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

Abstract

Chlamydiae contain a rough-type lipopolysaccharide (LPS) of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid residues (Kdo). Two Kdo trisaccharides, 2.8/2.4- and 2.4/2.4-linked, and a branched 2.4[2.8]2.4-linked Kdo tetrasaccharide occur in Chlamydiaceae. While the 2.8/2.4-linked trisaccharide contains a family-specific epitope, the branched Kdo oligosaccharide occurs only in Chlamydophila psittaci and antibodies against it will be useful in human and veterinarian diagnostics. To overcome the generation of cross-reactive antibodies that bind with high affinity to a dominant epitope formed by 2.4/2.4-linked Kdo, we immunized mice with a synthetic 2.4[2.8]-linked branched Kdo trisaccharide and used phage display of scFv to isolate recombinant antibody fragments (NH2240-31 and SAG506-01) that recognize the branched Kdo oligosaccharide with a K(D) of less than 10 nM. Importantly, although these antibodies used germline genes coding for an inherited Kdo recognition site, they were able clearly to distinguish between 2.4[2.8]2.4- and 2.4/2.4-linked Kdo. Sequence determination, binding data, and X-ray structural analysis revealed the basis for the improved discrimination between similar Kdo ligands and indicated that the alteration of a stacking interaction from a phenylalanine residue in the center of the combining site to a tyrosine residue facing away from the center favors recognition of branched 2.4[2.8]2.4-linked Kdo residues. Immunofluorescence tests of infected cell monolayers using this antibody show specific staining of C. psittaci elementary bodies that allow it to be distinguished from other pathogenic chlamydiae.

Bactericidal Monoclonal Antibodies Specific to the Lipopolysaccharide O Antigen from Multidrug-Resistant Escherichia coli Clone ST131-O25b:H4 Elicit Protection in Mice

“…The affinity of all of the MAbs was in the range of 10 Ϫ7 to 10 Ϫ8 M (Table 1), which is in good agreement with values published for other anti-carbohydrate MAbs (31,32). Importantly, no polyreactive characteristic for any of the MAbs was detected based on lack of binding to unrelated antigens, such as DNA, gelatin, fetuin, and dextran (Table 1).…”

Immunoglobulin gene repertoire in ocular adnexal lymphomas: hints on the nature of the antigenic stimulation

“…This case had a mutational rate lower than the expected experimental error frequency due to Taq DNA polymerase (2 Â 10 5 per base pair per cycle); thus, it was considered as if it did not show intraclonal diversity. Interestingly, no structural similarities between IGs expressed by OAMZL in the present study and the structure of Abs specific for Chlamydiae 27,28 could be demonstrated.…”

“…26 We also utilized IGHV-D-J sequences of previously reported antiChlamydia Abs. 27,28 To comprehensively identify possible restrictions in the VH CDR3 amino acid composition of the complete data set (OAMZL þ other entities), we used the TEIRESIAS algorithm, 29 a computational tool developed by the Bioinformatics and Pattern Discovery group at the IBM Computational Biology Center, freely available from http://cbcsrv.watson.ibm.com/ download.phtml.html. The parameters used in this analysis are as previously reported.…”

Structural Evaluation of a Mimicry-Recognizing Paratope: Plasticity in Antigen–Antibody Interactions Manifests in Molecular Mimicry

“…This could be perceived as a failure of the immune system to discriminate on the basis of molecular structure or may be seen as a way of tackling different types of invading pathogens simultaneously. Ab cross-reactivities have been described in the past, particularly involving primary Abs where degeneracy appears to be a physiological requirement (41)(42)(43)(44)(45)(46)(47)(48)(49)(50). Abs have been shown earlier to achieve binding to diverse Ags through flexible paratope structures involving either partially overlapping residues or achieving Ag-specific structures through induced fit processes (51)(52)(53).…”