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synthesis, antigen, core, Pseudomonas, Acinetobacter, antigens, Chlamydia, inner core

Journal NLM ID: 9433350
Publisher: Maney Publishing
Institutions: Institute of Chemistry, University of Agricultural Sciences, Vienna, Wien, Austria

High-resolution structures reveal how a germline antibody can recognize a range of clinically relevant carbohydrate epitopes. The germline response to a carbohydrate immunogen can be critical to survivability, with selection for antibody gene segments that both confer protection against common pathogens and retain the flexibility to adapt to new disease organisms. We show here that antibody S25-2 binds several distinct inner-core epitopes of bacterial lipopolysaccharides (LPSs) by linking an inherited monosaccharide residue binding site with a subset of complementarity-determining regions (CDRs) of limited flexibility positioned to recognize the remainder of an array of different epitopes. This strategy allows germline antibodies to adapt to different epitopes while minimizing entropic penalties associated with the immobilization of labile CDRs upon binding of antigen, and provides insight into the link between the genetic origin of individual CDRs and their respective roles in antigen recognition

Lipopolysaccharide, genetic, antigen, lipopolysaccharides, structure, common, disease, gene, Bacterial, role, carbohydrate, antibodies, antibody, epitope, recognition, response, epitopes, region, inner core, pathogen, pathogens, binding, binding site, site, monosaccharide, protection, high-resolution, flexibility, origin, high resolution, immobilization, immunogen, labile, selection

NCBI PubMed ID: 14625588
Journal NLM ID: 9421566
Publisher: Nature Publishing Co.
Correspondence: svevans@uvic.ca
Institutions: Institute of Chemistry, University of Agriculture, A-1190 Vienna, Austria, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada, National Research Council of Canada, Institute for Biological Sciences, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada, Division of Medical and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, Parkallee 1-40, D-23845 Borstel, Germany, Department of Biochemistry and Microbiology, University of Victoria, PO Box 3800, STN CSC, Victoria, British Columbia V8W 3P6, Canada.

Identification of protective epitopes is one of the first steps in the development of a subunit vaccine. One approach to accomplishing this is to identify structures or epitopes by using monoclonal antibodies (MAb) that can attenuate infectivity in vitro and in vivo. To date attempts to use this approach with Chlamydia pneumoniae have failed. This report is the first description of a MAb directed to the lipopolysaccharide (LPS) of Chlamydia that neutralizes both in vitro and in vivo the infectivity of C. pneumoniae. MAb CP-33, an immunoglobulin G2b (IgG2b), was identified from a fusion using splenocytes from mice immunized with C. pneumoniae TW-183. By Western blot analysis, MAb CP-33 exhibited genus-specific reactivity in that it recognized the LPSs of C. pneumoniae, Chlamydia trachomatis, and Chlamydia psittaci. MAb CP-33 did not react with 15 genera of gram-negative and gram-positive bacteria and Candida albicans. By using isolated LPS of Re mutants of Escherichia coli, Salmonella enterica serovar Minnesota, and recombinants expressing the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase gene kdtA of C. trachomatis, MAb CP-33 was shown to require for binding the presence of the genus-specific trisaccharide epitope αKdo(2→8)αKdo(2→4)αKdo. By employing synthetic oligosaccharides and neoglycoconjugates in an enzyme immunoassay (EIA) and EIA inhibition, it was further shown that MAb CP-33 differed from the extensively investigated prototype chlamydial LPS MAb S25-23. Most likely, MAb CP-33 recognizes a conformational epitope in which the αKdo(2→8)αKdo(2→4)αKdo trisaccharide is an essential structural component. When tested in an in vitro neutralization assay, MAb CP-33 gave a 50% neutralization titer of 8 ng/ml against C. pneumoniae TW-183. However, this MAb did not neutralize other C. pneumoniae strains, C. trachomatis, or C. psittaci. C. pneumoniae TW-183 was treated with either MAb CP-33 or a control IgG and then used to inoculate mice by the respiratory route. Five days after inoculation, there was a difference between the mice inoculated with the control IgG-treated inoculum and those inoculated with the MAb CP-33-treated organisms as to the number of mice infected as well as the number of inclusion-forming units recovered from lung cultures (P < 0.05). In summary, a Chlamydia-specific LPS MAb was able to neutralize in vitro the infectivity of C. pneumoniae TW-183.

