Delineation of a neutralizing subregion within the immunodominant epitope (GH loop) of foot-and-mouth disease virus VP1 which does not contain the RGD motif Fred Brown a , Nadia Benkirane b , David Limal b , Hubert Halimi b , John F.E. Newman a , Marc H.V. Van Regenmortel b , Jean-Paul Briand b , Sylviane Muller b, * a Plum Island Animal Disease Center, USDA/ARS, P.O. Box 848, Greenport, NY 11944-0848, USA b Institut de Biologie Mole Âculaire et Cellulaire, UPR 9021 CNRS, 15 rue Rene  Descartes, F67084 Strasbourg, France Received 16 December 1998; accepted 8 April 1999 Abstract The major immunogenic site of foot-and-mouth disease virus (FMDV) is contained in a disordered loop comprising residues 134±158 of capsid protein VP1, located on the surface of the viral particle. Peptides corresponding to this sequence generally elicit protective levels of neutralizing antibodies in guinea pigs. In some instances, however, the level of neutralizing antibodies is low although the level of antibodies against the peptide, determined by ELISA, is as high as that in the sera with high neutralizing antibody titres. In an attempt to ascertain the reason for this dierence, we have synthesized on a cellulose membrane 10 overlapping decapeptides, oset by one residue, covering the segment 141±159 of VP1 of two viruses belonging to serotypes A12 and O1, and tested them with guinea pig antisera raised against peptide 141±159, VP1 and FMDV particles (SPOTscan method). With type A, some peptides which were strongly positive with highly neutralizing antisera did not include the RGD triplet located at residues 145±147. In contrast, antisera with low neutralization titres reacted only with decapeptides which included the RGD motif. Moreover, peptide 147±156 coupled to keyhole limpet haemocyanin, but not peptide 141±149 coupled to the same carrier, elicited high levels of neutralizing antibodies in guinea pigs. In the case of serotype O, highly neutralizing antisera to virus reacted in ELISA with peptides 141±150 (containing the RGD motif) and 135±144 (located upstream from the RGD motif). The results suggest that the RGD triplet is not an indispensable constituent of peptides able to elicit a neutralizing antibody response against the virus. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Foot-and-mouth disease virus; Neutralization; Synthetic peptide 1. Introduction The major immunogenic site of FMDV is contained in a disordered loop comprising amino acids 134±158 of capsid protein VP1, located on the surface of the particle [1]. Peptides corresponding to this loop have been shown by several authors to elicit good levels of neutralizing antibodies in mice, guinea pigs, swine and cattle which can protect them against challenge infec- tion (reviewed in Ref. [2]). The loop includes the triplet RGD at residues 145±147 which is involved in cell attachment [3±5]. Since this triplet is present in the great majority of the many FMDV isolates for which the VP1 sequence is known, it has been assumed that the neutralizing activity of the sera elicited by the loop peptide was related to the attachment of the virus to susceptible cells. However, neutralizing antibodies against these peptides are serotype-speci®c, indicating that the amino acids surrounding the RGD motif, and not the triplet itself, are responsible for eliciting neu- tralizing antibodies. This view is supported by the Vaccine 18 (2000) 50±56 0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0264-410X(99)00169-3 www.elsevier.com/locate/vaccine * Corresponding author. Tel.: +33-3-8841-7027; fax +33-3-8861- 0680. E-mail address: smuller@ibmc.u-strasbg.fr (S. Muller)