Ubiquitin conjugation of hepatitis B virus core antigen DNA vaccine leads to enhanced cell-mediated immune response in BALB/c mice

Abstract

Background: Nearly 350 million persons worldwide are chronically infected with hepatitis B virus (HBV). Ubiquitin (Ub) is a highly conserved small regulatory protein, ubiquitous in eukaryotes, that usually serves as a signal for the target protein that is recognised and degraded in proteasomes . The Ub-mediated processing of antigens is rapid and efficient and stimulates cell-mediated immune responses. Accordingly, Ub-mediated processing of antigens has been widely used in chronic-infection and cancer studies to improve immune response.

Objectives: Many clinical trials have shown that DNA vaccine potency needs to be greatly enhanced. Here, we report a new strategy for designing an HBV DNA vaccine using the ubiquitin (Ub) sequence. The aim of this study was to investigate a novel DNA vaccination, based on the expression of HBV core antigen (HBcAg), fused to Ub to enhance DNA vaccine potency.

Materials and methods: Mouse ubiquitin fused to the HBcAg gene and cloned into the eukaryotic vector pcDNA3.1 (-). BALB/c mice were immunized with recombinant pUb-HBcAg or pHBcAg DNA vaccine. Lymphocyte proliferation assay, intracellular IFN-γ assay, CTL cytotoxicity assay, and antibody assay were performed to analyze the cellular and humoral immune responses to our DNA constructs.

Results: HBcAg was expressed effectively in the COS-7 cells that were transiently transfected with pUb-HBcAg. Strong anti-HBc IgG responses were elicited in mice that were immunized with pUb-HBcAg. The endpoint titers of anti-HBc peaked at 1:656100 on the 42nd day after the third immunization. pUb-HBcAg stimulated greater lymphocyte proliferation and induced higher levels of IL-2 and IFN-γ and a greater percentage of HBcAg-specific CD8+ T cells in mice than pHBcAg. In the CTL assay, the specific lysis rate reached 56.5% at an effector:target ratio of 50:1 in mice that were immunized with pUb-HBcAg.

Conclusions: pUb-HBcAg elicits specific anti-HBc responses and induces HBc-specific CTL responses in immunized BALB/c mice. Our results imply that Ub can be used as a molecular adjuvant that enhances the potency of DNA vaccines.

Keywords: DNA vaccine; Hepatitis B core antigen; Ubiquitin.

Figure 1

Electrophoresis of pUb-HBcAg Digested by EcoR I and Hind III. Lane 1: pUb-HBcAg Digested by EcoR I and Hind III; Lane M1: DNA Marker 2000; Lane M2: DNA Marker 15000.

Figure 2

Expression of the Plasmids. (A) Gene expression of HBcAg and Ub-HBcAg, Lane M: DNA marker 2000; Lane 1: blank plasmid pcDNA3.1 (-) transfecting COS-7 cells; Lane 2,3,5: β-actin;Lane 4: plasmid pHBcAg transfecting COS-7 cells; Lane 6: plasmid pUb-HBcAg transfecting COS-7 cells. (B) Protein expression of HBcAg (about 21 kDa), Lane 1: COS-7 cells transfected with pcDNA3.1 (-); Lane 2 and Lane 3 are the same sample: COS-7 cells transfected with pUb-HbcAg; Lane 4: COS-7 cells transfected with pHBcAg

Figure 3

HBcAg-Specific IgG Titer in the Sera of Mice Immunized with Various Formulations. The initial dilution of each serum from immunized mouse was 1:100 and followed with serial of three-fold dilution for anti-HBc IgG detection. Data is from one representative experiment of three performed and is presented as the geometric mean titer ± SD (n = 8). **P < 0.01 pUb-HBcAg 100μg group.

Figure 4

Detection of T Lymphocyte Proliferation Response. Data shown represent the mean and SD. **P < 0.05, *P <0.01.

Figure 5

Cytokines Production in the Supernatant of Splenocytes Harvested from Immunized Mice after in Vitro Re-stimulation. Data Represent the Means ± SD (n = 8).

Figure 6

Intracellular Cytokine Expression in Spleen Cells. The whole cell population was doubly stained with fluorescent material labeled using FITC-CD8α and PE-IFN-γ antibodies. The doubly stained cells were counted and analyzed by flow cytometry. The data are the mean ± SD from eight mice per group.