Varied immunity generated in mice by DNA vaccines with large and small hepatitis delta antigens

Abstract

Whether the hepatitis delta virus (HDV) DNA vaccine can induce anti-HDV antibodies has been debatable. The role of the isoprenylated motif of hepatitis delta antigens (HDAg) in the generation of immune responses following DNA-based immunization has never been studied. Plasmids p2577L, encoding large HDAg (L-HDAg), p2577S, expressing small HDAg (S-HDAg), and p25L-211S, encoding a mutant form of L-HDAg with a cysteine-to-serine mutation at codon 211, were constructed in this study. Mice were intramuscularly injected with the plasmids. The anti-HDV antibody titers, T-cell proliferation responses, T-helper responses, and HDV-specific, gamma interferon (IFN-gamma)-producing CD8(+) T cells were analyzed. Animals immunized with p2577S showed a strong anti-HDV antibody response. Conversely, only a low titer of anti-HDV antibodies was detected in mice immunized with p2577L. Epitope mapping revealed that the anti-HDV antibodies generated by p2577L vaccination hardly reacted with epitope amino acids 174 to 194, located at the C terminus of S-HDAg. All of the HDAg-encoding plasmids could induce significant T-cell proliferation responses and generate Th1 responses and HDV-specific, IFN-gamma-producing CD8(+) T cells. In conclusion, HDAg-specific antibodies definitely exist following DNA vaccination. The magnitudes of the humoral immune responses generated by L-HDAg- and S-HDAg-encoding DNA vaccines are different. The isoprenylated motif can mask epitope amino acids 174 to 195 of HDAg but does not interfere with cellular immunity following DNA-based immunization. These findings are important for the choice of a candidate HDV DNA vaccine in the future.

FIG. 1.

Viral protein expression. Huh-7 cells were transfected with p2577L, p2577S, and p25L-211S. Cell lysates were harvested 48 h after transfection. Equal volumes of samples were loaded for SDS-PAGE (lanes 1 to 3). HDAg was also detected in lysates of P815/2577L cells (lane 4).

FIG. 2.

Kinetics of anti-HDV antibodies in mice immunized with plasmid p2577L (•), p2577S (▴), p25L-211S (○), or pcDNA3.1(−) (▵). Titers of anti-HDV antibodies were assayed by an ELISA and determined by serial dilution of sera. Titers below 50:1 were considered representative of nonresponders.Data are presented as the mean and standard deviation for all immunized animals per time point.

FIG. 3.

Western blotting with sera from immunized mice. L-HDAg (lanes 1 and 4) and S-HDAg (lanes 2 and 5) obtained from lysates of p2577L- and p2577S-transfected Huh-7 cells were loaded at the same volumes for SDS-PAGE. Lanes 3 and 6 represented a nontransfected cell lysate used as a negative control. After blotting onto nitrocellulose membranes, the antigens were stained with p2577L (lanes 1 to 3)- or p2577S (lanes 4 to 6)-immunized mouse sera (200:1 dilution).

FIG. 4.

Epitope mapping. (A) Serum samples with anti-HDV antibody titers equal to or greater than 400:1 at week 9 were analyzed for epitope mapping. Two major epitopes, at amino acids 96 to 122 and 174 to 195, were identified. The antibodies generated after immunization with p2577L could not bind to the epitope at amino acids 174 to 195. (B) Anti-HDV (amino acids 174 to 195) antibody titers at week 9 determined by serial dilution of sera. The results were considered significant when the OD of the tested sera was higher than the mean OD and 3 standard deviations of the control sera.

FIG. 5.

T-cell proliferation responses. BALB/c mice were given an intramuscular injection of p2577L (□), p2577S (▪), p25L-211S (▤), or pcDNA3.1(−) (▨). At 7 days after the last immunization, splenocytes from three immunized mice were used in proliferation assays. Stimulated wells received purified L-HDAg (10 μg/ml), purified S-HDAg (10 μg/ml), purified MBP (10 μg/ml), and transferrin (120 μg/ml). Data are presented as the mean and standard deviation SI. An SI of greater than 2 was defined as significant.

FIG. 6.

IFN-γ ELISPOT assay for quantification of T-helper responses in mice immunized with various plasmid constructs. Symbols: ▨, L-HDAg; ▩, S-HDAg; □, MBP. Data are presented as the mean and standard deviation.

FIG. 7.

CD8⁺ cytotoxic-T-lymphocyte responses determined by intracellular IFN-γ staining. At 2 weeks after the last immunization, immune splenocytes were cultured in the presence of irradiated P815/2577L cells. The cells were surface stained with fluorescein isothiocyanate-conjugated rat anti-mouse CD8a monoclonal antibody. Finally, the cells were stained with R-phycoerythrin-conjugated rat anti-mouse IFN-γ monoclonal antibody. Samples were acquired for flow cytometry analysis. Data are presented as the mean and standard deviation.