Hepatitis B virus core antigen epitopes presented by HLA-A2 single-chain trimers induce functional epitope-specific CD8+ T-cell responses in HLA-A2.1/Kb transgenic mice

Abstract

The potency of CD8+ cytotoxic T lymphocyte (CTL) responses toward core antigen has been shown to affect the outcomes of hepatitis B virus (HBV) infection. Since single-chain trimers (SCT) composed of peptide epitope beta2-microglobulin (beta2m) and major histocompatibility complex (MHC) class I heavy chain covalently linked together in a single molecule have been shown to stimulate efficient CTL responses, we investigated the properties of human leucocyte antigen (HLA)-A2 SCTs encoding the HBV core antigen (HBcAg) epitopes C(18-27) and C(107-115). Transfection of NIH-3T3 cells with pcDNA3.0-SCT-C(18-27) and SCT-C(107-115) leads to stable presentation of HBcAg epitopes at the cell surface. HLA-A2.1/Kb transgenic mice vaccinated with the SCT constructs, either as a DNA vaccine alone or followed by a boost with recombinant vaccinia virus, were shown to generate HBcAg-specific CTL responses by enzyme-linked immunospot assay (ELISPOT) and in vitro interferon-gamma release experiments. HBcAg-specific CTLs from vaccinated HLA-A2.1/Kb transgenic mice were able to inhibit HBV surface and e antigen expression as indicated by HepG2.2.15 cells. Our data indicate that a DNA vaccine encoding a human HLA-A2 SCT with HBV epitopes can lead to stable, enhanced HBV core antigen presentation, and may be useful for the control of HBV infection in HLA-A2-positive HBV carriers.

Figure 1

Diagrams depicting structure of HBV SCTs and chimeric DNA and vaccinia virus constructs. (a) SCT which includes a signal peptide, epitope, β₂m and human MHC-I molecule HLA-A2 was linked by a 15 amino acid GS linker. (b) SCTs containing HBcAg epitope C₁₈₋₂₇ (pcDNA3.0-SCT-C₁₈₋₂₇, FLPSDFFPSV), epitope C_107−115 (pcDNA3-SCT-C_107−115, CLTFGRETV) and a control epitope from HIV gag (SLYNTVATL) were cloned into pCDNA3.0 vector separately to make the DNA vaccine in the study. (c) Diagram of transfer vector AE-SCT-C₁₈₋₂₇, which includes an EGFP gene and SCT-C₁₈₋₂₇ gene. (d) Transfer vectors AE (control vaccinia vector with EGFP), AE-SCT-C₁₈₋₂₇ and AE-SCT-C_107−115 were constructed and recombined with wild-type vaccinia virus TianTan strain (VTT) to make recombinant vaccinia virus (rVTT) in this study.

Figure 2

Cell surface expressions of SCTs. (a) Flow cytometry analysis of cell surface expression of SCTs. The pcDNA3.0-SCT constructs were transiently transfected to NIH-3T3 cells. Cell surface expression was determined by FITC-labelled anti-HLA-A2 antibody (clone BB7.2). (b) Confocal microscopic detection of the cell surface expression of SCTs on NIH-3T3 cells. After transient transfection, NIH-3T3 cells were stained with FITC-labelled HLA-A2 antibody (clone BB7.2) and analysed using confocal microscopy. NIH-3T3 (negative control), NIH-3T3-pcDNA-SCT-HIVgag (NIH-3T3 cells transfected with pcDNA3.0-SCT-HIVgag).

Figure 3

Construction and detection of recombinant vaccinia virus. (a) Example of recombinant vaccinia virus rVTT-AESCT-C₁₈₋₂₇ selection. Vero cells were transfected with 0·1 MOI wild-type VTT for 90 min in six-well plates, followed by transfection of 1 μg transfer vector AESCT-C₁₈₋₂₇ plasmids by Effectene transfection reagent. Cells were left to grow for 24 hr and then frozen and thawed three times. Supernatants were planted on six 60-mm plates and cultured for 24 hr. EGFP-expressing cells were selected using a fluorescent microscopy and further purified for six rounds. (b) Purification of rVTT-AESCT-C₁₈₋₂₇ was performed using 20–60% sucrose-gradient centrifugation at 18 000 g and diluted in phosphate-buffered saline. Purified recombinant virus was subjected to transmission electron microscopy.

Figure 4

DNA vaccine with SCT constructs elicits epitope-specific CTL responses in HLA-A2.1/Kb transgenic mice. (a) Four groups of HLA-A2.1/Kb transgenic mice, each contain four to eight mice, were vaccinated with 100 μg pcDNA-3.0-SCTs intramuscularly at 2-week intervals for three injections. Splenocytes were dissected 2 weeks after the last vaccination. Epitope-specific IFN-γ-releasing cells were counted by ELISPOT experiments. pcDNA3.0-SCT vaccines were able to prime CD8⁺ T-cell responses with statistical significance (P = 0·002, P = 0·009 and P = 0·007 for C₁₈₋₂₇, C_107−115 and HIV gag, respectively, compared to pcDNA3.0 control group vaccination; SFC, spot-forming cells). (b) Splenocytes were prestimulated with 10 μg/ml epitopes for 2 days and then tested by flow cytometry using FITC-CD8a and APC-IFN-γ antibodies. CD8⁺ IFN-γ-expressing cells were gated against CD8⁺ T cells.

Figure 5

Epitope-specific CTLs from HLA-A2.1/Kb transgenic mice inhibit HBV surface and e antigen expression from HepG2.2.15 cells. Splenocytes were immediately cocultured with HepG2.2.15 cells at ratios of 20 : 1 and 40 : 1 for 2 days. S and e antigen expression in the culture media was tested with HBV S and e antigen detection kits by ELISA. (a) Splenocytes from pcDNA3.0-SCT-C₁₈₋₂₇-vaccinated and pcDNA3.0-SCT-C_107−115-vaccinated mice were shown to inhibit HBV S antigen expression of HepG2.2.15 cells with statistical significance (P < 0·05 compared to mice from control pcDNA3.0- and pcDNA3.0-SCT-HIVgag-vaccinated groups). (b) HBV e antigen expression was inhibited by CTLs from core-antigen-specific SCT-vaccinated mice with statistical significance compared to control groups.

Figure 6

A vaccinia virus boost greatly enhanced the magnitude of HBV core-antigen-specific CTL responses primed by DNA vaccine. HLA-A2.1/Kb transgenic mice were vaccinated with pcDNA-SCTs twice and then boosted with 2 × 10⁷ EFU recombinant vaccinia virus rVTT-AE and rVTT-AE-SCTs. Splenocytes were dissected 2 weeks after the last vaccination. (a) ELISPOT analysis of HBcAg₁₈₋₂₇-specific and HBcAg_107−115-specific CTLs was performed immediately after the splenocyte dissection. Vaccinia virus boost greatly enhanced the CTL responses in an epitope-specific manner (with P = 0·01, P =0·002 and P = 0·005 for pcDNA-AE, pcDNA-AE-SCTC₁₈ and pcDNA-AE-SCTC₁₀₇, respectively, compared to the pcDNA3.0-vaccinated group; P = 0·0003 and P = 0·02 for pcDNA-AE-SCTC₁₈ and pcDNA-AE-SCTC₁₀₇, respectively, compared to the pcDNA-AE-vaccinated group). (b) Splenocytes were stimulated with corresponding epitopes for 2 days and were then subjected to in vitro IFN-γ staining analysis.