(a) Groups of five nave HBcAg- and HBeAg-Tg mice were injected s.c. mechanism of selection of Glucagon receptor antagonists-2 HBeAg-negative variants is unknown. The finding that hepatocytes expressing wild-type HBV (made up of both HBcAg and HBeAg) are more susceptible to CTL-mediated clearance than hepatocytes expressing only HBcAg suggest that the HBeAg-negative variant may have a selective advantage over wild-type HBV within the livers of patients with chronic contamination during an immune response and may represent a CTL escape mutant. Hepatitis B computer virus (HBV) is an enveloped computer virus with a partially double-stranded circular DNA genome of approximately 3.2 kb encoding structural and nonstructural proteins. Control and clearance of acute and chronic HBV infections are thought to be dependent on multispecific T-cell responses directed to several HBV-encoded antigens (6,31,38,42,43). HBV expresses two forms of the nucleoprotein: the 21-kDa intracellular nucleocapsid (hepatitis core antigen [HBcAg]), which self-assembles into particles and encapsidates the viral genome and polymerase, and the secreted nonparticulate form (hepatitis e antigen [HBeAg]). HBeAg and HBcAg are translated from two distinct RNA species that have different 5 initiation sites (19). The HBeAg or precore mRNA encodes a hydrophobic signal sequence that directs the HBeAg to the endoplasmic reticulum, where it undergoes N- and C-terminal cleavage within the secretory pathway and is secreted as an 18-kDa monomeric protein (32,41,44,56). Glucagon receptor antagonists-2 Because of the structural differences between the HBcAg and HBeAg (referred to below as the HBc/HBeAgs), they are distinctly recognized by antibodies (24), but due to extensive amino acid homology, they are highly cross-reactive at the CD4+and CD8+T-cell levels (6,28,37,55). In contrast to the well-established structural and replicative functions of HBcAg, the function of the secreted HBeAg in the viral life cycle is less clear because it is not required for assembly, contamination, or replication (10,11,46). However, studies in a number Rabbit polyclonal to ZMAT5 of murine transgenic (Tg) systems indicate that secreted HBeAg functions as an immunoregulatory protein that downregulates the immune response to HBcAg via a variety of mechanisms, including deletional, nondeletional, central, and peripheral immune tolerance (12,13,33-36). The cytotoxic T-lymphocyte (CTL) response is usually believed to be involved in both viral clearance and liver disease during HBV contamination (14). CTL responses directed against HBcAg have been suggested to be of major importance in the clearance of HBV infections in humans (6). Several reports have indicated that both HBcAg and HBeAg expressed as endogenous proteins can primary and be the targets of CTL effector cells (27,28,52,55). The ability of the HBeAg, as well as the intracellular HBcAg, to primary and be recognized as a target of CTL effector cells indicates that intracellular HBeAg and/or its precursors are processed and presented in the context of major histocompatibility complex (MHC) class I molecules for recognition by CTL effector cells. Furthermore, previous studies (27,28,52,55) and the experiments reported here indicate that this HBc/HBeAgs appear to be indistinguishable in terms of priming CTLs and CTL target recognition in vitro. In the current study the comparative abilities of HBc/HBeAg-based genetic vaccines and/or HBc/HBeAg-expressing tumor cell lines to induce CTL responses in wild-type and HBc/HBeAg-Tg mice and to induce liver injury were examined. These studies indicated that a unique two-step immunization protocol was necessary to elicit maximal CTL priming in vivo and that endogenously expressed HBc/HBeAgs can function as tolerogens at the CTL level. Most importantly, although the HBc/HBeAgs were indistinguishable in terms of priming CTLs and as targets for CTL recognition in vitro, CTL recognition of the HBc/HBeAgs expressed in hepatocytes in vivo was significantly different and resulted in different phenotypes of liver injury. == MATERIALS AND METHODS == == Plasmid DNA, recombinant proteins, and synthetic peptides. == An HBcAg gene fragment (552 nucleotides) of theaywsubtype was amplified and cloned into the eukaryotic expression vector pVAX1 (Invitrogen, San Diego, CA) as previously described (29). The HBcAg expression plasmid was designated HBcAg-pVAX1. An HBeAg gene fragment (639 nucleotides) was amplified by PCR from tail DNA extracted from an HBeAg-Tg mouse (23). The amplified gene fragment was ligated into a HindIII- and ApaI-digested pVAX1 vector. The HBeAg expression plasmid was designated HBeAg-pVAX1. Sequencing of Glucagon receptor antagonists-2 the HBcAg-pVAX1 and HBeAg-pVAX1 expression plasmids showed that this inserted genes had.