoP/PhoQ and PmrA/ PmrB. Such systems are also involved in the structural modification of LPS and have been suggested to play a role in structural changes of LPS upon intracellular growth (Guo et al., 1997; Gunn et al., 1998, 2000; Kawasaki et al., 2004; Vinogradov et al., 2004; Moskowitz et al., 2004; Tamayo et al., 2005). Mammalian pathogens have to cope with the evolutionary pressure exerted by the innate and particularly the adaptive immune systems of their hosts. Trying to escape the immunological defense, surface molecules such as LPS are structurally altered to evade recognition. With respect to the establishment of structure–function relationships, it is important to realize that despite a common overall architecture, all bacteria, even of the same strain, produce a variety of different LPS molecules with potentially very different biological activities. The strong biological activity of certain LPS requires the identification of even minor components in these preparations to fully understand biological effects, and LPS preparations of highest purity are needed. Despite advances in knowledge and technology, the chemical analysis of LPS is still difficult due to the amphiphilic nature of LPS, the heterogeneity of LPS preparations due to non-stoichiometric structural variations, and the chemical lability of certain substituents. The chemical structures of LPS of enterobacteria such

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Fig. 3. Schematic representation of LPS from S. enterica. The LPS molecule consists of a lipid A, an inner and outer core region, and an O-polysaccharide [Smooth(S)-type LPS]. The O-polysaccharide often is made up from smaller oligosaccharides (repeat units).

as Salmonella (Olsthoorn et al., 1998, 2000), E. coli (Holst, 1999; Vinogradov et al., 1999; Mu¨ller-Loennies et al., 2002, 2003b; Stenutz et al., 2006), and Klebsiella (Su¨sskind et al., 1995, 1998; Vinogradov and Perry, 2001; Vinogradov et al., 2001, 2002; Frirdich et al., 2004; Regue et al., 2005) have been investigated in great detail (see below) and for the purpose of this review are presented in more detail below. LPS obtained from wildtype bacteria of these genera are composed of three domains, a lipid A, a core oligosaccharide (core-OS), and an O-polysaccharide (O-PS, Fig. 3). Bacteria which contain an O-PS have a smoothcolony appearance when grown on agar plates and therefore this type of LPS is referred to as smooth(S)type LPS. A length polymorphism of the O-PS leads to a ladder-like banding pattern in polyacrylamide gel electrophoresis. LPS molecules that are devoid of the O-PS are called rough(R)-type LPS. Mutant bacteria (rough mutants) with a defect in certain biosynthetic steps involved in LPS biosynthesis produce LPS with short oligosaccharide chains and are unable to produce or attach an O-PS. Chemical analysis of LPS from such Salmonella mutants led to the differentiation of several chemotypes (R-chemotypes) that could be distinguished serologically (R-factors) (Galanos et al., 1977) and are referred to as Ra to Re chemotypes. Ra describes the largest core structure and Re was assigned to the smallest core structure (Fig. 3), which is devoid of all core sugars except for an (a2-4)-linked 3-deoxy-Dmanno-oct-2-ulopyranosonic (Kdo) disaccharide. Apart from these mutants, certain non-enteric pathogenic bacteria such as Neisseria, Haemophilus, Chlamydia, and Yersinia pestis produce only R-type LPS which are naturally devoid of O-PS (Holst, 1999).

The lipid A The lipid anchor of LPS is called lipid A, and several reviews of the chemical structures of lipid A and their

Fig. 4. Chemical structure of the main lipid A from E. coli and S. enterica. Under certain growth conditions, structural variants are found which contain in the 1- or 40 -position 4amino-4-deoxy-b-L-arabinose (Ara4N) or 2-amino-ethanol phosphate (PEtN) which are connected to the 1- and 40 monophosphates. A diphosphate may be present in the 1position instead of a monophosphate. Palmitate (C16:0) may be present as secondary fatty acid attached to the 3hydroxymyristic acid [14:0(3-OH)] in position 2. The secondary myristic acid (14:0) may be replaced by S-2-hydroxymyristic acid [14:0(2-OH)]. When grown at lower temperature, a 16:1 fatty acid replaces the secondary 12:0 at position 2’ in E. coli.

biosynthesis have been published recently (Za¨hringer et al., 1999; Gronow and Brade, 2001; Raetz and Whitfield, 2002; Trent, 2004). A lipid A structure which is widely distributed in nature has first been elucidated in LPS from Salmonella and E. coli (Fig. 4) and consists of an N- and O-hexaacylated (b1-6)-linked D-glucosamine (GlcN) disaccharide, which is phosphorylated in positions 1 and 40 (P-GlcNII-GlcNI’P), lipid A backbone). b-Hydroxylated fatty acids are characteristic components of lipid A which, for biosynthetic reasons (Raetz and Whitfield, 2002; Trent, 2004; Trent et al.,

