Engineering parvovirus-like particles for the induction of B-cell, CD4 + and CTL responses Paloma Rueda a , Jorge L. Martı´nez-Torrecuadrada a , Javier Sarraseca a , C. Sedlik b , Manuel del Barrio a , Alicia Hurtado a , Claude Leclerc b , J. Ignacio Casal a, * a Immunologia y Genetica Apl. S.A. (INGENASA), Hnos, Garcı´a Noblejas 41, 48. 28037 Madrid, Spain b Unite´ de Biologie des Re´gulations Immunitaires, Institute Pasteur, 25 rue du Dr. Roux, 75015 Paris, France Abstract An antigen delivery system based on hybrid recombinant parvovirus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of porcine (PPV) or canine parvovirus (CPV) expressed in insect cells with the baculovirus system has been developed. PPV:VLPs containing a CD8 + epitope from the LCMV nucleoprotein evoked a potent CTL response and were able to protect mice against a lethal infection with the virus. Also, PPV:VLPs containing the C3:T epitope from poliovirus elicited a CD4 + response in mice. These T-cell epitopes were located in the N-terminus of the VP2 protein. Unfortunately, the N-terminus is not adequate for antibody responses because it is inside the particle. Previous attempts to modify the surface-exposed regions of the capsid were unsuccessful. Deletions in the four loops of the surface resulted in the complete disruption of the particles except those in loop 2. However, preliminary insertions of the C3:B epitope of poliovirus in VP2 residue 225 of the loop 2 were not able to elicit antibody responses. To optimise the insertion site, the epitope was inserted consecutively in positions 226, 227 and 228, which seem to be more accessible on the capsid. Mice immunised with these chimeric C3:B CPV:VLPs were able to elicit a strong neutralising antibody response (>3 log 10 units) against poliovirus. The possibility of combining dierent types of epitopes in dierent positions of a single particle to stimulate dierent branches of the immune system paves the way to the production of more potent vaccines in a simple and cheap way. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Parvovirus-like particles; VLPs; Delivery systems; Vaccines 1. Introduction Canine (CPV) and porcine (PPV) parvovirus are autonomously replicating members of the feline parvo- virus subgroup of the genus Parvovirus within the family Parvoviridae [1,2]. Parvoviruses have a single- stranded DNA genome encapsidated by a non-envel- oped icosahedral particle of 25 nm in diameter that is composed of three structural proteins: VP1, VP2 and VP3, of which VP2 is the major component of the virus capsid [3]. VP1 and VP2 are dierent splicing products from the same gene and VP3 is derived from VP2 by proteolytical cleavage of about 20 amino acids from the N terminus. The structure of VP2 consists of an eight-stranded antiparallel b-barrel motif with four large insertions between the b-strands called loops, which make up much of the surface of the virus capsid [4]. These loops contain many of the virus B-cell epi- topes, being the immunodominant domains of the cap- sid [5]. Other antigenic regions were also found at the N- and C-termini of the VP2 subunits [6,7]. Recently, we have described the production of par- vovirus-like particles (VLPs) based on the expression of the major structural protein VP2 of CPV and PPV in insect cells using the baculovirus expression system [8,9]. These VLPs mimic morphologically authentic vir- ions, are very stable in extreme environmental con- ditions and are highly immunogenic, providing full protection against the diseases in the target animal Vaccine 18 (2000) 325–332 0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0264-410X(99)00202-9 www.elsevier.com/locate/vaccine * Corresponding author. Tel.: +34-91-368-0501; fax: +34-91-408- 7598. E-mail address: icasal@ingenasa.es (J.I. Casal)