Lipopolysaccharide, core, characterization, antibodies, antibody, epitope, monoclonal, monoclonal antibodies, monoclonal antibody, Chlamydia, protective, neutralizing

NCBI PubMed ID: 9673271
Journal NLM ID: 0246127
Publisher: American Society for Microbiology
Correspondence: epeterso@uci.edu
Institutions: Department of Pathology, University of California, Irvine , Ervine, California 92697-4800, Forschungsinstitut Borstel, Zentrum fur Medizin und Biowiddenschaften, Medizinische und Biochemische Mikrobiologie, 23845 Borstel, Germany
Methods: serological methods

The 1H- and 13C-NMR parameters, chemical shifts and coupling constants, for the pentasaccharide of the genus-specific epitope of Chlamydia lipopolysaccharide and related di-, tri-, and tetra-saccharides have been measured and assigned completely using 1D and 2D techniques, and their structures have been confirmed. NOE experiments indicated the preferred conformation of the pentasaccharide and the component oligosaccharides. The 3JH,H demonstrate a change in conformation by rotation of the C-6-C-7 bond of the side chain of the (2→8)-linked Kdo (unit b) in α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo-(2→6)-β-GlcN-(1→6)-GlcNol, α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo-(2→6)-β-GlcNAc-(1→O)-allyl, and α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo-(2→O)-allyl relative to that preferred in α-Kdo-(2→4)-α-Kdo-(2→6)-β-GlcNAc-(1→O)-allyl, α-Kdo-(2→8)-α-Kdo-(2→O)-allyl, α-Kdo-(2→4)-α-Kdo-(2→O)-allyl, and α-Kdo-(2→6)-β-GlcNAc-(1→O)-allyl, irrespective of the size of the aglycon, e.g., allyl or β-D-GlcN residues. The conformational results have been substantiated by computer calculations using the HSEA approach.

NCBI PubMed ID: 1382853
Publication DOI: 10.1016/s0008-6215(00)90571-8
Journal NLM ID: 0043535
Publisher: Elsevier
Institutions: Department of Chemistry, Carlsberg Laboratory, Valby, Denmark
Methods: 13C NMR, 1H NMR, NMR-2D

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 α-3-deoxy-D-manno-2-octulosonic acid (KDO)-(2-8)-α-Kdo-(2-4)-α-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.

NCBI PubMed ID: 2294050
Journal NLM ID: 0246127
Publisher: American Society for Microbiology
Institutions: Division of Biochemical Microbiology, Forschungsinstitut Borstel, Federal Republic of Germany

The structure of the antigen-binding fragment of mAb S25-26 determined to 1.95 A 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 germline-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 germline residues to recognize the entire trisaccharide antigen and so confer strict specificity. Interest in S25-23 was sparked by its rare high muM affinity and strict specificity for the family-specific trisaccharide antigen; however, only the related antibody S25-26 proved amenable to crystallization. The structures of three unliganded forms of S25-26 have a labile complementary determining region H3 adjacent to significant glycosylation of the variable heavy chain on asparagine 85 in Framework Region 3. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the few reported structures of glycosylated mAbs containing these epitopes is the therapeutic antibody Cetuximab; however, unlike Cetuximab, one of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three dimensional structure of an αGal containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as they have been implicated in allergic response in patients receiving therapeutic antibodies.

antibodies, epitopes, MAb, Chlamydiaceae, N-linked glycan, lipopolysaccharide antigen

NCBI PubMed ID: 24682362
Publication DOI: 10.1074/jbc.M113.528224
Journal NLM ID: 2985121R
Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: sml@fz-borstel.de; svevans@uvic.ca
Institutions: From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 3P6, Canada, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 22, Borstel D-23845, Germany
Methods: X-ray, ELISA, ESI-MS, genetic methods, HPLC, crystallization, ITC