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2006), always quantitatively substitute the 2 and 20 positions of the backbone in an amide linkage. They always possess the R-configuration and may be further located at the 3 and 30 -positions in ester linkage. Secondary fatty acids may be present in ester linkage at the b-hydroxyl groups. In E. coli and Salmonella, b-hydroxy tetradecanoic acid [14:0(3-OH)] is the only primary fatty acid, which carries tetradecanoic acid and dodecanoic acid in positions 20 and 30 of the lipid A backbone, respectively, leading to an asymmetrically hexaacylated lipid A (Za¨hringer et al., 1999). In S. enterica, a heptaacylated lipid A species is synthesized in non-stoichiometric amounts which carries hexadecanoic acid (16:0) as secondary fatty acid at position 2 of GlcNI. In several lipid A from diverse species including Salmonella, Pseudomonas, Klebsiella, Bordetella, and Legionella a-hydroxy fatty acids have been identified. These always possess the S-configuration (Rietschel et al., 1972; Rietschel, 1976). So far, these have neither been found in primary positions nor further esterified. The degree of a-hydroxylation appeared to be regulated by environmental factors such as growth temperature, and structural analysis suggested that a-hydroxylation takes place at the fully acylated lipid A (Za¨hringer et al., 1994). The biosynthetic pathway of lipid A in E. coli and Salmonella has been elucidated (Raetz and Whitfield, 2002) and it could be shown that the non-stoichiometric substitution with a-hydroxymyristate instead of myristate as secondary fatty acid in the 30 -position in S. enterica sv. Typhimurium is due to a hydroxylation reaction carried out by the LpxO enzyme (Gibbons et al., 2000). This enzyme is missing in E. coli, but homologs have been identified in the abovementioned bacteria which contain 2-hydroxy fatty acids (Trent, 2004). Further structural modifications of lipid A in Salmonella, such as the addition of 16:0 and the removal of the 14:0(3-OH) fatty acid at the 3-position of GlcN I are the result of the PagP (Bishop et al., 2000; Raetz, 2001) and PagL (Trent et al., 2001; Trent, 2004) enzymes, respectively, which are both located in the outer membrane. Homologs of the PagL enzyme have been identified in a variety of other genera, including Bordetella, Pseudomonas, Ralstonia, Burkholderia, and Azotobacter (Geurtsen et al., 2005). Attached to the 60 -position of the lipid A backbone is a heteropolysaccharide of varying length via a ketosidic linkage involving Kdo in most bacteria (Holst, 2002). The ketosidic linkage between Kdo and the lipid A is labile towards mild acid, a procedure that is often applied to cleave the lipid A from the remaining saccharides in order to isolate the bioactive lipid A component. Alternatively, strong alkaline hydrolysis may be applied for complete deacylation which renders the molecule biologically inactive, however, allows the chromatographic isolation of homogeneous oligosaccharide preparations which are amenable to a detailed

structural analysis by, e.g., NMR (Seltmann and Holst, 2001). Structural modifications of the lipid A backbone comprise the addition of organic phosphate, 2-aminoethanol (EtN), 2-aminoethanol phosphate (PEtN), 2-aminoethanol diphosphate (PPEtN), GlcN, GalN, GalA, 4-amino-4-deoxy-b-L-arabinopyranose (Ara4N), D-arabinofuranose (Araf), or the enzymatic removal of phosphate by phosphatases (Za¨hringer et al., 1999; Raetz and Whitfield, 2002). Two-component regulatory systems such as the PhoP/PhoQ and PmrA/PmrB systems have been shown to be involved in the control of several of these modifications (Gunn and Miller, 1996; Soncini and Groisman, 1996). Structural modifications of LPS can thus be induced by low external Mg2+ concentration and low pH, which mimic the situation encountered inside of RAW 264.7 cells (Gibbons et al., 2005). Groisman et al. (1997) have suggested that LPS may serve as a reservoir for divalent cations, which under limiting concentrations of Mg2+ and Ca2+ are mobilized by reducing the net negative charge of LPS. The hexaacylated E. coli lipid A as depicted in Fig. 4 has been shown to exert the highest endotoxic activity in vitro (Rietschel et al., 1994), and almost every structural modification leads to a drastic reduction in activity (Rietschel et al., 1994). Biophysical measurements have connected endotoxicity to a conical three-dimensional shape of the single molecule in aqueous systems which adopt inverted supramolecular structures in aqueous phases (Brandenburg et al., 1996; Seydel et al., 1999; Schromm et al., 2000; Gutsmann et al., 2007).

The core oligosaccharide Chemical analysis of LPS obtained from S. enterica revealed that the same subset of carbohydrates was present in similar amounts in different serovars, whereas other carbohydrates were present in variable amounts. This led to the assumption that Salmonella LPS possess a common structure, which carry the O-PS (Galanos et al., 1977). This LPS domain, which connects the lipid A domain with the O-PS, has been termed core region. In some bacteria, based on genetic and structural differences, the core-OS can be further subdivided into an inner and an outer core and typical examples are LPS from Enterobacteriaceae and Pseudomonadaceae. Characteristic components of the inner core-region are heptose (Hep) and Kdo (Fig. 5). In E. coli and Salmonella Hep mostly possesses the L-glycero-D-manno configuration (L,D-Hep; Fig. 2), but also D,D-Hep has been found in LPS of E. coli (Bradley et al., 1991) and other bacteria (Holst, 1999, 2002). The inner-core region is rich in negative charges which come from Kdo and

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also phosphate substituents which are often located on Hep residues. In some core-OS, e.g. from Klebsiella, phosphate substituents are absent and uronic acids are found instead (Holst, 1999). Distal sugars form the outer-core region which in these bacteria is composed of ubiquitous hexoses such as

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glucose (Glc), galactose (Gal) and glucosamine (GlcN). The chemical structures of core-OS from diverse bacteria have been summarized in recent reviews (Holst, 1999, 2002). For a review on the structural modifications observed in core-OS of pathogenic enterobacteria and their impact on outer-membrane stability, the interested reader is referred to a recent article by Frirdich and Whitfield (2005).

