Molecular Immunology xxx (2006) xxx–xxx Signal sequences modulate the immunogenic performance of human hepatitis C virus E2 gene Irina Sominskaya a , Ekaterina Alekseeva a , Dace Skrastina a , Vladislav Mokhonov b,c , Elizaveta Starodubova b , Juris Jansons a , Mikael Levi b , Alexei Prilipov c , Tatyana Kozlovska a , Valeri Smirnov c , Paul Pumpens a , Maria G. Isaguliants b,c,∗ a Biomedical Research and Study Centre, University of Latvia, LV-1067 Riga, Latvia b Swedish Institute of Infectious Disease Control, SE-17182 Solna, Stockholm, Sweden c D.I. Ivanovsky Institute of Virology, 123098 Moscow, Russia Received 14 October 2005; accepted 30 November 2005 Abstract Envelope protein E2 of human hepatitis C virus (HCV) is an attractive component of a prototype HCV vaccine. Delivered by DNA immunogens, E2 evokes specific immune response of Th1-type, failing to induce either considerable antibody production, or T-helper cell proliferation. We aimed at modulating the immunogenic performance of E2 gene by changing the mode of protein expression in eukaryotic cells. Plasmids were constructed encoding full-length E2 and nonstructural protein 1 (p7) fused to either 13 or 38 C-terminal amino acids (aa) of HCV E1 that contain second hydrophobic segment of E1 stop-transfer signal, or a complete E1 stop-transfer signal with duplicated second hydrophobic segment. Injected into BALB/c mice, E2/p7 genes induced potent antibody and T-helper cell response targeted against hypervariable region 1, aa 472–586 of E2, and a novel epitope at aa 774–796 of p7. Profile of cytokines secreted by proliferating mouse splenocytes stimulated in vitro with E2- and p7-derived peptides, indicated mixed Th1/Th2 type of immune response. Thus, the full-length E2 and p7 genes supplied in one cassette were both immunogenic. E2/p7 containing a complete E1 stop-transfer signal with prolonged membrane spanning domain was superior to the shorter E2/p7 version in terms of both antibody and cellular immunogenicity. Optimal performance of HCV E2 could thus be achieved without the aid of external/heterologous signals by easing, through modification of the E2 signal sequence, the release of E2 from the rough ER while retaining full-length E2 and p7 sequences. This finding may help to improve the Th2 performance of HCV envelope genes as prototype vaccines. © 2006 Elsevier Ltd. All rights reserved. Keywords: HCV envelope E2; p7; Eukaryotic expression; Signal sequence; Stop-transfer signal; Cellular localization; DNA immunization 1. Introduction Hepatitis C virus (HCV) is a positive strand RNA virus that belongs to the Hepaciviridae genus within the Flaviviridae family. HCV infection has a wide spectrum of clinical presen- tations; 55–85% of patients do not clear the virus, but develop chronic hepatitis C (Hoofnagle, 2002). The chronic sequelae of hepatitis C include progressive hepatic fibrosis and cirrho- sis, the major risk factors of hepatocellular carcinoma (HCC), and eventually HCC (Saito et al., 1990). Extensive efforts con- ∗ Corresponding author at: Department of Virology, Swedish Institute for Infectious Disease Control, SE-17182 Solna, Stockholm, Sweden. Tel.: +46 8 4572609; fax: +46 8 337272. E-mail address: maria.isaguliants@smi.ki.se (M.G. Isaguliants). tinue to develop a vaccine for prevention and treatment of HCV infection (Inchauspe and Feinstone, 2003; Koff, 2003). A break through was recently made by the envelope protein E1-based immunotherapeutic vaccine. Delivered to patients with chronic hepatitis C, it enhanced anti-E1 cellular immune response and improved liver histology (Nevens et al., 2003). The vaccine was well tolerated and immunogenic in healthy volunteers (Leroux- Roels et al., 2004). Our research focused on envelope protein E2 and nonstruc- tural protein p7 of HCV. E2 encompasses amino acids (aa) 384–746 of HCV polyprotein. Fifty to 70% of aa residues in E2 are variable, and aa 383–414 (HVR1) and 470–480 (HVR2) are hypervariable (Weiner et al., 1991; Kato, 2001). E2 variability reflects an inherent low fidelity of the viral replication machin- ery and provides a large reservoir of biologically different 0161-5890/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2005.11.018 MIMM-1920; No. of Pages 12