Mouse models of orthopoxvirus disease provide great promise for probing basic questions regarding host responses to this group of pathogens, which includes the causative agents of monkeypox and smallpox. However, some essential tools for their study that are taken for granted with other mouse models are not available for these viruses. Here we map and characterize the initial CD8 ؉ T-cell determinants for poxviruses in H-2 d -haplotype mice. CD8 ؉ T cells recognizing these three determinants make up around 40% of the total responses to vaccinia virus during and after resolution of infection. We then use these determinants to test if predicted conservation across orthopoxvirus species matches experimental observation and find an unexpectedly cross-reactive variant peptide encoded by ectromelia (mousepox) virus.

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.Hepatitis C virus (HCV) is now recognized as the major cause of non-A, non-B hepatitis. It has been estimated that about 170 million people worldwide are infected with HCV, of whom 70 to 80% will develop chronic liver disease, leading to cirrhosis in 10 to 20% and liver cancer (hepatocellular carcinoma) in 1 to 5% of chronically infected individuals (6). The linear, single-stranded, positive-sense HCV RNA genome of ca. 9.5 kb contains a single open reading frame (ORF) encoding a polyprotein which is cleaved into the individual mature viral proteins by host-and virus-specific proteinases. Three structural proteins have been identified, the core protein and two envelope proteins, E1 and E2.It has been reported that cellular and humoral immune responses play a pivotal role in the host defense mechanism against HCV (8, 36). Most HCV carriers have circulating antibodies to the virus envelope proteins and to a region located in the extreme amino terminus of E2, hypervariable region 1 (HVR1), which has been reported to contain neutralizing Bcell epitopes and a T-cell epitope (16,17,44). HVR1 probably represents the major site of HCV genetic drift, with amino acid substitutions leading to escape from recognition by existing anti-HVR1 antibodies. Due to the variability within the HVR1 region, it has been proposed that these mutations are responsible for the persistence of HCV infection through neutralizing antibody escape mutants (25,46,52). Qualitative antibody changes accompany HVR1 epi...

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen-and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.Vaccination strategies capable of safely and effectively inducing cytotoxic T-lymphocyte (CTL) responses which are acceptable in humans have not proven easy to develop (18). The hepatitis B virus (HBV) surface antigen (HBsAg) small envelope protein (HBsAg-S) self-assembles into highly organized virus-like particles (VLPs) (11). HBsAg VLPs are extremely efficient at inducing long-lived HBsAg-specific CTL responses when administered as a vaccine, comparing favorably with traditional strong CTL inducers in this regard (40). This likely relates to the presentation of the CTL epitopes as multiple copies in repetitive arrays, a format known to enhance induction of CTL responses (21). HBsAg may also be delivered as a DNA vaccine. By use of this strategy, CTL induction by intracellular translated HBsAg protein occurs through the classical endogenous pathway as well as through the alternative exogenous pathway via secreted HBsAg particles (28, 38, 39).HBsAg-S polypeptide is composed of 226 residues, is synthesized at the endoplasmic reticulum anchored at the lipid membrane and is secreted as empty 22-nm VLPs, with each VLP consisting of 100 to 150 polypeptides. The HBsAg molecule has a complex configuration and contains at least two transmembrane regions, an internal luminal domain and a hydrophilic external domain (the a determinant). The C-terminal domain is hydrophobic and it probably contains two additional transmembrane regions. HBsAg CTL epitopes are located primarily in the transmembrane and luminal domains (2, 42).Although HBsAg per se i...