Structural Mimicry of O-Antigen by a Peptide Revealed in a Complex with an Antibody Raised against Shigella flexneri Serotype 2a

Abstract

The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4–antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.

Introduction

Surface polysaccharides of pathogenic bacteria are major targets of protective humoral immune responses against reinfection and are thus commonly used as basis for vaccine development against these microorganisms.¹ By themselves, however, polysaccharides are T-cell-independent antigens and, as such, cannot induce B-cell memory responses.² Consequently, vaccines of this kind that are in current use or under development are formulated with either bacteria (attenuated or inactivated) or glycoconjugates, which are made by covalently coupling the bacterial polysaccharide [detoxified lipopolysaccharide (LPS)], or synthetic fragments thereof, to a carrier protein or peptide.¹ Mimicry of carbohydrate antigens by peptides or proteins offers an alternative approach. For example, peptides presented in phage display systems can be selected using protective antibodies specific for the targeted bacterial polysaccharide. These peptides are tested, in turn, as immunogens for their capacity to induce an anti-carbohydrate response that protects against bacterial infection. The objective is thus to identify an antigenic mimic of the bacterial antigen that is also an immunogenic mimic.³ Although a number of immunogenic mimics for different bacterial polysaccharides have been obtained from phage-displayed peptides4, 5 (some of which have been shown to induce protection against infection),6, 7 factors that determine the success of this method are difficult to decipher, not only because of the conformational flexibility characteristic of carbohydrate and peptide antigens but also because of the complex relationship between the antigen and the induction of protective immune response.

For several years, we have examined these different approaches to develop vaccine candidates for shigellosis, or bacillary dysentery, for which no clinically accepted vaccine is currently available.⁸ Shigellosis is caused by inflammation and subsequent destruction of the colonic mucosa following intestinal infection by the Gram-negative bacterium Shigella. The major protective immune response to Shigella arises from antibodies directed against the O-antigen (O-Ag), the serotype-specific polysaccharide component of bacterial LPS.⁹ Our efforts have focused on the species Shigella flexneri, which is endemic in developing countries, and more specifically on serotype 2a (Fig. 1), which is the most prevalent.¹⁰ Serotype specificity is determined by the repeat unit structure of the O-Ag,11, 12 which is the branched pentasaccharide AB(E)CD for serotype 2a (Fig. 1).

We have previously reported a panel of specific IgG monoclonal antibodies (mAbs) produced by immunizing mice with S. flexneri serotype 2a and have shown in a mouse model that they protect against homologous, but not heterologous, challenge.¹³ More recently, we determined the crystal structure of the fragment antigen binding (Fab) of one of these mAbs, designated F22-4, in complex with synthetic [AB(E)CD]₂ and [AB(E)CD]₃,¹⁴ representing a dimer and a trimer of the serotype 2a O-Ag repeat unit, respectively, which act as functional mimics of the bacterial polysaccharide.13, 15, 16 We now report the selection of phage-displayed peptides using F22-4 and their subsequent screening for immunogenic mimicry of serotype 2a O-Ag. Although none induced anti-carbohydrate antibodies, we pursued a structural investigation of the dodecapeptide B1, which showed the highest affinity for F22-4 among those selected. This was motivated by the paucity of detailed studies reported to date that combine immunogenic and structural analyses of carbohydrates and their peptide mimics.5, 17, 18 Here, we analyze the binding of B1 to F22-4 by isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and saturation transfer difference (STD) NMR; describe the conformation of the free peptide in solution by NMR; and present the crystal structure of Fab F22-4 in complex with B1. Antigenic mimicry of O-Ag by B1 is analyzed by comparing the oligosaccharide and peptide complexes of F22-4, and factors that might contribute to the apparent lack of immunogenic mimicry are discussed.