Antiviral Research 81 (2009) 113–122 Contents lists available at ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral Chimeric virus-like particles for the delivery of an inserted conserved influenza A-specific CTL epitope Wan-Shoo Cheong a , Jessica Reiseger b , Stephen John Turner c , Richard Boyd b , Hans-Jürgen Netter a,∗ a Department of Microbiology, Monash University, Clayton, Victoria, Australia b Monash Immunology and Stem Cell Laboratory, Monash University, Clayton, Victoria, Australia c Department of Microbiology and Immunology, The University of Melbourne, Victoria, Australia article info Article history: Received 11 August 2008 Received in revised form 8 October 2008 Accepted 10 October 2008 Keywords: Hepatitis B virus envelope protein Modified virus-like particles Influenza A-specific epitope Vaccination study abstract The small hepatitis B virus surface antigens (HBsAg-S) have the ability to self-assemble with host-derived lipids into empty non-infectious virus-like particles (VLPs). HBsAg-S VLPs are the sole component of the licensed hepatitis B vaccine, and they are a useful delivery platform for foreign epitopes. To develop VLPs capable of transporting foreign cytotoxic T lymphocyte (CTL) epitopes, HBsAg-S specific CTL epitopes at various sites were substituted with a conserved CTL epitope derived from the influenza matrix protein. Depending on the insertion site, the introduction of the MHC class I A2.1-restricted influenza epitope was compatible with the secretion competence of HBsAg-S indicating that chimeric VLPs were assembled. Immunizations of transgenic HHDII mice with chimeric VLPs induced anti-influenza CTL responses prov- ing that the inserted foreign epitope can be correctly processed and cross-presented. Chimeric VLPs in the absence of adjuvant were able to induce memory T cell responses, which could be recalled by influenza virus infections in the mouse model system. The ability of chimeric HBsAg-S VLPs to induce anti-foreign CTL responses and also with the proven ability to induce humoral immune responses constitute a highly versatile platform for the delivery of selected multiple epitopes to target disease associated infectious agents. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Envelope and capsid proteins from various viral agents can be synthesized as virus-like particles (VLPs) in the absence of nucleic acid. Due to their repetitive structure, VLPs are highly immuno- genic and have the ability to induce anti-viral humoral and cellular immune responses (Jondal et al., 1996; Pumpens and Grens, 2001; Ruedl et al., 2002). As exogenous antigens, VLPs have the ability to enter the major histocompatibility complex (MHC) class I path- way in contrast to less structured, exogenous antigens, which are processed via the MHC class II pathway (Schirmbeck et al., 1995a, 1996). Normally, only endogenously synthesized antigens are effi- ciently presented via the MHC class I pathway inducing cytotoxic T lymphocyte (CTL) reactions. Because of the ability of exogenous VLPs to induce CTL reactions in the absence of viral replication and gene expression, they represent attractive tools for developing therapeutic vaccine approaches. ∗ Corresponding author at: Department of Microbiology, Clayton Campus, Wellington Road, Monash University, Victoria 3800, Australia. E-mail address: hans.netter@med.monash.edu.au (H.-J. Netter). The small envelope protein (HBsAg-S) encoded by the human hepatitis B virus (HBV) has the capacity to self-assemble with host-derived lipids into VLPs. HBsAg-S VLPs are the sole anti- genic component of one of the most successful vaccines (Bruss and Ganem, 1991; Maupas et al., 1981; Stirk et al., 1992; Szmuness et al., 1980). Research outcomes and clinical trials have shown that they are a successful delivery system for foreign B-cell epi- topes or protein domains (Berkower et al., 2004; Bisht et al., 2002; Bojang, 2006; Delpeyroux et al., 1986, 1990; Eckhart et al., 1996; Michel et al., 2007; Netter et al., 2001; Neurath et al., 1989; Phogat et al., 2008; Pumpens et al., 2002). HBsAg-S VLPs containing for- eign B-cell epitopes in the external hydrophilic loop (Delpeyroux et al., 1986; Eckhart et al., 1996; Netter et al., 2001,2003; Phogat et al., 2008) induced anti-foreign antibody responses. Immuniza- tions also induced potentially neutralizing antibodies indicating that the inserted sequences have retained a native conformation (Delpeyroux et al., 1986; Vietheer et al., 2007). The preparation of the antigen and the mode of delivery are critical for the elicited immune response. In the presence of the adjuvant aluminium hydroxide, HBsAg-S VLPs lack the ability to induce a CTL response but induce a humoral response. In the absence of adjuvant, wild- type HBsAg-S VLPs primed CD8 + CTL responses (Schirmbeck et al., 1996). Endogenous synthesis of modified HBsAg-S proteins after 0166-3542/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.antiviral.2008.10.003