Molecular Immunology 40 (2004) 1237–1247 Functional, molecular and structural characterisation of five anti-Brucella LPS mAb Thierry Cl. Laurent a,∗ , Pascal Mertens a,1 , Jean-François Dierick b , Jean-Jacques Letesson a , Christophe Lambert a , Xavier De Bolle a a Unité de Recherche en Biologie Moléculaire, Facultés Universitaires Notre-Dame de la Paix, Rue de Bruxelles 61, B-5000 Namur, Belgium b BioVallée—Proteomics Unit, Rue des Professeurs Jeener et Brachet 12, 6041 Gosselies, Belgium Received 10 September 2003; received in revised form 18 November 2003; accepted 26 November 2003 Abstract The O-antigen of the gram negative bacteria Brucella is composed of an homopolymer of 4,6-dideoxy-4-formamido--d-mannopyranosyl (or perosamine). Several mAb interact specifically with only the O-antigen of certain Brucella species. Although, many studies show that this specific recognition results mainly from the ratios of 1-2 and 1-3 link between the different Brucella strain perosamine residues, little is known about the mAb recognising this O-antigen. In this paper, we describe the binding profile of five anti-Brucella O-antigen mAb to the LPS of two Brucella strains and a bacteria possessing a nearly identical O-antigen: Yersinia enterocolitica O:9. We show that the specificity of these five mAb can be correlated to their germ line gene usage. Besides, their relative affinity to the different LPS is correlated to their ability to protect against Brucella infection by passive transfer in a mouse model. The analysis of their 3D structure gives new hypothesis of the epitopes recognised. © 2004 Elsevier Ltd. All rights reserved. Keywords: Monoclonal antibody; Brucella; Homology modelling; Sequence analysis; Germline; Main text 1. Introduction Brucellae are gram-negative facultative intracellular bac- teria infecting humans and domestic animals. The impor- tance of the humoral immune response for a protective im- munity against brucellosis was demonstrated (Elzer et al., 1994; Vizcaino and Fernandez-Lago, 1994). Most antibodies produced following Brucella infection are directed against its lipopolysaccharide (Rojas et al., 2001). Moreover, several monoclonal antibodies (mAb) directed against the O-chain of the lipopolysaccharide (LPS) are able to reduce the bac- terial charge in spleen and liver after a passive transfer in mice (Cloeckaert et al., 1992; Limet et al., 1987; Limet et al., 1989). The O-chain of Brucella LPS bears two distinct anti- gens, designated A and M. The variable ratio of A and M antigen in smooth Brucella strains defined their A- and/or M-dominant character. The structure of the these antigens was elucidated by NMR as repeating units of a pentasac- ∗ Corresponding author. Tel.: +32-817-24416; fax: +32-817-24297. E-mail address: thierry.laurent@fundp.ac.be (T.Cl. Laurent). 1 Present address: CorisBioconcept, Science Park Crealys, Rue Phocas Lejeune no. 30-9, 5032 Gembloux, Belgium. charide consisting of four 1,2-linked and one 1,3-linked 4,6-dideoxy-4-formamido--d-mannopyranosyl (-d-Rha4- NFo or perosamine) for the M antigen (Bundle et al., 1987) and a homopolymer of 1,2-linked -d-Rha4NFo with fewer 1,3 link (2–3%) for the A-antigen (Caroff et al., 1984a). Yersinia enterocolitica O:9, the most important source of false positive reactions in the serological diagno- sis of cattle (Weynants et al., 1995), possesses a very similar O-chain with only 1,2-linked -d-Rha4NFo (Caroff et al., 1984b). All these O chains share common epitopes but possess also specific epitopes. The results obtained with the use of eight mAb and synthetic oligosaccharides in a competitive Enzyme Immnuo Sorbent Assay (ELISA) al- lowed to propose the composition of A, M and C (common) epitope present on the LPS and recognised specifically by these mAb. The A epitope was defined as a pentasaccha- ride or a larger oligosaccharide composed of 1,2-linked 4,6-dideoxy-4-formamido--d-mannopyranosyl, the M epitope should be a disaccharide -d-Rha4NFo(1 → 3) -d-Rha4NFo that require adjacent 1,2-linked residues for full activity. Finally, the C epitope was defined as a linear 1-2-linked tri- or tetra-saccharide (Bundle et al., 1989). However, little is currently known about the sequence and the structure of mAb recognising the Brucella O chain since 0161-5890/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2003.11.037