APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY A chimeric baculovirus displaying bovine herpesvirus-1 (BHV-1) glycoprotein D on its surface and their immunological properties A. Peralta & P. Molinari & D. Conte-Grand & G. Calamante & O. Taboga Received: 22 September 2006 / Revised: 20 December 2006 / Accepted: 22 December 2006 / Published online: 7 February 2007 # Springer-Verlag 2007 Abstract The ability of a recombinant baculovirus con- taining the ectodomain of the mature sequence of glyco- protein D (gD) fused to the amino-terminus of baculoviral glycoprotein gp64 to display gD on its surface and to serve as an improved immunogen against bovine herpesvirus-1 was tested. The gD–gp64 fusion protein was correctly expressed on the virus particles as revealed by immunomi- croscopy assays. Mice immunized with 5×10 8 plaque forming units developed antibodies that specifically reacted in an enzyme-linked immunosorbent assay with recombi- nant gD and whole bovine herpesvirus-1. These antibodies were able to neutralize bovine herpesvirus-1 in vitro, whereas those elicited by a version of gD expressed in Escherichia coli did not. Our data demonstrated that the display on the virion surface of recombinant baculovirus can provide a tool for the development of recombinant vaccines against bovine herpesvirus-1. Keywords Baculovirus display . BHV-1 vaccines . Viral vectors Introduction To improve the immunogenic properties of soluble anti- gens, multimeric, self-assembling proteins have been used as carriers for the delivery of foreign epitopes (Gamvrellis et al. 2004; Noad and Roy 2003; Roy 1996; Tobin et al. 1996). Based on their multimeric nature and due to the presence of T cell epitopes, these carriers elicit humoral and cellular responses. Besides, these macromolecules can be easily purified by simple centrifugation. However, prob- lems associated with toxicity of recombinant products in bacteria, loss of multimerization of core-like particles carrying foreign sequences and restrictions in size of antigenic sites that could be expressed have been reported (Tami et al. 2000). It has been previously shown that the in-frame fusion of foreign sequences between the signal sequence and ecto- domain of the mature sequence of gp64, an outer glycoprotein of Autographa californica nuclear polyhedro- sis virus (AcNPV), drives the chimeric protein to the surface of the baculovirus and facilitates its purification and concentration (Boublik et al. 1995). Proteins as large as the P1 precursor from foot-and-mouth disease virus (FMDV), a polypeptide of about 74 kDa, could be successfully displayed on the surface of recombinant baculoviruses. This strategy, known as baculovirus display, has been used to develop recombinant vaccines against FMDV (Tami et al. 2000), Plasmodium berghei (Yoshida et al. 2003), rubella (Mottershead et al. 1997), and Theileria parva p67 antigen (Kaba et al. 2003). Some of them have induced high titers of antigen-specific antibodies. The baculovirus display system has several potential advantages as a vaccine vehicle: its particulate nature, the intrinsic immunostimulatory effect of many viral proteins, the multimeric presentation of the epitopes, and the proper Appl Microbiol Biotechnol (2007) 75:407–414 DOI 10.1007/s00253-006-0825-4 A. Peralta : P. Molinari : D. Conte-Grand : G. Calamante : O. Taboga (*) Instituto de Biotecnología, CICVyA, INTA, Castelar CC25 (1712) Buenos Aires, Argentina e-mail: otaboga@cnia.inta.gov.ar A. Peralta : P. Molinari : D. Conte-Grand : O. Taboga Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Rivadavia 1917 (1033) Ciudad Autónoma de Buenos Aires, Argentina