E. coli

Fig. 5. Chemical structures of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), L-glycero-D-manno-heptose (L,D-Hep), and Dglycero-D-manno-heptose (D,D-Hep).

In E. coli, chemical and serological data led Schmidt et al. (1969) to conclude that several different core types had to be distinguished due to structural differences in the outer core. On the contrary, the lipid A-proximal inner core was very similar or even identical in the different core types. Five core types are currently

Fig. 6. Core-oligosaccharide structures after alkaline degradation of E. coli LPS. Shown are the structures of all currently recognized E. coli core types. In LPS, the inner-core monophosphate on the first heptose is further derivatized with PEtN. Nonstoichiometric substitutions known to occur in the R2 core type are a third Kdo in a2-4-linkage and aGal in E. coli strain EH100 (Frirdich and Whitfield, 2005).

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distinguished in E. coli (R1 to R4, and K-12; Fig. 6) and the chemical structures including minor glycoforms could be identified apart from the main components of these LPS (Holst, 1999; Vinogradov et al., 1999; Mu¨llerLoennies et al., 2002, 2003b). Atypical core types have been suggested to occur, and it was reported that the elaboration of an atypical core is related to the adherent phenotype of enteropathogenic E. coli O119 (Bradley et al., 1991). Unfortunately, no structural data have been reported yet, apart from the presence of equal amounts of L,D- and D,D-Hep. The genes involved in the biosynthesis of the E. coli core-OS have been mostly identified and their functions have been assigned (Raetz and Whitfield, 2002; Frirdich et al., 2003; Kaniuk et al., 2004; Frirdich and Whitfield, 2005). The phosphorylation of the inner-core region of E. coli LPS is identical to the one found in S. enterica, and the kinases involved which generate the monophosphates have been identified (Raetz and Whitfield, 2002). Like in Salmonella the phosphates of the inner core may be further derivatized to PPEtN. E. coli R1 and R3 core types contain a structural variant in which the inner core is partially substituted at the side-chain heptose with GlcN in a1-7-linkage (Vinogradov et al., 1999; Mu¨llerLoennies et al., 2002). These two core types are also the most frequent core types found in clinical E. coli isolates (Appelmelk et al., 1994; Amor et al., 2000). Whereas this residue was previously assigned to be partially N-acetylated (Haishima et al., 1992), a later performed mass-spectrometric analysis on de-O-acylated LPS identified this residue as free GlcN (Mu¨ller-Loennies et al., 2002). The same was found for the E. coli J-5 Rc-mutant strain which is a derivative of E. coli O111:B4 (R3-core type) (Mu¨ller-Loennies et al., 1994). On the contrary, the GlcN attached to the second hexose of the outer core in the R3 core-OS is Nacetylated stoichiometrically. Concomitant with the GlcN modification of the inner core heptose the second heptose is devoid of phosphate. According to the current view of the E. coli R3 LPS biosynthetic pathway (Kaniuk et al., 2004), this phosphate must be removed by a phosphatase, since addition of the side-chain heptose depends on the presence of phosphate at this position, providing evidence for structural trimming during LPS biosynthesis. The gene (WabA) for the non-stoichiometric aGlcN-(1-7)-L-a-D-Hep substitution was identified on the large virulence plasmids of E. coli O157 and Shigella flexneri serotype 2a (Kaniuk et al., 2004). Surprisingly, overexpression of WabA resulted in the incorporation of GlcNAc rather than GlcN and this was confirmed in in vitro assays (Frirdich and Whitfield, 2005). Thus, it appears that an N-deacetylase generates GlcN after incorporation of GlcNAc. In LPS from Enterobacteriaceae, e.g. Yersinia, Salmonella, E. coli, Shigella, and Citrobacter, the tetrasaccharide D-Glc-(a1-3)-L,D-Hep-(a1-3)-L,D-

Hep-(a1-5)-Kdo represents a common partial structure (Holst, 1999). Further non-stoichiometrical structural modifications have been observed for the inner-core Kdo region of E. coli K-12 and R2 core types and also in S. enterica. In these, a third Kdo is present also in a-(2-4)-linkage. The third Kdo residue is presumed to be transferred by a dedicated Kdo transferase. Cloning and expression of the waaZ gene in the E. coli R1 background resulted in the generation of the expected Kdo trisaccharide (Frirdich et al., 2003). Surprisingly, concomitantly an incomplete outer-core OS was observed in these molecules. The structural characterization of E. coli K-12 LPS revealed that molecules containing three Kdo residues also had a truncated outer core and were stoichiometrically substituted with L-Rha in a-(1-5)-linkage at the middle Kdo (Mu¨ller-Loennies et al., 2003b). In these molecules the attachment site of the O-PS is lost. Therefore, there is evidence that the generation of certain inner-core structures influences the biosynthesis of the outer core and also the attachment of the O-PS. Upon deletion of the first two heptosyltransferases (WaaC and WaaF) in E. coli, an increased PEtN substitution of the terminal Kdo residue in position 7 was observed (Brabetz et al., 1997). It was later shown that this structural modification was due to an increased Ca2+ concentration used in the growth medium to suppress colonic acid production, and the corresponding enzyme could be identified (Kanipes et al., 2001). Thus, contrarily to modifications seen in lipid A molecules induced at suboptimal Mg2+ concentration, stress induced by high Ca2+ led to the introduction of a positively charged PEtN group into the inner core.

