Enhanced hepatitis C virus NS3 specific Th1 immune responses induced by co-delivery of protein antigen and CpG with cationic liposomes Xuanmao Jiao, Richard Yan-Hui Wang, Qi Qiu, Harvey J. Alter and J. Wai-Kuo Shih Correspondence J. Wai-Kuo Shih jshih@mail.cc.nih.gov Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, Building 10, Room 1C711, National Institutes of Health, Bethesda, MD 20892-1184, USA Received 11 December 2003 Accepted 3 February 2004 Mice were immunized intramuscularly with free recombinant hepatitis C virus (HCV) NS3 (non-structural protein 3) protein, liposomes encapsulating rNS3 or rNS3 and CpG mixture, liposomes co-encapsulating rNS3 and CpG or liposomes co-encapsulating rNS3 and GpC. Liposomes co-encapsulating rNS3 and CpG induced a much higher titre of anti-HCV NS3 IgG and the dominant IgG subtype was IgG2a. Liposomes co-encapsulating rNS3 and GpC also induced high levels of anti-HCV NS3 IgG antibody, but the dominant IgG subtype was still IgG1, the same as in free HCV/NS3 immunized mice. Liposomes encapsulating rHCV NS3 and the mixture of rHCV NS3 and CpG did not increase the antibody response but switched the IgG subtype. A cytokine profile analysis revealed that the levels of Th1 cytokines in the mice immunized with liposomes co-encapsulating rHCV NS3 and CpG were significantly higher than in other mice while the levels of Th2 cytokine were significantly lower than in the mice immunized with naked rNS3. IL-12 in the mice immunized with liposome-NS3-CpG was significantly higher than in other mice. In conclusion, liposomes co-encapsulating HCV NS3 and CpG are a good candidate vaccine to induce strong Th1 immune responses against hepatitis C viruses. INTRODUCTION Hepatitis C virus (HCV ) infects approximately 1 % of the world’s population. Over 75 % of acutely infected indivi- duals progress to a persistent infection that can result in cirrhosis, liver failure and hepatocellular carcinoma (Choo et al., 1989; Cohen, 1999). The development of a screening test in 1990 has virtually eliminated the spread of HCV through blood transfusion, but 36 000–150 000 new cases of HCV infection continue to occur in the USA every year through other transmission routes (Cohen, 1999; Stratton et al., 2000). Currently, the most effective treatment for chronic HCV infection is a combination therapy with interferon-a and ribavirin, but across all genotypes only 50 % of treated patients have sustained benefit from antiviral therapy (Poynard et al., 1998; McHutchison et al., 1998). Treatment efficiency might be considerably enhanced by direct stimulation of HCV-specific T-cell responses using a therapeutic vaccine. Clinical observations of HCV-specific immune responses in patients with acute self-limited HCV infection or patients who have recovered from chronic HCV infection suggest that T-cell-mediated immune responses may determine the outcome of HCV infection (Rehermann & Chisari, 2000). During the first few weeks of acute hepatitis C infection, HCV-specific CD4 + T cells that proliferate after in vitro stimulation with recombinant HCV core, NS3 (non- structural protein 3) and NS4 (non-structural protein 4) antigens have been detected (Rehermann & Chisari, 2000). An NS3-specific CD4 + T cell immune response is much stronger and more frequently found in patients who resolve acute hepatitis than in patients who develop chronic infection and this response may be necessary for virus clearance (Diepolder et al., 1995). Similarly, IFN plus ribavirin treatment-induced control of hepatitis C viraemia is associated with the development of HCV-specific T-cell responses with enhanced IFN-c and low IL-10 production (Cramp et al., 2000). The effective immune response frequently displays a Th1 or Th0 cytokine profile while the activation of Th2 responses seems to be involved in the development of chronic hepatitis (Tsai et al., 1997). An immunodominant epitope recognized by NS3-specific Th cells has been described at aa 1251–1259 within HCV NS3 (Diepolder et al., 1997) and is completely conserved within HCV 1a, 1b, 1c, 2a and 2b genotypes (Rehermann & Chisari, 2000). These findings suggest that a vigorous HCV-specific CD4 + Th1 response, particularly against the non-structural proteins of the virus, may be associated with virus clearance and protection from disease progression. Accordingly, the development of a vaccine that increased Th1 immune 0007-9896 G 2004 SGM Printed in Great Britain 1545 Journal of General Virology (2004), 85, 1545–1553 DOI 10.1099/vir.0.79896-0