MHC Class I Presented T Cell Epitopes as Potential Antigens for Therapeutic Vaccine against HBV Chronic Infection

Abstract

Approximately 370 million people worldwide are chronically infected with hepatitis B virus (HBV). Despite the success of the prophylactic HBV vaccine, no therapeutic vaccine or other immunotherapy modality is available for treatment of chronically infected individuals. Clearance of HBV depends on robust, sustained CD8(+) T activity; however, the limited numbers of therapeutic vaccines tested have not induced such a response. Most of these vaccines have relied on peptide prediction algorithms to identify MHC-I epitopes or characterization of T cell responses during acute infection. Here, we took an immunoproteomic approach to characterize MHC-I restricted epitopes from cells chronically infected with HBV and therefore more likely to represent the true targets of CD8(+) T cells during chronic infection. In this study, we identified eight novel MHC-I restricted epitopes derived from a broad range of HBV proteins that were capable of activating CD8(+) T cells. Furthermore, five of the eight epitopes were able to bind HLA-A2 and A24 alleles and activated HBV specific T cell responses. These epitopes also have potential as new tools to characterize T cell immunity in chronic HBV infection and may serve as candidate antigens for a therapeutic vaccine against HBV infection.

Figure 1

Immunoproteomics method work flow for the identification and characterization of HBV specific T cell epitopes.

Figure 2

HBV protein expression in infected cell lines. (a) HepG2, DE19 (lacks sAg), and 2.2.15 (complete HBV genome) were harvested, fixed and permeabilized, and stained with HBV specific antibodies directed against core or sAg. (b) Median fluorescent intensity (MFI) derived from the flow plots shown in (a).

Figure 3

Validation of naturally presented MHC peptides from HBV infected cells. Mass spectrometry (MS/MS) spectra of the experimentally identified peptides (top spectra) versus their synthetic analogs (bottom spectra). Fragment masses that match are denoted.

Figure 4

HBV specific peptides stimulate CD8⁺ T cell activation in vitro. (a) HLA-A2 restricted CTLs directed against the identified peptides (peptides are represented as first 3 residues of the sequence) were generated using peripheral blood from healthy donors. PBMCs containing the epitope specific CTLs were harvested, washed, and cultured with the peptide pulsed or HBV expressing cells overnight in an IFN-gamma ELISpot assay. Data is represented as % increase over background. (b) PBMCs containing epitope specific CTLs were harvested, washed, and cultured with uninfected or HBV expressing cells overnight in an IFN-gamma ELISpot assay. Normal liver cells served as a negative, nonspecific control.

Figure 5

HBV specific peptides are able to activate CD8⁺ T cells in vivo in both an HLA-A2 and HLA-A24 restricted fashion. HLA-A2 (a) or HLA-A24 (b) transgenic mice were primed and boosted with peptides as previously described. Spleens were harvested, homogenized into single cell suspensions, and cultured with peptide pulsed (peptides are represented as first 3 residues of the sequence) HepG2 cells or HBV expressing cells overnight in an IFN-gamma ELISpot assay. T cell activation was also measured by examining CD107a upregulation on HLA-A2 (c) or HLA-A24 (d) CD8⁺ T cells. Splenocytes were cultured for 6 hours with peptide pulsed or HBV expressing cells in the presence of anti-CD107a and subsequently stained for CD8⁺ expression. Data is presented as the percent of cells in culture that are CD8⁺ CD107a⁺.

Figure 6

HBV peptide specific CD8⁺ T cells activated in vivo secrete cytotoxic effector molecules. In an assay that mirrored the setup described in Figure 5, splenocytes were cultured with peptide pulsed (peptides are represented as first 3 residues of the sequence) or HBV expressing cells overnight. Supernatant was harvested and used in the Milliplex magnetic bead assay to detect granzyme B secretion in response to specific stimulation. Splenocytes from PBS primed, naïve mice were used as a negative control.