Salmonella enterica In S. enterica only a single core-OS structure was recognized in all LPS investigated (Fig. 7), until recently. Olsthoorn et al. (1998) identified in S. enterica sv. Arizonae a structural modification in the replacement of a terminal GlcNAc residue by a Glc, establishing a second outer core. The inner-core region in S. enterica is phosphorylated at the two inner heptoses in position 4. The phosphate at the first heptose may be further derivatized with ethanolamine phosphate (PEtN) to form ethanolamine diphosphate. Whereas this substitution has been known for a long time (Holst, 1999), only recently a PmrA-regulated gene was identified, which is involved in the transfer of PEtN to the first heptose in S. enterica sv Typhimurium (Tamayo et al., 2005).

Klebsiella As for S. enterica, only two core types have been described for K. pneumoniae (Fig. 8). The structural

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Fig. 7. Core-oligosaccharide structure after alkaline degradation of S. enterica LPS. Upon alkaline treatment phosphodiesters are cleaved off and diphosphodiesters are converted into mono-phosphates. Thus, depending on the presence of Ara4N, the 40 -position in the lipid A may lack phosphate. In LPS, PPEtN is present at the 4-position of the inner two L,D-Hep residues instead of monophosphates depicted here. A third Kdo in a2-4-linkage is present non-stoichiometrically.

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analysis of this LPS was hampered by the presence of a 4-substituted galacturonic acid in the core-OS. This led to the b-elimination of the outer-core sugars which were subsequently destroyed under the alkaline conditions used for the deacylation of LPS. Thus, only the inner core of K. pneumoniae R20 O1:K20 could be analyzed (Su¨sskind et al., 1995). It was found that in this LPS phosphate substitution of the core-OS was absent and that negative charges were only supplied by galacturonic acid residues. A subsequent analysis of this mutant revealed the substitution of the inner core by a novel heptoglycan (Su¨sskind et al., 1998). Vinogradov and coworkers realized that a deamination reaction could be employed to obtain intact fragments of the outer-core residues and the O-PS (Vinogradov and Perry, 2001; Vinogradov et al., 2001, 2002) which led to the discovery of a novel common structural element in Klebsiella strains (Fig. 9). This a-Kdo-(2-6)-a-GlcN disaccharide, containing a nonstoichiometric substitution of Kdo by L,D-Hep in a1-4-linkage, connects the O-PS to the core. Only from K. pneumoniae R20 LPS a heptoglycan was isolated which was later shown to be a minor component and is lacking in all other Klebsiella strains.

Fig. 8. Core-oligosaccharide structures of Klebsiella LPS (Su¨sskind et al., 1998; Vinogradov and Perry, 2001; Vinogradov et al., 2001, 2002; Frirdich et al., 2004). (A) Most common core oligosaccharide; (B) a second core type was described for K. pneumoniae 52145 sv O1:K2; (C) K. pneumoniae R20 O1:K20, this structure is a minor component in K. pneumoniae R20 and absent from all other Klebsiella strains investigated.

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Fig. 9. Common structural element identified in Klebsiella LPS. Chemical cleavage of the O-polysaccharide and core may be achieved by treatment with mild acid which cleaves the ketosidic linkage of Kdo or nitrous acid which leads to the deamination of GlcN and its release.

A second core type in K. pneumoniae 52145 sv O1:K2 has been described recently which differs by the replacement of the outer-core Kdo by a b-Glc-(1-6)a-Glc disaccharide connected to position 4 of the a-GlcN (Regue et al., 2005). Based on genetic evidence, the O-PS has been suggested to substitute the terminal b-Glc residue in this case.

The O-PS The O-PS in many cases is a heteropolymer made up from repeating units (Fig. 3) that are smaller oligosaccharides of 2 to 8 monomeric sugar residues (Jann and Jann, 1984; Knirel and Kochetkov, 1994; Jansson, 1999; Stenutz et al., 2006). In enterobacterial LPS, these polysaccharide chains may contain up to 50 repeating units. In other cases, the O-PS may also be a large homopolysaccharide, and a representative is the O:54 antigen of S. enterica sv. Borreze (Keenleyside et al., 1994). One of the biological functions attributed to the O-PS is the shielding of the bacteria against the serum attack probably by keeping dangerous molecules away from the essential and vulnerable membrane surface. The loss of the O-PS is accompanied by a significant decrease in virulence due to an increased susceptibility to complement-mediated killing. Temperature-dependent variations in the chemical structure of LPS have been observed for Yersinia enterocolitica, where the OPS expression is downregulated in vitro at a temperature of 37 1C. However, under in vivo conditions where the O-PS likely plays a protective role, the O-PS may be expressed normally (Skurnik and Bengoechea, 2003). Whereas in other bacteria the expression of an O-PS leads to enhanced resistance against the bactericidal activity of serum and CAMP, this does not seem to be the case for Y. enterocolitica (Bengoechea et al., 1998). Various monosaccharides are used to synthesize the O-PS, which are subject to further modification by, e.g., acetylation, methylation, phosphorylation, amidation, and substitution with amino acids (Knirel and Kochetkov, 1994). Therefore, bacterial cells produce a tremen-

