Unit: Structural Immunology
Director: BENTLEY Graham
Research projects of the Unit of Structural Immunology focus on the structural biology of proteins from pathogenic agents. Structure determination of protein molecules by X-ray crystallography is the principle activity of the Unit, but a variety of immunochemical and physicochemical techniques are also employed to extend and complement our studies on structure-function correlations. Certain of our projects concern proteins implicated in the development of vaccines and medicaments. We are particularly interested in surface antigens from the malaria parasite Plasmodium that are vaccine candidates
Plasmodium surface antigens
Surface proteins of Plasmodium are potential malaria vaccine candidates because they are targets of the humoral immune response by the host. Since all pathologies associated with malaria are caused by the asexual blood stage in the Plasmodium life cycle, antigens expressed during this critical phase are particularly favoured candidates. Two kinds of surface antigen enter into consideration here: those present on the surface of the merozoite, or free blood-stage form of the parasite, and those expressed by the parasite inside the infected erythrocyte but presented on the surface of the host cell. Antibodies induced by certain of these proteins can confer immune protection by interfering with functions critical for the survival of the parasite. The molecular functions of many Plasmodial blood-stage surface antigens identified to date, however, are poorly, if at all, characterised. We study Plasmodium antigens present on the merozoite and the surface of infected erthrocytes.
(i) Apical Membrane Antigen 1: (G. Bentley, M.L. Chesne-Seck, G. Faure, S. Igonet and B. Vulliez-Le Normand in collaboration with A. Thomas, Biomedical Primate Research Centre, Rijswijk, the Netherlands, and M. Blackman, National Institute of Medical Research, MillHill, UK)
Apical Membrane Antigen 1 (AMA1), a surface antigen present in all species of Plasmodium, plays an important role in erythrocyte invasion by the merozoite. Nonetheless, the function of this protein at the molecular level is poorly characterised. AMA1 is a malaria vaccine candidate currently undergoing clinical trials. The protein comprises an ectoplasmic region, a transmembrane segment and a cytoplasmic domain. We have expressed and crystallised the ectoplasmic domain, and the structure has been determined in two different crystalline forms at 1.8 Å and 2.0 Å, respectively. In collaboration with the groups of the B.P.R.C., Rijswijk, and the N.I.M.R, Mill Hill, we have characterised the epitope recognised by a monoclonal antibody that inhibits erythrocyte invasion and have related this to the 3-dimensional structure of AMA1. Although the folding of AMA1 had not been previously identified from analysis of the sequence, our structural results show that two of the domains belong to a protein fold already characterised. These structural and immunological results, together, have allowed us to identify a region of the ectoplasmic domain that could be critical for biological function.
(ii) P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1): (C. Badaut, G.A. Bentley, S. Igonet, Hélène Souchon et L. Larret, in collaboration with O. Puijalon, Immunologie Moléculaire des Parasites, I.P., M. Klinkert, Bernhard Nocht Institute, Hamburg, P. Deloron, Institut de Recherche pour le Développement, Université Paris V, and D. Arnot, University of Edinburgh.
Inside the infected erythrocyte, P. falciparum expresses PfEMP1, a virulence factor that is transported to, and presented on, the surface of the host cell. PfEMP1 forms family of adhesins that confers upon infected erythrocytes the capacity to auto-agglutinate, to adhere to non-infected red blood cells or to sequester in the vascular beds of divers tissues. These adhesion phenomena are directly linked to many of the pathological consequences of P. falciparum infection. PfEMP1 is encoded by the var (variable) genes, present in about 60 copies (depending on the parasite strain); however, only one variant is presented on the erythrocyte surface at any given time, thus giving different parasite isolates their particular receptor specificity for agglutination or sequestration. PfEMP1 molecules are organised in a modular fashion, with the extra-cellular region composed mainly of DBL (Duffy-Binding Like) and CIDR (Cysteine-rich Interdomain region) domains belonging to different classes. We are studying the structural and functional properties of different variants of PfEMP1.
Pregnancy-associated malaria arises from the sequestration of infected erythrocytes in the placenta. P. falciparum isolates implicated in this form of malaria express PfEMP1 variants that show a binding specificity to chondroitin sulphate A (CSA) present on certain proteoglycans localised in the placenta. This specificity is due, in large part, to a sub-group of the domain DBL class γ. We are studying a PfEMP1 variant isolated from a placental parasite isolate with the objective of characterising the structure of its DBLγ domain, and to study its interaction with CSA and specific antibodies. We have expressed this domain in the baculovirus/insect cell system, demonstrated that the recombinant protein is recognised by the serum of women who have had placental malaria and have shown that it binds CSA and CSA-bearing proteolglycans.
Figure 1. The structure of the ectoplasmic region of Apical Membrane Antigen 1 from Plasmodium vivax.
Keywords: structural biology, X-ray crystallography, antigenic recognition, antibody structure, Plasmodium antigens