This may be due to the intracellular retention of LHBs in transfected muscle cells with a subsequent delay in accessibility of the antigen for the initiation of the immune response. plasmids encoding wild-type LHBs or nonsecreted mutant SHBs and IL-2 had no significant effects on immune responses. Interestingly, mice immunized with cytokine expression plasmids 14 days after the injection of the wild-type LHBs plasmid showed augmented immune responses compared to animals simultaneously injected with both expression constructs. Anti-HBs responses in mice injected with plasmids encoding secreted forms of HBsAgs were detectable about 10 days earlier than those in mice immunized with plasmids encoding nonsecreted forms of HBsAgs. Based on these observations, we conclude that cytokines produced by DNA plasmids at the initial site of antigen presentation cannot augment LHBs specific immune responses because LHBs is not produced at high enough levels or is not accessible for uptake by antigen-presenting cells. Hepatitis B virus (HBV) is a noncytopathic, hepatotropic virus. Worldwide, more than 350 million individuals are infected (21). HBV is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (3, 19). CREB4 The cellular immune response to HBV is thought to be responsible for viral clearance and pathogenesis of liver disease, including hepatocellular carcinoma. The observation of spontaneous HBV clearance IV-23 in some chronically infected individuals implies that the suboptimal cellular immune response may be reversible. Therefore, strategies designed to boost the HBV-specific T-cell immune response, to alter the balance between the cytopathic and the regulatory components of the response, or to mimic the regulatory functions of the T-cell response in the liver may terminate persistent infection. For these reasons, we chose genetic immunization as an immunotherapeutic approach to chronic HBV infection because this approach offers the potential advantage of inducing cellular and humoral immune responses against conserved viral epitopes because vaccination is based on DNA expression plasmids rather than proteins. IV-23 This strategy involves the transfer of a viral gene into muscle cells and antigen-presenting cells by a plasmid vector with subsequent endogenous production and intracellular processing of the viral structural proteins into smaller antigenic peptides. Such peptides are subsequently expressed on the cell surface in the context of major histocompatibility complex molecules (23, 25) and therefore have been shown to induce CD8+ cytotoxic T-lymphocyte (CTL) and helper T-cell type 1 (TH1) responses against various viral antigens (24). Using this approach, several groups have demonstrated that HBV surface and nucleocapsid antigens are highly immunogenic at both the T-cell and B-cell levels in mice (6, 9, 12, 15, 22). Immunogenicity of the secreted middle HBV surface protein (MHBs) was significantly better than that of the nonsecreted large IV-23 HBV surface protein (LHBs) (12). In addition, recent studies demonstrated that coimmunization of interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA expression plasmids enhanced humoral and cellular immune responses to rabies glycoprotein (26), HBV small surface protein (SHBs) and MHBs (5) and the hepatitis C virus core protein (8, 10). Different from the findings with MHBs DNA, coimmunizations of LHBs encoding DNA with either IL-2 or GM-CSF expression plasmids did not augment cellular and humoral immune responses to HBV envelope proteins (11). This finding was not due to an inhibition of the secretion of IL-2 and GM-CSF by LHBs. The effects of LHBs on the immune response augmenting properties of IL-2 and GM-CSF in vivo, therefore, were not related to inhibition of their secretion from the cell by LHBs. Conversely, IL-2, gamma interferon (INF-) and tumor necrosis factor alpha (TNF-) did not down-regulate HBV surface gene expression in several mouse cell lines with different genetic backgrounds (11). We and others have recently demonstrated that the anti-HBs response to an LHBs DNA expression construct is IV-23 detectable about 10 to 14 days later than the responses to MHBs (6, 12). This may be due to the intracellular retention of LHBs in transfected muscle cells with a subsequent delay in accessibility of the antigen for the initiation of the immune response. To test this hypothesis, in the present study we designed plasmids producing a secreted and a nonsecreted form of LHBs and SHBs, respectively, without changing antigenicity and determined the immunogenicity of these proteins in vivo using the genetic immunization approach. MATERIALS AND METHODS DNA expression vectors. pSVL encodes wild-type LHBs and carries mutations of both the MHBs and SHBs start codons to the threonine codon ACG. pSVblaL carries a bacterial -lactamase secretion signal sequence upstream from the LHBs coding sequence and encodes a secreted LHBs. MHBs and SHBs start codons are mutated in the same manner as in pSVL. pSVs25L corresponds to pSVblaL but contains a truncated nonfunctional signal sequence as well as wild-type MHBs and SHBs start codons. These plasmids have been described in detail (1). The pSVBX24H vector encodes SHBs (14). The.
