Intramuscular injection of plasmid DNA expression vectors encoding the three envelope proteins of the hepatitis B virus (HBV) induced humoral responses in C57BL/6 mice specific to several antigenic determinants of the viral envelope. The first antibodies appeared within 1-2 weeks after injection of DNA and included antibodies of the IgM isotype. Over the next few weeks, an IgM to IgG class switch occurred, indicating helper T-lymphocyte activity.Peak IgG titers were reached by 4-8 weeks after a single DNA injection and were maintained for at least 6 months without further DNA injections. The antibodies to the envelope proteins reacted with group-and subtype-specific antigenic determinants of the HBV surface antigen (HBsAg). Expression vectors encoding the major (S) and middle (preS2 plus S) envelope proteins induced antibodies specific to the S protein and the preS2 domain, and preS2 antibodies were prominent at early time points. In general, the expression vectors induced humoral responses in mice that mimic those observed in humans during the course of natural HBV infection.

In a pilot study, it was established that specific therapy by standard anti-hepatitis B virus (HBV) vaccination may be effective in reducing HBV replication and canceling the immune tolerance to hepatitis B surface antigen (HBsAg) particles in about 50% of persons with chronic active HBV replication. In the present study, the vaccine-induced immune responses were analyzed during an ongoing controlled multicenter vaccine trial. Vaccination elicited peripheral blood mononuclear cell proliferative responses specific for envelope antigen in 7 of 27 subjects given HBsAg. The responses induced by the vaccines were mediated by CD4+ T lymphocytes, and at least three different epitopes were recognized. HBV-specific CD4+ T lymphocytes produced high levels of interferon-gamma [corrected] and belonged to a T helper 1 subset. Reduction of serum HBV DNA in some of these persons suggests that induction of CD4+ T cell responses could be important in controlling viremia during vaccine therapy of chronic HBV carriers.

Transgenic mice expressing the sequences coding for the envelope proteins of the hepatitis B virus (HBV) in the liver have been used as a model of the HBV chronic carrier state. We evaluated the possibility of inducing a specific immune response to the viral envelope antigens and thus potentially controlling chronic HBV infection. Using HBV-specific DNA-mediated immunization in this transgenic model, we show that the immune response induced after a single intramuscular injection of DNA resulted in the complete clearance of circulating hepatitis B surface antigen and in the long-term control of transgene expression in hepatocytes. This response does not involve a detectable cytopathic effect in the liver. Adoptive transfer of fractionated primed spleen cells from DNA-immunized mice shows that T cells are responsible for the down-regulation of HBV mRNA in the liver of transgenic mice. To our knowledge, this is the first demonstration of a potential immunotherapeutic application of DNA-mediated immunization against an infectious disease and raises the possibility of designing more effective ways of treating HBV chronic carriers.Hepatitis B virus (HBV) is the most common etiologic agent for infectious liver disease. Following initial infection, some individuals fail to resolve their infection and thereby become chronic carriers. The proportion of HBV-infected persons proceeding to the chronic carrier state is 5-20% of those infected as adults and as high as 95% with perinatal transmission such as occurs in areas where HBV is endemic. There are estimated to be more than 250 million chronic HBV carriers in the world today and there is a significant association between persistent infection and liver cirrhosis or hepatocellular carcinoma (1). The control of HBV infection is thought to be mediated by both humoral and cellular immune responses involving neutralizing antibodies as well as class I and class II major histocompatibility complex (MHC)-restricted T cells (2-4). Patients who successfully clear the virus following acute infection mount a multispecific polyclonal immune response to several HBV antigens, whereas those who remain persistently infected develop only a weak and more restricted antiviral response (5-9). In infected newborns, a diminished antiviral immune response that may be due to neonatal tolerance mechanisms (see ref. 10) probably plays an important role in viral persistence. In chronically infected patients empty viral particles carrying the HBV surface antigen (HBsAg) are produced and secreted in large amounts by the hepatocytes, whether or not the virus replicates in the liver. These particles persist in the serum and the corresponding HBsAg-specific neutralizing antibodies (anti-HBs) are either not induced or remain undetectable by conventional techniques due to the formation of immune complexes (11).We have used transgenic (Tg) mice that constitutively express the HBsAg (12) as a model of chronic HBV carriers to study the possibility of inducing an immune response to HBsAg and thus contr...