dously large number of different chemical structures. Each O-PS contains specific chemical groups (Ofactors), which form epitopes recognized by specific antibodies and determine the serotype of a particular strain. Such antibodies have been successfully used for the development of serotyping schemes used in epidemiological studies. Therefore, the O-PS is synonymously called the O-antigen or O-specific chain. In order to escape preformed serum antibodies, an extremely large number of different O-PS structures is formed by different strains, often within a single species. Details on the biosynthesis of O-PS have been reviewed recently (Raetz and Whitfield, 2002). Whereas in most cases the enzymatic equipment for the biosynthesis of O-PS is encoded in the genome, a phenomenon called seroconversion results from phage-encoded structural modifications of the O-PS ((Raetz and Whitfield, 2002) and references therein). The genetic basis for the seroconversion by glucosylation of the O-PS among S. flexneri has been reviewed recently (Allison and Verma, 2000). West et al. (2005) were able to show that this phage-encoded glucosylation of the O-PS resulted in the expression of shorter O-PS, which was about half the length of nonglucosylated O-PS and enhanced the function of the type III secretion system. The latter is involved in bacterial invasion which is accompanied by a strong inflammatory response contributing to pathogenesis. Thus, by retaining the O-PS the bacteria are still protected; however, the modulation of the chemical structure shortening the O-PS increased the efficiency of invasion and infection.

E. coli In E. coli, at present more than 180 different O-PS are recognized (Stenutz et al., 2006), and an E. coli database (ECODAB) of these structures has been assembled (http://www.casper.organ.su.se/ECODAB/) which contains structures, NMR data and to some extent serological information (Stenutz et al., 2006). As an indication of molecular mimicry for immune evasion in E. coli, the O-PS O:86, O:90, O:127, and O:128 show structural similarities to mammalian blood-group determinants.

Salmonella enterica According to the current taxonomy the genus Salmonella is divided into two species, S. enterica and S. bongori, which are further subdivided into several subspecies (Brenner et al., 2000). In S. enterica, 46 O-serogroups are distinguished but serological differences as a result of structural modifications of the O-PS lead to approximately 2500 serotypes (Brenner et al., 2000). Immunodominant factors within the repeating

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Fig. 10. O-polysaccharide from four different S. enterica serovars. In these, on a common polysaccharide backbone the substitution with side branches involving dideoxy-hexoses determine the serotype.

units of the O-PS are frequently 3,6-dideoxy-hexoses, e.g. abequose, tyvelose and paratose (Fig. 10), which are connected to the main chain as side branches, and crystal structures of an antibody in complex with such an O-PS have been published (Cygler et al., 1991).

Klebsiella In the genus Klebsiella 11 O-PS structures are known; however, due to serological cross-reactions only nine serotypes are distinguished. Several of the O-PS are based on a galactan structure (D-galactans I and II), and genetic (Clarke and Whitfield, 1992) and chemical (Whitfield et al., 1991; Kol et al., 1992) evidences indicate that galactan I is linked directly to the core-OS, whereas galactan II is present at the distal end of some galactan I chains (Kol et al., 1991). A recent thorough reinvestigation of O-PS from K. pneumoniae revealed that in O-PS of serogroups O:4 and O:12, a b-Kdo residue is found at the non-reducing end of the molecule, whereas in O:5 a methyl group terminates the O-PS (Vinogradov et al., 2002) Both have been suggested to represent termination signals for O-PS biosynthesis. Similarly, a b-Kdo residue has been identified in Serratia marcescens O:19, supporting this notion and providing evidence that this structural modification may be widespread also among other bacteria (Vinogradov et al., 2003).

Neutralizing and cross-reactive endotoxin antibodies It has been known for a long time that after passive immunization LPS antisera are able to protect mice and

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rabbits against subsequent LPS challenge (Tate et al., 1966; Davis et al., 1969; Schiff et al., 1993). Unfortunately, the protection is highly specific for a given bacterial O-serotype. The vast number of different serotypes (see above) and the rapid onset of septic shock do not permit to serotype the sepsis-causing bacteria, and thus prevents the use of such antisera in the treatment of septic patients. Nevertheless, these studies have shown the beneficial effects of neutralizing antibodies. Antibodies directed against conserved epitopes in the core-OS or lipid A which are present in all or a large number of clinically relevant Gram-negative bacteria therefore appeared to be a promising alternative for LPS neutralization in an O-serotype-independent manner. The structural similarity of lipid A molecules of almost all Gram-negative bacteria (Za¨hringer et al., 1999) prompted studies to generate and investigate the cross-reactive and protective effects of anti-lipid A antibodies (Olsthoorn et al., 1998). These attempts failed because epitopes which are bound by these antibodies are not accessible in LPS of wild-type bacteria due to the masking by the core-OS and O-PS (Brade et al., 1997). We have shown that antibodies against lipid A can be generated and their epitopes are located exclusively in the lipid A backbone. Fatty acids are not part of the epitope but may have an influence on the binding (Brade et al., 1987; Kuhn et al., 1992, 1993; Kuhn, 1993). As a matter of fact, the presence of a methyl group (-CH3) in the 60 -position of the lipid A backbone was sufficient to abrogate binding to anti-lipid A antibodies (Brade et al., 1997). Therefore, such antibodies cannot be used for the neutralization of LPS which is released during infections with wild-type bacteria which contain mostly an S-type LPS. A number of unsuccessful clinical studies using anti-lipid A and anti-Re LPS antibodies have led to the questioning of the concept of antibodybased sepsis treatment in general (Cross, 1994). Nevertheless, the beneficial use of a polyclonal IgM preparation in septic patients (Rodriguez et al., 2005) may indicate that an antibody therapy based on crossreactive monoclonal antibodies (mAbs) may be successfully developed. As a prerequisite such antibodies must be able to bind S-type LPS and must possess protective properties. Similarities between lipid A of different pathogenic bacterial species generated the hope that it might be possible to identify a single antibody which neutralizes LPS from diverse pathogens such as E. coli, Neisseria, Klebsiella, Pseudomonas, and others. However, knowing that anti-lipid A antibodies do not react with LPS, this is likely impossible due to the structural differences in the remaining parts of the LPS of these species. However, a cross-reactive and neutralizing mAb against all serotypes of E. coli would cover a large percentage of clinically relevant bacteria.

