Viral antigens presented on the cell surface in association with MHC class I molecules are recognized by CD8⁺ T cells. MHC restricted peptides are important in eliciting cellular immune responses. As peptide antigens have a weak immunigenicity, pH-sensitive liposomes were used for peptide delivery to induce effective cytotoxic T lymphocyte (CTL) responses. In the previous study, as the HBx peptides could induce specific CTLs in vitro, we tested whether the HLA-A2/K^b transgenic mice that were immunized by HBx-derived peptides could be protected from a viral challenge.

HBx-peptides encapsulated by pH-sensitive liposomes were prepared. A2K^b transgenic mice were immunized i.m. on days one and seven with the indicated concentrations of liposome-encapsulated peptides. Three weeks later, mice were infected with 1X10⁷ pfu/head of recombinant vaccinia virus (rVV)-HBx via i.p. administration. The ovaries were extracted from the mice, and the presence of rVV-HBx in the ovaries was analyzed using human TK-143B cells. IFN-γ secretion by these cells was directly assessed using a peptide-pulsed target cell stimulation assay with either peptide-pulsed antigen presenting cells (APCs), concanavalin A (2µ/ml), or a vehicle. To generate peptide-specific CTLs, splenocytes obtained from the immunized mice were stimulated with 20nicrog/ml of each peptide and restimulated with peptide-pulsed APC four times. The cytotoxic activity of the CTLs was assessed by standard (51)Cr-release assay and intracellular IFN-γ assay.

Immunization of these peptides as a mixture in pH-sensitive liposomes to transgenic mice induced a good protective effect from a viral challenge by inducing the peptide-specific CD8⁺ T cells. Mice immunized with 50µ/head were much better protected against viral challenge compared to those immunized with 5µ/head, whereas the mice immunized with empty liposomes were not protected at all. After in vitro CTL culture by peptide stimulation, however, specific cytotoxicity was much higher in the CTLs from mice immunized with 5µ/head than 50µ/head group. Increase of the number of cells that intracellular IFN-γ secreting cell among CD8⁺ T cells showed similar result.

Mice immunized with XEPs within pH-sensitive liposome were protected against viral challenge. The protective effect depended on the amount of antigen used during immunization. XEP-3-specific CTLs could be induced by peptide stimulation in vitro from splenocytes obtained from immunized mice. The cytotoxic effect of CTLs was measured by ⁵¹Cr-release assay and the percentage of accumulated intracellular IFN-γ secreting cells after in vitro restimulation was measured by flow cytometric analysis. The result of ⁵¹Cr-release cytotoxicity test was well correlated with that of the flow cytometric analysis. Viral protection was effective in immunized group of 50µ/head, while in the in vitro restimulation, it showed more spectific response in 5µ/head group.