Hepatitis B Surface Antigen Vector Delivers Protective Cytotoxic T-Lymphocyte Responses to Disease-Relevant Foreign Epitopes

Abstract

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...

Fusion of HBsAg and prime/boosting augment Th1 and CTL responses to HCV polytope DNA vaccine

“…Hence, activated HBsAg-specific effector cell populations may influence the strength and quality of HCV epitope-specific cellular immune responses by cytokines. Accordingly, immunization with HBsAg plasmid DNA was previously shown to evoke Th1 cytokines probably due to the HBsAg-specific helper epitopes [19]. This observation is also consistent with the increased secretion of IFN-in pcHPOL immunized mice of our study compared to the group vaccinated with pcPOL plasmid, without any significant change in IL4 secretion (data not shown).…”

“…Besides, in vitro analysis of pcPOL and pcHPOL plasmids by normalized RT-PCR indicated the equal transcription levels for both constructs (data not shown), therefore, it is unlikely that the increased immune responses observed for pcHPOL were the consequence of increased expression. In contrast, these augmented responses may be most likely explained by secretion of HBsAg-based chimeric particles, which allows their efficient uptake and presentation not only to CD4 + but to CD8 + T cells by MHC-I molecules via a cross priming pathway [19] and leads to the induction of higher immune responses compared to intracellular or membrane-anchored proteins [29]. Of note, the partial secretion of HBsAg-based particles from Cos-7 cells transfected with pcHPOL plasmid was already reported [20].…”

“…3A). Absence of detectable responses against irrelevant CTL peptides (H-2 d ; core [16][17][18][19][20][21][22][23][24][25] and HLA-A2; E 6 and N) in both CTL and ELISpot assays indicated the specificity of the responses against HCVspecific C, E 4 and CE 4 E 6 N (IFN--ELISpot only) peptides. Results of IFN--ELISpot assay ( Fig.…”

“…Peptides corresponding to CD8 + -epitopic peptides (C, E 4, E 6 , N) and an irrelevant H-2 drestricted epitope (core [16][17][18][19][20][21][22][23][24][25] ; NRRPQDVKFP), besides the synthetic CE 4 E 6 N long polytopic peptide were synthesized by solid phase Fmoc chemistry with 97% purity (Institute de Biochimie, Universite de Lausanne, Swiss). In this study, HLA-A2-restricted CD8 + -epitopic peptides (E 6 and N) were also used as irrelevant (negative control) peptides for all assays in BALB/c mice.…”

“…To this end, different strategies such as prime/boost [15;16] and particle-based immunizations have been employed. Chimeric viruslike particle (VLP)-forming DNA vaccines, which encode viral structural proteins capable of assembling into particulate repeated arrays (such as hepatitis B surface antigen; HBsAg), are recently considered as a promising approach for the efficient delivery of antigenic targets [17][18][19]. This so called "plasmo-VLP" strategy combines the ease of production of plasmid DNA and the improved immunogenicity of VLPs [17].…”