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Generation of cross-reactive antibodies against E. coli LPS Using E. coli bacteria of all core types that lack the OPS but express a complete LPS core-OS as immunogen, antibodies could be induced in mice which bound to a large number (4100 isolates) of clinical E. coli and S. enterica isolates representing different serotypes. They also bound to the isolated LPS of these bacteria, and as shown in blotting studies, the binding was independent of the length and the presence of the O-PS (Di Padova et al., 1993). The characterization of the specificities of these antibodies revealed that they did not bind to bacteria which expressed highly truncated LPS molecules of the Rd and Re chemotype. None of these antibodies bound to Klebsiella or Pseudomonas. The mAb WN1 222-5 (IgG2ak) was selected for its apparent high affinity in binding assays and further investigated in detail for its binding characteristics and its neutralizing properties in models of endotoxic shock (Di Padova et al., 1993). It was shown that mAb WN1 222-5 was able to bind LPS even when complexed to high-density lipoprotein, intercalated into plasma membranes or when coated onto erythrocytes (Di Padova et al., 1994). Also living E. coli O18:K1 cells were bound in an ELISA, and this binding was further enhanced upon prior antibiotic treatment. However, in flow cytometry, WN1 222-5 failed to opsonize the latter strain whereas E. coli O111:B4 was efficiently recognized. Thus, it appears that some viable bacteria are able to escape WN1 222-5 binding, at least under certain conditions. MAb WN1 222-5 was able to neutralize the endotoxic activity of LPS in vitro and, more importantly, also in in-vivo models of septic shock (Di Padova et al., 1994). The protective effect was dose dependent and correlated well with TNFa levels in the blood of treated animals.

Identification of the WN1 222-5 epitope in E. coli and S. enterica LPS Since mAb WN1 222-5 was able to offer protection in models of septic shock against bacteria containing different LPS, we initiated studies to characterize the structural determinants that are required for binding to be able to explain the observed cross-reactivity. The identification of the antigenic determinants that harbor the epitope and the factors that influence binding would furthermore offer the possibility to identify a potential candidate structure for the development of a conjugated vaccine that may be applicable to human vaccination. Clearly, the immunization scheme which was used for the generation of the mAb WN1 222-5 in mice is not applicable to humans.

As described above, individual bacterial cells produce a variety of LPS molecules differing in the degree of polymerization of the O-chain, acylation, phosphorylation, and sugar decoration, as well as in the chemical structures of the core-OS. So far it has been impossible to separate individual molecular species from each other due to their amphiphilic properties. In order to understand the cross-reactive properties and to define the epitope specificity in detail, we have therefore developed a methodology for the isolation of oligosaccharides from individual molecular species of LPS after deacylation by alkaline treatment (Mu¨ller-Loennies et al., 1994, 1999). The applied chemical degradation procedures left the carbohydrate chains intact, and the subsequent purification by HPLC allowed the preparation of highly purified oligosaccharides from E. coli and S. enterica rough mutants (Olsthoorn et al., 1998; Vinogradov et al., 1999; Mu¨ller-Loennies et al., 2002, 2003b). The purified oligosaccharides were amenable to a detailed structural analysis by modern analytical techniques such as NMR spectroscopy and electrospray ionisationFourier transform-mass spectrometry (ESI-FT-MS) and allowed their investigation in antibody binding assays using surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC) (Mu¨llerLoennies et al., 2003a). These well-characterized highly homogeneous oligosaccharides were tested in inhibition ELISAs and were conjugated to proteins for the investigation of their reactivity in ELISAs (Mu¨llerLoennies et al., 2003a). In a first experiment, we tested the reactivity of mAb WN1 222-5 in an ELISA with immobilized acylated isolated Ra LPS, representing core types of R1 to R4 of E. coli and R1 of S. enterica. As expected, mAb WN1 222-5 bound to all these LPS. After chemical deacylation of LPS and conjugation of the resulting oligosaccharide mixtures to bovine serum albumin, we could show that fatty acids were not required for binding. From previous ELISA studies it was known that the Rc mutant strain J-5 of E. coli contained the smallest LPS bound by mAb WN1 222-5 (Di Padova et al., 1993). This strain is unable to produce Gal (Elbein and Heath, 1965) leading to a mixture of truncated LPS molecules in the outer membrane (Mu¨ller-Loennies et al., 1994, 1999). To obtain further insight into the epitope specificity of mAb WN1 222-5, we separated the individual molecules and elucidated their structures (Fig. 11; Mu¨ller-Loennies et al., 1999). Five naturally occurring oligosaccharides were isolated, the structures of which are depicted in Fig. 6: one nonasaccharide containing three phosphate residues (nonasaccharide P3), one octasaccharide which lacked the side-chain Kdo which was otherwise identical to nonasaccharide P3, one octasaccharide containing four phosphates but lacked the side-chain GlcN (octasaccharide P4), and two heptasaccharides which did not contain the

