Arch Virol (2005) 150: 327–340 DOI 10.1007/s00705-004-0402-z Expression of multiple foreign epitopes presented as synthetic antigens on the surface of Potato virus X particles K. Uhde 1 , R. Fischer 1,2 , and U. Commandeur 1 1 RWTH Aachen, Institute for Molecular Biotechnology, Aachen, Germany 2 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Schmallenberg, Germany Received March 10, 2004; accepted June 8, 2004 Published online October 20, 2004 c  Springer-Verlag 2004 Summary. We describe the construction of recombinant Potato virus X (PVX) vectors expressing two different epitopes, ep4 and ep6, from Beet necrotic yel- low vein virus (BNYVV). The seven-amino-acid epitopes were expressed as N- terminal coat protein fusions and were displayed on the surface of PVX particles. Particle assembly into full virions was successful even though no wild type coat protein subunits were present, and the epitopes could be detected in crude extracts and purified virus preparations with appropriate antibodies. A construct containing both epitope sequences in tandem was also prepared. The resulting PVX parti- cles could be detected by antibodies against ep4 and ep6, either individually or simultaneously, showing that both epitopes were accessible. In addition mixed infections with PVX vectors containing the individual ep4 and ep6 sequences were carried out. This resulted in the formation of PVX particles displaying ep4 alone, ep6 alone, or both epitopes. These experiments demonstrate for the first time that PVX can be utilized to present multiple epitopes, either tandemly on every coat protein subunit or as heteromultimeric assemblies, both of which could be useful vaccination strategies. The production of epitope-presenting viruses in which every coat protein subunit contains a foreign epitope allows the high-level expression of defined numbers of foreign antigen sites, making such viruses useful standards for immune detection. Introduction Viruses are widely used as vectors for the transient expression of foreign proteins in plants [27]. While the production of full-length proteins is often the objective of such expression experiments, another important application is the presentation of