Antigen binding by conformational selection in near-germline antibodies.

Conformational flexibility in antibody-combining sites has been hypothesized to facilitate polyspecificity toward multiple unique epitopes and enable the limited germline repertoire to match an overwhelming diversity of potential antigens; however, elucidating the mechanisms of antigen recognition by flexible antibodies has been understandably challenging. Here, multiple liganded and unliganded crystal structures of the near-germline anticarbohydrate antibodies S25-2 and S25-39 are reported, which reveal an unprecedented diversity of complementarity-determining region H3 conformations in apparent equilibrium. These structures demonstrate that at least some germline or near-germline antibodies are flexible entities sensitive to their chemical environments, with conformational selection available as an evolved mechanism that preserves the inherited ability to recognize common pathogens while remaining adaptable to new threats.

Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody-Carbohydrate Binding Affinity by an Order of Magnitude.

Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26.

The Combining Sites of Anti-lipid A Antibodies Reveal a Widely Utilized Motif Specific for Negatively Charged Groups.

Lipopolysaccharide dispersed in the blood by Gram-negative bacteria can be a potent inducer of septic shock. One research focus has been based on antibody sequestration of lipid A (the endotoxic principle of LPS); however, none have been successfully developed into a clinical treatment. Comparison of a panel of anti-lipid A antibodies reveals highly specific antibodies produced through distinct germ line precursors.

Structural Basis for Antibody Recognition of Lipid A: INSIGHTS TO POLYSPECIFICITY TOWARD SINGLE-STRANDED DNA.

Septic shock is a leading cause of death, and it results from an inflammatory cascade triggered by the presence of microbial products in the blood. Certain LPS from Gram-negative bacteria are very potent inducers and are responsible for a high percentage of septic shock cases. Despite decades of research, mAbs specific for lipid A (the endotoxic principle of LPS) have not been successfully developed into a clinical treatment for sepsis.

Groove-type recognition of chlamydiaceae-specific lipopolysaccharide antigen by a family of antibodies possessing an unusual variable heavy chain N-linked glycan.

The structure of the antigen binding fragment of mAb S25-26, determined to 1.95 Å resolution in complex with the Chlamydiaceae family-specific trisaccharide antigen Kdo(2→8)Kdo(2→4)Kdo (Kdo = 3-deoxy-α-d-manno-oct-2-ulopyranosonic acid), displays a germ-line-coded paratope that differs significantly from previously characterized Chlamydiaceae-specific mAbs despite being raised against the identical immunogen. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the entire trisaccharide antigen and so confer strict specificity.

Cellular distribution of lipid A and LPS R595 after in vitro application to isolated human monocytes by freeze-fracture replica immunogold-labelling.

We have performed freeze-fracture replica immunogold labelling of endotoxin preparations (lipid A and deep rough mutant LPS Re from Salmonella enterica sv. Minnesota), i.e.

Exploring the cross-reactivity of S25-2: complex with a 5,6-dehydro-Kdo disaccharide.

The near-germline antibody S25-2 exhibits a remarkable cross-reactivity for oligosaccharides containing the bacterial lipopolysaccharide carbohydrate 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The recent synthesis of a variety of Kdo analogues permits a detailed structural analysis of the importance of specific interactions in antigen recognition by S25-2. The Kdo disaccharide analogue Kdo-(2→4)-5,6-dehydro-Kdo lacks a 5-OH group on the second Kdo residue and has been cocrystallized with S25-2.

Antibody WN1 222-5 mimics Toll-like receptor 4 binding in the recognition of LPS.

Escherichia coli infections, a leading cause of septic shock, remain a major threat to human health because of the fatal action to endotoxin (LPS). Therapeutic attempts to neutralize endotoxin currently focus on inhibiting the interaction of the toxic component lipid A with myeloid differentiating factor 2, which forms a trimeric complex together with Toll-like receptor 4 to induce immune cell activation. The 1.

