Downloaded from www.microbiologyresearch.org by IP: 54.237.16.148 On: Sun, 31 Jul 2016 18:55:59 Journal of General Virology (2001), 82, 1299–1308. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Humoral and CD4 M T helper (Th) cell responses to the hepatitis C virus non-structural 3 (NS3) protein : NS3 primes Th1-like responses more effectively as a DNA-based immunogen than as a recombinant protein Una Lazdina, 1 Catharina Hultgren, 1 Lars Frelin, 1 Margaret Chen, 1 Karin Lodin, 1 Ola Weiland, 2 Geert Leroux-Roels, 3 Juan A. Quiroga, 4 Darrell L. Peterson, 5 David R. Milich 6 and Matti Sa  llberg 1 1,2 Divisions of Clinical Virology, F68, and Biomedical Laboratory Technology 1 and Division of Infectious Diseases, Department of Medicine 2 , Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden 3 Department of Vaccinology, University of Ghent, Ghent, Belgium 4 Department of Hepatology, Fundacion Jimenez Diaz, Madrid, Spain 5 Department of Biochemistry, Virginia Commonwealth University, Richmond, VA, USA 6 Vaccine Research Institute of San Diego, San Diego, CA, USA The non-structural 3 (NS3) protein is one of the most conserved proteins of hepatitis C virus, and T helper 1 (Th1)-like responses to NS3 in humans correlate with clearance of infection. Several studies have proposed that DNA-based immunizations are highly immunogenic and prime Th1-like responses, although few head-to-head comparisons with exogenous protein immunizations have been described. A full-length NS3/NS4A gene was cloned in eukaryotic vectors with expression directed to different subcellular compartments. Inbred mice were immunized twice in regenerating tibialis anterior (TA) muscles with either plasmid DNA or recombinant NS3 (rNS3). After two 100 μg DNA immunizations, specific antibody titres of up to 12 960 were detected at week 5, dominated by IgG2a and IgG2b. NS3-specific CD4 M T cell responses in DNA-immunized mice peaked at day 13, as measured by proliferation and IL-2 and IFN-γ production. Mice immunized with 1–10 μg rNS3 without adjuvant developed antibody titres comparable to those of the DNA-immunized mice, but dominated instead by IgG1. CD4 M T cell responses in these mice showed peaks of IL-2 response at day 3 and IL-6 and IFN-γ responses at day 6. With adjuvant, rNS3 was around 10-fold more immunogenic with respect to speed and magnitude of the immune responses. Thus, immunization with rNS3 in adjuvant is superior to DNA immunization with respect to kinetics and quantity in priming specific antibodies and CD4 M T cells. However, as a DNA immunogen, NS3 elicits stronger Th1-like immune responses, whereas rNS3 primes a mixed Th1/Th2-like response regardless of the route, dose or adjuvant. Introduction Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. HCV can be divided into different genotypes, of which genotypes 1, 2 and 3 are the most common in Europe. Today’s therapies based on IFN-α and ribavirin can induce a sustained virological and biochemical response in 20–60 % of treated patients, depending on the Author for correspondence : Matti Sa  llberg. Fax 46 8 5858 79 33. e-mail misglabd01.hs.sll.se infecting genotype, the virus load and the age of the patient (Reichard et al., 1998 ; Schvarcz et al., 1995). In patients infected by HCV of genotype 1b, response rates as low as 10 % are not uncommon (Schalm et al., 1996). Thus, additional compounds or treatment strategies are urgently needed to improve the treatment of chronic HCV infections. Several studies have stressed the importance of HCV- specific CD4 + T cell responses in clearance of HCV infections (Diepolder et al., 1995 ; Missale et al., 1996). It has been suggested that T helper 1 (Th1) CD4 + T cell responses to the non-structural 3 (NS3) protein are present in patients who clear 0001-7559  2001 SGM BCJJ