Structural Studies of Merozoite Surface Protein 1 (MSP1) from Plasmodium(G.A.Bentley, G. Boulot, V. Chitarra, D. Verger, in collaboration with S. Longacre, Immunologie Moléculaire des Parasites I.P., and F. Nato, Ingénierie des Anticorps I.P.)
Surface proteins of the merozoite, the erythrocyte-invading form of Plasmodium, are promising anti-malaria vaccine candidates. In this regard, Merozoite Surface Protein 1 (MSP1) is one of the most widely studied of the Plasmodium surface antigens. This protein, which is implicated in the erythrocyte invasion process of the parasite, is subjected to several proteolytic cleavages during maturation of the merozoite. During the first phase, it is cleaved to yield four peptide fragments, of which the 42 kDa C-terminal fragment (MSP1-42) remains fixed to the merozoite membrane. During the second phase, occurring at the moment of erythrocyte invasion, MSP1-42 itself is cleaved to yield a polypeptide of molecular weight 11 kDa (MSP1-19). The latter cleavage is essential for erythrocyte invasion, although the mechanism of this process has yet to be elucidated. In order to understand the biological role of MSP1 in the infection of erythrocytes by Plasmodium, we are studying the structure of the recombinant fragments of MSP1. Firstly, the structural comparison of MSP1-19 with MSP1-42 could help to explain the importance of the second cleavage step for the penetration of the red blood cell by the merozoite. Secondly, their 3-dimensional structure would give the spatial distribution of polymorphic (dimorphic) residues and the position of protecting epitopes, which is essential information for the optimal conception of candidate vaccine products. We have determined the structure of MSP1-19 from P. cynomolgi and MSP1-19 from P. falciparum as a complex with the Fab fragment of a specific monoclonal antibody.
Figure 1. Two mutually orthogonal views of MSP1-19 from P. falciparum complexed to the Fab fragment of a specific monoclonal antibody
Studies of the preS Antgenic Determinants of Hepatitis B Virus(G.A. Bentley, F. Lema, J.P. Pizarro, M.M. Riottot, F. Saul, B. Vulliez-Le Normand)
The immune response to hepatitis B virus (HBV) is directed in large part against the viral surface antigen (HBsAg). The preS region of HbsAg (comprising segments preS1 and preS2) is very immunogenic and contains neutralising epitopes which play an important role in the elimination of the virus during infection. In addition, the segment preS1 plays a role in the attachment of the virus to the hepatocyte, the principle target cell of HBV. Characterisation of the conformation of this antigen and the 3-dimensional distribution of epitopes should improve our understanding of HBV and the mechanisms of immune protection afforded by these neutralising antibodies. Our studies are centred on the structural and immunochemical analysis of the preS region in order to characterise its antigenic properties.
Structural Studies of a Superantigen that activates T Cells in the presence of MHC Molecules of both Class I and II.(G.A. Bentley, G. Boulot, F. Saul, in collaboration with P. Truffa-Bachi and P. Rovira, Immunophysiologie Moléculaire, I.P., and E. Van Damme and W. Peumans, Katholieke Universiteit, Leuven)
Urtica Dioica Agglutinin (UDA), a lectin found in the rhizomes of the stinging nettle, acts as a superantigen by activating the sub-family of murine T cells carrying the Vb-8.3 domain in their antigen receptor. In contrast to " classical " superantigens, which activate only through presentation by class II molecules of the Major Histocompatibility Comlex (MHC), UDA induces the stimulation of T cells through presentation by MHC molecules of both class I and class II. The structure of UDA has been determined in both the free form as well as complexes with tri-acetylchitotriose and tetra-acetylchitotetraose. UDA comprises two domains, each possessing a saccharide-binding site. This observation suggests that the superantigenic properties of the lectin are due to the simultaneous fixation of carbohydrate ligands present on the T cell receptor and the MHC molecules, respectively.
Figure 2. The structure of UDA as a complex with tetra-acetylchitotetraose.
Inhibition Studies of HIV Protease by Monoclonal Antibodies(G. Bentley, V. Chitarra, M.M. Riottot, in collaboration with the laboratory of Dr. J. Sedlacek, Institute of Molecular Genetics, Czech Academy of Sciences, Prague)
The protease of Human Immunodeficiency Virus (HIV) is essential for the maturation and consequent infectivity of the virus because of its role in the enzymatic cleavage of the viral polyprotein precursors, Gag/Pol and Gag. The object of this project is to study monoclonal antibodies that inhibit the proteolytic activity in order to analyse the dynamic behaviour of the enzyme. This year, we have continued our studies of the antibody 1696 which inhibits the protease by dissociation of the active homodimer into its inactive monomer subunits. This is achieved by the antibody binding to the N-terminal segment of the protease, a region that is important for the formation of the active homodimer. Although 1696 was raised against the HIV-1 protease, it cross-reacts with the HIV-2 enzyme since the epitope it recognises is highly conserved. We have determined the crystal structure of the recombinant Fv fragment of 1696 as a complex with the N-terminal peptide segment of HIV-1 protease.
Figure 3. Stereo view of Fv1696 seen from above the antigen-bonding site showing the bound N-terminal peptide segment of HIV-1 protease.