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Fig. 11. Chemical structures of E. coli J-5 core oligosaccharides. *Octasaccharide 1 P3 has been obtained by a chemical deamination reaction from nonasaccharide P3 and is not produced by E. coli J-5.

GlcN-(1-7)-Hep side chain of the inner core and differed in their phosphorylation (heptasaccharide P3 and heptasaccharide P4). In addition, we have prepared and isolated one oligosaccharide by chemical means (deamination reaction), which was a derivative of nonasaccharide P3 and which did not contain the sidechain GlcN residue (Fig. 11, octasaccharide P3; Mu¨llerLoennies et al., 2003a). After conjugation, WN1 222-5 showed strongest binding to octasaccharide P4 and no binding to hepta- and nonasaccharide P3 in ELISA. An intermediate affinity was observed for heptasaccharide P4 and octasaccharide P3 (Mu¨ller-Loennies et al., 2003a). This result indicated that major contributions to the binding energy for WN1 222-5 originated from the side-chain heptose and the phosphate in position 4 of the second, lipid A-distal heptose. LPS molecules in which the side-chain heptose is absent or not accessible for antibody binding due to the presence of an additional sugar (e.g. a GlcN linked to position 7 of the side-chain heptose) are not recognized by mAb WN1 222-5. In order to investigate the influence of the outer coreOS on the binding of mAb WN1 222-5 by ELISA, SPR, and ITC (Mu¨ller-Loennies et al., 2003a), we have isolated and structurally characterized phosphorylated core-OS from all E. coli core-types (Fig. 6). These OS were conjugated to bovine serum albumin, immobilized

and used in ELISAs. In these assays, a tight binding of mAb WN1 222-5 to all antigens was observed. SPR experiments revealed that the affinities towards oligosaccharides with an outer core (i.e. core oligosaccharides from Ra mutants and wild-type LPS) are generally higher than those towards oligosaccharides in which the outer core sugars are absent, such as in the rough mutant E. coli J-5. The oligosaccharide obtained from E. coli F576 (R2 core type) displayed the highest affinity in SPR with a dissociation constant (KD) of 32 nM for the monovalent binding. When determined by microcalorimetry the KD was as low as 6 nM. The affinity towards the other E. coli core types was lower by a factor of 5–10 and to the best binding oligosaccharide from E. coli J-5 (octasaccharide P4) lower by a factor of 29. In comparison with other carbohydrate–protein interactions which usually occur with KD values in the micromolar range, these values proved an extraordinary high affinity. For the antibody with two combining sites, the avidity effect would lead to an apparent affinity even up to two orders of magnitudes higher, thus resembling protein–protein interactions. Finally, we wanted to know whether the presence of the carbohydrate backbone of the lipid A component influenced mAb WN1 222-5 binding. We treated isolated LPS with acetic acid which cleaved the acidlabile ketosidic linkages of Kdo but left all other

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glycosidic linkages and the phosphorylation sites intact. After this treatment, the released free lipid A was removed by precipitation. The chemical structures of the resulting oligosaccharides in the supernatant differed from the ones obtained by alkaline deacylation by the absence of the carbohydrate backbone of lipid A and also the side-chain Kdo. These isolated oligosaccharides were tested for their ability to inhibit the binding of mAb WN1 222-5 to LPS in ELISA. Even at high concentrations these oligosaccharides were unable to inhibit the binding of mAb WN1 222-5. Therefore, and despite the fact that the bound epitope apparently resides in sugars distant from the lipid A, an intact Kdolipid A region is required for binding. This may be explained either by a direct structural contribution of this region to the binding or as a result of conformational changes of the inner-core region.

Conclusions and perspectives The mAb WN1 222-5 was obtained after immunization of mice with bacteria which express an LPS containing a complete outer core. This mAb showed neutralizing properties against LPS and bacteria in various endotoxin models in vitro and in vivo, and was broadly cross-reactive with bacteria representing different O-serotypes and all core types which were isolated

from patients. Binding assays using different E. coli LPS and partial structures thereof allowed us to identify an epitope in enterobacterial LPS which is accessible to high-affinity antibody binding on viable wild-type bacteria despite the presence of an O-PS. It was shown previously (Di Padova et al., 1994) that this epitope is also recognized in isolated LPS, LPS-high-density lipoprotein complexes, and in LPS intercalated into membranes. The most important structural determinants of this epitope are the phosphorylation in position 4 of the lipid A-distal catenal heptose and a nonsubstituted side-chain heptose (Fig. 12). Such a carbohydrate structure is present in all E. coli and S. enterica LPS core-OS (Figs. 6 and 7) and explains the observed cross-reactivity of mAb WN1 222-5. In general, the presence of outer-core sugars, independently of the core type, increases the affinity whereas sugars of the Kdolipid A region are necessary for mAb WN1 222-5 recognition. These contributions are currently not understood at the molecular level. Since it is the lipid A component which is responsible for the endotoxic effects, the LPS-neutralizing properties of mAb WN1 222-5 and also mAbs against the O-PS are not understood, since the epitopes are located in domains distal from lipid A, the endotoxic center of LPS. Thus, it seems unlikely that mAb WN1 222-5 exerts its protective effects by interfering with the binding to receptor molecules such as CD14. However,

