Molecular Immunology, Vol. 33, No. I l/12, pp. 909-916, 1996 Pergamon PII: SO161-589O(%)OOOSs-2 Copyright IC!1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0161-5890196 $15.00+ 0.00 REPRODUCING THE IMMUNE RESPONSE AGAINST THE zyxwvutsrqponmlkjihgfedcbaZYX Plasmodium viuax MEROZOITE SURFACE PROTEIN I WITH MIMOTOPES SELECTED FROM A PHAGE-DISPLAYED PEPTIDE LIBRARY C. DEMANGEL, P. LAFAYE and J. C. MAZIE Laboratoire d’Hybridolab, Institut Pasteur, Paris. France zyxwvutsrqponmlkjihgfedcbaZYXWVU (Received 6 February 1996; accepted in rezised,fhm 6 June 1996) Abstract-We have used phage display technology to identify peptides binding D14-3, a monoclonal antibody raised against the A4, 42000 C-terminal fragment of Plasmodium uivax merozoite surface protein 1 (PvMSPl). By screening a constrained hexapeptide library, seven independent clones binding D14-3 were isolated. The reactivity of D14-3 for these peptides was lower than for the natural antigen and the antibody binding was strictly associated with the viral context and the peptide conformation. Sequence analysis showed that five of them shared homology with the M, 42000 C- terminal fragment (Pv42) and therefore appears to identify the D14-3 epitope. However, the other two peptides. while related to each other, did not correspond to any sequence in the Pv42 molecules. To evaluate their immunological interest, these phagotopes were injected into mice belonging to Balb/c, lC57B1/6 and Biozzi strains. All animals developed a strong immune response against phage particles but only Biozzi mice produced antibodies cross-reacting with Pv42. All phagotopes in Biozzi mice elicited a specific response against Pv42. even those sharing no sequence similarity with the antigen. Moreover. the avidities of these immune sera and the polyclonal response against Pv42 were comparable, suggesting phagotopes could be used as components of a subunit vaccine based on the C-terminal fragment of MSPl. Copyright c 1996 Elsevier Science Ltd Key words: MSP I, phage display, epitope, mimotope, immunization. vaccine INTRODUCTION The identification of protective epitopes and their local- ization on antigens can sometimes be an important step in vaccine development, but often requires much time and experience. The affinity panning of phage peptide libraries offers an attractive alternative technique for identifying the ligands of target molecules such as mono- clonal antibodies (mAbs). When large repertoires of bacteriophages displaying random peptides are used to determine linear epitopes (Cwirla et al., 1990; Scott and Abbreviations: A, absorbance, BSA, bovine serum albumin, Carb, carbenicillin, ELISA, enzyme-linked immunosorbant assay, lC50%, 50% inhibition concentration, Ig, immu- noglobulin, IgG, immunoglobulin G, IgM, immunoglobulin M, Kan, kanamycin, LB, Luria Bertani medium, mAb, mon- oclonal antibody, MSPl, merozoite surface protein 1, pIII, minor coat protein, pVII1, major coat protein, Phagotope. peptide displayed on phage, PvMSPl, merozoite surface protein 1 of Plasmodium uivax, Pv42, M, 42 000 C-terminal region of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA PvMSPl , Pc42, equivalent protein of Pv42 in Plas- modium cy nomolgi, Pv19, M, 19000 C-terminal region of PvMSPl, PBS, phosphate-buffered saline, PEG, poly- ethylene glycol, PMSF, phenylmethylsulfonyl fluoride, TBS. Tris-buffered saline, SB, super broth medium, Tet, tetra- cycline. TU. transducing unit(s), UV, ultraviolet. Smith, 1990; Stephen and Lane, 1992) the selection pro- cedure leads both to sequences that resemble the natural epitope, and to mimotopes, which are totally different from the natural epitope but are nevertheless highly spec- ific. The affinities of the selected clones are extremely variable and depend on the ability of phage-displayed peptides to reproduce the natural epitope conformation (O’Neil et al., 1992; McConnell ef al., 1994). One additional advantage of this technology is the use of recombinant phages as antigens. Short peptides from the Plasmodium falciparum circumsporozoite antigen dis- played on filamentous bacteriophages were found to be highly immunogenic (De la Cruz et al., 1988; Greenwood et a/., 1991). Moreover, selected phage-displayed mim- otopes are capable of mimicking an epitope for antibody binding (Felici et al., 1993; Hoess et al., 1994) and recent works report that they can even represent immunogenic mimics of the natural antigen (Orlandi et al., 1994; Motti et al., 1994; Folgori et al., 1994). In a vaccine development strategy, this system is therefore of great interest, since it allows the identification of peptide ligands in vitro and a rapid screening of their in viva properties. We chose to investigate epitopes of the Plasmodium vivax merozoite surface protein 1 (PvMSPl) by this method. This protein plays an essential role in the red cell invasion and is a leading malaria vaccine candidate 909