Evaluation of a LPS-based glycoconjugate vaccine against bovine Escherichia coli mastitis: Formation of LPS Abs in cows after immunization with E. coli core oligosaccharides conjugated to hemocyanine.

The immune response of cows against the core oligosaccharide of Escherichia coli rough mutants (core types R1-R4, K-12 and J-5) was investigated after immunization with a synthetic glycoconjugate composed of deacylated LPS conjugated to hemocyanine (22 animals). Ab formation was measured by ELISA using LPS or deacylated LPS conjugated to BSA as an Ag. The glycoconjugate immunogens were used to vaccinate cows (36 animals), which were then challenged intramammarily with E.

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits.

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS.

Structural insights into parallel strategies for germline antibody recognition of lipopolysaccharide from Chlamydia.

The structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide has been determined by x-ray diffraction to 2.6 Å resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment.

A common NH53K mutation in the combining site of antibodies raised against chlamydial LPS glycoconjugates significantly increases avidity.

The crystal structures of the antigen-binding fragment of the murine monoclonal antibody (mAb) S25-39 in the presence of several antigens representing chlamydial lipopolysaccharide (LPS) epitopes based on the bacterial sugar 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) have been determined at resolutions from 2.4 to 1.8 Å.

Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci.

Chlamydiae contain a rough-type lipopolysaccharide (LPS) of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid residues (Kdo). Two Kdo trisaccharides, 2.8/2.

Monoclonal antibody S60-4-14 reveals diagnostic potential in the identification of Pseudomonas aeruginosa in lung tissues of cystic fibrosis patients.

The lipopolysaccharide (LPS) of Pseudomonas aeruginosa has been identified to contain an inner-core structure expressing a Pseudomonas-specific epitope. This target structure is characterized by a highly phosphorylated and 7-O-carbamoyl-l-glycero-alpha-d-manno-heptopyranose (CmHep) and was found to be present in all human-pathogenic Pseudomonas species of the Palleroni (RNA)-classification I scheme. We raised and selected the monoclonal antibody S60-4-14 (mAb S60-4-14, subtype IgG1) from mice immunized with heat-killed Pseudomonas bacteria.

Antibodies raised against chlamydial lipopolysaccharide antigens reveal convergence in germline gene usage and differential epitope recognition.

The structures of antigen-binding fragments from two related monoclonal antibodies have been determined to high resolution in the presence of several carbohydrate antigens raised against chlamydial lipopolysaccharide. With the exception of CDR H3, antibodies S54-10 and S73-2 are both derived from the same set of germline gene segments as the previously reported structures S25-2 and S45-18. Despite this similarity, the antibodies differ in specificity and the mechanism by which they recognize their cognate antigen.

The role of CDR H3 in antibody recognition of a synthetic analog of a lipopolysaccharide antigen.

In order to explore the structural basis for adaptability in near germline monoclonal antibodies (mAb), we have examined the specificity of the promiscuous mAb S67-27 to both naturally derived carbohydrate antigens and a variety of synthetic nonnatural antigens based on the bacterial lipopolysaccharide component 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (Kdo). One such analog, a 7-O-methyl (7-O-Me) Kdo disaccharide, was found to bind to the antibody with at least 30-fold higher affinity than any other antigen tested. The structure of S67-27 in complex with this analog and three other naturally occurring Kdo antigens revealed that the enhanced affinity of the mAb for the synthetic analog was accomplished by the strategic positioning of CDR H3 away from a conserved Kdo binding pocket that allowed the formation of new antibody-antigen contacts.

Structural and immunochemical analysis of the lipopolysaccharide from Acinetobacter lwoffii F78 located outside Chlamydiaceae with a Chlamydia-specific lipopolysaccharide epitope.

Chemical analyses, NMR spectroscopy, and mass spectrometry were used to elucidate the structure of the rough lipopolysaccharide (LPS) isolated from Acinetobacter lwoffii F78. As a prominent feature, the core region of this LPS contained the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo=3-deoxy-d-D-manno-oct-2-ulopyranosonic acid), which so far has been identified only in chlamydial LPS. In serological investigations, the anti-chlamydial LPS monoclonal antibody S25-2, which is specific for the epitope alpha-Kdo-(2-->8)-alpha-Kdo, reacted with A.