Fig. 12. Chemical structure of the E. coli F653 (R3 core type) core oligosaccharide. Important contributions to the binding of mAb WN1 222-5 come from the side chain heptose and the phosphate at the middle heptose, depicted in red.

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it was shown that mAb WN1 222-5 is indeed able to inhibit the recognition and uptake of LPS by target cells expressing membrane-associated CD14 (Pollack et al., 1997). The inhibition is seen in the presence and the absence LBP. One possible explanation might be the interference with a correct assembly of the signaltransducing receptor complex within the membrane. Therefore, it appears that LPS bound to mAb WN1 2225 is taken up by macrophages via Fc receptors or after opsonization by complement receptors and, once internalized, is unable to activate cellular responses which lead to the production and secretion of proinflammatory cytokines. It is not yet clear whether LPS internalization is necessary to enable signaling. However, there is evidence that blocking internalization and endosome fusion does prevent LPS-induced signaling (Van Amersfoort et al., 2003). It has been suggested that the intracellular trafficking of LPS to the Golgi leads to activation whereas transport to lysosomes inhibits activation. This assumption warrants further investigations to clarify whether LPS complexed to mAb WN1 222-5 takes a different intracellular route than free LPS. An important factor for the neutralizing activities of mAb WN1 222-5 is certainly its high affinity with a KD in the low nanomolar range for free LPS. When several LPS molecules create a multivalent surface, as it is the case on the surface of bacteria or in vesicles, an affinity in the picomolar range can be expected due to avidity effects. Thus, the affinity of mAb WN1 222-5 exceeds that determined for the binding of LPS to CD14 (27–48 nM; Kirkland et al., 1993) and to LBP (1–58 nM; Tobias et al., 1989; Gazzano-Santoro et al., 1994). Although we have not determined values in the presence of serum, it is likely that most LPS molecules are seen by CD14 complexed to mAb WN1 222-5. The protective effects against experimentally induced bacteremia and infection may be explained by an accelerated clearance of bacteria due to a rapid opsonization and destruction by, e.g., complement activation. A humanized version of the mouse mAb WN1 222-5, termed SDZ 219-800, which was created for a potential therapeutic application in man, showed the same specificity in binding assays and the same protective effects (Di Padova et al., 1994; Bailat et al., 1997). Although an artificial experimental setup was used to analyze the protective effects of mAb WN1 222-5, where the antibody was given prior to endotoxin and bacterial challenge, the protective effect could clearly be shown. In a clinical scenario, however, it may be difficult to decide which group of patients would benefit from antibody treatment. The identification of the E. coli R2 core-OS, which was bound by mAb WN1 222-5 with highest affinity and the identification and description of the epitope, now opens the possibility to develop a conjugated vaccine aiming at the induction of WN1

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222-5-type antibodies which bind and neutralize all E. coli and S. enterica LPS. Our results indicate that antibodies of even broader cross-reactivity against LPS from diverse genera such as Klebsiella and Pseudomonas will be difficult, if not impossible, to obtain. This is concluded from the elucidation of Klebsiella and Pseudomonas LPS core structures (see above and Pier, 2007) that differ in important aspects from E. coli and S. enterica LPS (Vinogradov and Perry, 2001; Vinogradov et al., 2002). However, the identification of a different structurally highly conserved element in the outer-core region of LPS from all Klebsiella strains (Vinogradov et al., 2002) and P. aeruginosa (Beckmann et al., 1995; Reiter et al., 2001) may allow the induction and isolation of antibodies with neutralizing properties against these pathogens and possibly the development of carbohydrate-based vaccines. The combination of such antibodies may then contribute to a lowered death rate of patients at risk of developing septic shock. Due to the complexity of the disease, it seems unlikely that a monotherapeutic strategy alone will be able to overcome all the difficulties clinicians are currently facing in the battle against this serious condition. However, the development of an array of weapons to be used in a combination therapy, which might include anti-endotoxin antibodies, will hopefully lead to a significant reduction of the mortality in the future. It will be important to determine the natural variability of LPS structures and the importance of individual structural modifications for the biology of the bacteria, including their pathogenic potential. Naturally occurring modifications of LPS structures may enable pathogenic bacteria to escape recognition and modify host cell responses that could promote their survival. Such studies will contribute to a general understanding of the biology of Gram-negative bacteria and may allow treatment of infections with novel antibacterial drugs in the future. Identification of natural heterogeneity with respect to LPS structure and their biosynthetic pathways is thus of considerable biomedical and pharmaceutical interest due to the fact that LPS is essential for most Gram-negative bacteria and is responsible for the toxic effects.

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