Immunology and Cell Biology (2002) 80, 425–435 Research Article Synergy of type I interferon-A6 and interferon-B naked DNA immunotherapy for cytomegalovirus infection EMMALENE J BARTLETT, VANESSA S CULL, NATALIE L BREKALO, JASON C LENZO and CASSANDRA M JAMES Division of Veterinary and Biomedical Sciences, Murdoch University, Western Australian Biomedical Research Institute, Murdoch, Western Australia, Australia Summary Delivery of type I IFN transgenes by naked DNA immunization can protect against cytomegalovirus infection and myocarditis. Here, we investigate IFN transgene expression, antiviral efficacy, and immunomodula- tion of myocarditis using various treatment regimes in a mouse CMV model. In vivo expression of the IFN transgene was observed in the sera for 35 days post-DNA inoculation. Prophylactic IFN-A6 and IFN-B DNA treatment for 14 days prior to murine cytomegalovirus (MCMV) infection was more efficacious in significantly reducing viral titres, than 2 days prior to or 2 days post-virus infection. Similarly, IFN-A6 DNA treatment commencing 14 days prior to virus infection was superior in suppressing both acute and chronic myocarditis. Furthermore, reduction of autoantibody titres was more pronounced when IFN was administered 14 days prior to viral infection. Combina- tional IFN gene therapy was assessed for synergy between IFN subtypes. Combination treatment with either IFN- A6/A9 or IFN-A6/B greatly reduced spleen viral titres while IFN-A6/B and IFN-A9/B reduced virus replication in the liver. Only IFN-A6/A9 and IFN-A9/B reduced acute viral myocarditis, whereas IFNA6/B treatment was most efficacious for autoimmune chronic myocarditis. Finally, treatment with IFN-A6 DNA 2 weeks post-MCMV infection proved effective at inhibiting the development of chronic autoimmune myocarditis. These findings suggest that immunomodulation of both antiviral and autoimmune responses by IFN DNA immunization may be an avenue for improved viral immunotherapy. Key words: cytomegalovirus, immunotherapy, naked DNA, type I interferon. Introduction The advent of gene transfer technology provides an alter- native to conventional therapy. Direct intramuscular injection of plasmid or naked DNA, encoding a specific protein, leads to uptake of the DNA plasmid and protein expression in vivo. 1 Initially, vaccines encoding antigenic proteins were devel- oped for a number of disease models including influenza virus, 2 human immunodeficiency virus 1 (HIV-1), 3 hepatitis B virus, 4 hepatitis C virus, 5 and bacterial, parasitic and tumori- genic diseases. 6–8 More recently, plasmid DNA encoding cytokines for immunotherapy has been established as a novel and effective approach in the treatment of several viral and disease models. Cytokines such as the type I IFN, 9–13 IL-6, 14 IL-12, 15 GM-CSF 15 and IL-2 16 have proven effective in disease treatment. In addition, combination therapy including IL-12/ GM-CSF 15 or cytokine covaccination with immunogen 17–19 provide enhanced efficacy in the treatment of disease. Cytomegalovirus (CMV) infection is prevalent in human populations and, in the majority of cases, infection remains asymptomatic. However, CMV is a serious opportunistic path- ogen in immunocompromised individuals, including transplant recipients, AIDS patients, and neonates. 20,21 Cytomegalovirus has also been implicated in the pathogenesis of chronic heart inflammation and the subsequent development of myocarditis and cardiomyopathy. 21–26 Increased rates of severe allograft rejection and cardiac dysfunction, in heart transplant recipi- ents and AIDS patients, are thought to be a consequence of active CMV infection. 21,24 Heart transplant recipients experi- encing a primary CMV infection during transplantation have a 46% incidence of developing myocarditis, 25 while 50–65% of patients with active myocarditis have been found to be positive for CMV DNA in heart tissues. 26 Furthermore, myo- cardial damage postviral infection may persist in immuno- compromised patients and lead to dilated cardiomyopathy. Due to the strict host specificity of CMV, a direct animal model for human cytomegalovirus (HCMV) infection has not been established. However, murine cytomegalovirus (MCMV) provides an experimental model for CMV infection and disease that mimics HCMV pathogenesis. Murine cyto- megalovirus closely resembles HCMV tissue tropism, 27 latent infection 28 and the development of pathological sequelae, such as the development of pneumonitis, hepatitis and myo- carditis. 29–31 Murine cytomegalovirus infection of adult BALB/c mice induces a biphasal inflammatory disease in cardiac tissue characterized by a mononuclear cell infiltrate predominated by CD8 + T cells, macrophages and neutro- phils. 32 While MCMV is the trigger for myocarditis in this model, accumulating evidence suggests the disease is immunopathological in nature. The acute phase of the disease is observed from days 3 to 21 p.i., with infectious virus seen Correspondence: Dr CM James, Division of Veterinary and Bio- medical Sciences, Murdoch University, 6150, Western Australia, Australia. Email: casjames@central.murdoch.edu.au Received 19 February 2002; accepted 30 April 2002.