Exploration of specificity in germline monoclonal antibody recognition of a range of natural and synthetic epitopes.

To explore the molecular basis of antigen recognition by germline antibodies, we have determined to high resolution the structures of the near-germline monoclonal antibody S25-2 in complex with seven distinct carbohydrate antigens based on the bacterial sugar 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (Kdo). In contrast to previous findings, the inherited germline Kdo monosaccharide binding site is not restricted to this bacterial sugar but is able to accommodate an array of substitutions and chemical modifications of Kdo, including naturally occurring antigens containing the related monosaccharide d-glycero-alpha-d-talo-oct-2-ulosonic acid as well as nonterminal Kdo residues. However, we show by surface plasmon resonance and ELISA how antibody S25-2 specificity is so dependent on the context in which the antigen is presented that a free disaccharide displays strong binding while the same lipid-A-bound disaccharide does not bind.

Neutralizing and cross-reactive antibodies against enterobacterial lipopolysaccharide.

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.

A novel 40-kDa protein containing six repeats of an epidermal growth factor-like domain functions as a pattern recognition protein for lipopolysaccharide.

Determination of structures and functions of pattern recognition proteins are important for understanding pathogen recognition mechanisms in host defense and for elucidating the activation mechanism of innate immune reactions. In this study, a novel 40-kDa protein, named LPS recognition protein (LRP), was purified to homogeneity from the cell-free plasma of larvae of the large beetle, Holotrichia diomphalia. LRP exhibited agglutinating activities on Escherichia coli, but not on Staphylococcus aureus and Candida albicans.

Diuretic-induced hypokalaemia in patients with hypertension.

We describe a 55 year-old woman with longstanding hypertension who developed hypokalaemia following diuretic treatment. Investigations revealed primary hyperaldosteronism due to an adrenal adenoma, and normal blood pressure was restored after surgical removal of the tumour. Primary hyperaldosteronism is a potentially curable cause of hypertension and should be considered in hypertensive patients who present with diuretic - induced hypokalaemia.

A monoclonal antibody against a carbohydrate epitope in lipopolysaccharide differentiates Chlamydophila psittaci from Chlamydophila pecorum, Chlamydophila pneumoniae, and Chlamydia trachomatis.

Lipopolysaccharide (LPS) of Chlamydophila psittaci but not of Chlamydophila pneumoniae or Chlamydia trachomatis contains a tetrasaccharide of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid (Kdo) of the sequence Kdo(2-->8)[Kdo(2-->4)] Kdo(2-->4)Kdo. After immunization with the synthetic neoglycoconjugate antigen Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4) Kdo-BSA, we obtained the mouse monoclonal antibody (mAb) S69-4 which was able to differentiate C. psittaci from Chlamydophila pecorum, C.

Cell membrane array fabrication and assay technology.

Background: Microarray technology has been used extensively over the past 10 years for assessing gene expression, and has facilitated precise genetic profiling of everything from tumors to small molecule drugs. By contrast, arraying cell membranes in a manner which preserves their ability to mediate biochemical processes has been considerably more difficult.

Results: In this article, we describe a novel technology for generating cell membrane microarrays for performing high throughput biology.

Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity.

Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the 'endotoxic principle' of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition.

PAF-mediated pulmonary edema: a new role for acid sphingomyelinase and ceramide.

Platelet-activating factor (PAF) induces pulmonary edema and has a key role in acute lung injury (ALI). Here we show that PAF induces pulmonary edema through two mechanisms: acid sphingomyelinase (ASM)-dependent production of ceramide, and activation of the cyclooxygenase pathway. Agents that interfere with PAF-induced ceramide synthesis, such as steroids or the xanthogenate D609, attenuate pulmonary edema formation induced by PAF, endotoxin or acid instillation.