Structures of synthetic O-antigen fragments from serotype 2aShigella flexneriin complex with a protective monoclonal antibody

Abstract

The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes.antibody complex ͉ carbohydrate ͉ crystal structure ͉ polyliposaccharide ͉ shigellosis S higellosis (1), or bacillary dysentery, causes significant morbidity and mortality worldwide, particularly among young children (2). The disease arises from colonization and subsequent destruction of the colonic mucosa by the Gram-negative enteroinvasive bacteria Shigella. Immune protection induced by natural infection derives from antibodies directed against the bacterial surface antigen lipopolysaccharide (LPS) (3). Moreover, protection shows a serotype specificity that is determined by the repeat unit (RU) structure of the O-antigen (O-Ag), the polysaccharide moiety of LPS (4). In the species Shigella flexneri, which is responsible for endemic infections in developing countries, the serotype is defined by glucosyl and O-acetyl modifications added to the basic tri-rhamnose-N-acetyl-glucosamine tetrasaccharide (designated ABCD) of the O-Ag backbone (5). (Serotype 6 is an exception.) Of the 14 S. flexneri serotypes identified to date, the 2a serotype is the most prevalent in developing countries (2). The serotype 2a RU is characterized by a branching glucose (residue E) linked to the third rhamnose (residue C) to form the motif AB(E)CD (Fig. 1).The induction of protective immunity by natural infection with Shigella suggests that an effe...

A Synthetic Carbohydrate-Protein Conjugate Vaccine Candidate against Shigella flexneri 2a Infection

“…5 Besides, AB(E)CD, [AB(E)CD] 2 , and [AB(E)CD] 3 were similarly recognized by human sera from patients naturally infected with SF2a, demonstrating that O-Ag epitopes recognized by human sera are, to some extent, present in haptens corresponding to a small number of RUs. In addition, available structural data on [AB(E)CD] 2 and [AB(E)CD] 3 , in complex with one of the available protective mAbs, mIgG F22-4, demonstrated that the nine residue-epitopes that bind to mIgG F22-4 Fab are already present in the decasaccharide (35). As an opening to challenge the topic, additional structural and physicochemical investigations are in progress to unravel the molecular basis for accurate mimicking of SF2a O-SP by [AB(E)CD] 3 .…”

Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein–Polysaccharide Interactions

“…17,18 Virulence of S. flexneri is intimately linked to this specific and dynamic O-antigen composition. 8,19 The basic unbranched serogroup Y antigen is composed of tetrasaccharide repeat units (RU) with the structure…”

Structure of a protective epitope of group BStreptococcustype III capsular polysaccharide

“…Minimal epitopes can be composed of short, defined glycans comprising 2-3 monosaccharides, as for the β-(1→2) mannans of the Candida albicans cell wall (4), Vibrio cholerae O1 (5), Shigella flexneri variant Y (6), and Salmonella (7) O-antigens, or a tetrasaccharide, as for the repeating unit (RU) of S. pneumoniae type 14 PS (Pn14) (8,9), and even six sugar residues, as in the case of S. flexneri serotype 2a O-antigen (10). In contrast, the type III PS of Streptococcus agalactiae (group B Streptococcus, GBS) has been proposed as a prototype of a unique length-dependent conformational epitope (11).…”