Coimmunization with IL-15 plasmid enhances the longevity of CD8 T cells induced by DNA encoding hepatitis B virus core antigen

Abstract

Aim: To test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for improving the immune responses induced by hepatitis B virus core gene DNA vaccine.

Methods: We used RT-PCR based strategies to develop IL-15 expression constructs. We first confirmed that the gene could be expressed in Escherichia coli due to the poor expression of IL-15. Then the bioactivity of IL-15 plasmid expression product was identified by CTLL-2 proliferation assay. One hundred micrograms of DNA from each of the IL-15 eukaryotic expressed plasmid and the recombinant plasmid harboring DNA encoding the 144 amino acids of the N-terminus of HBV core gene (abbreviated pHBc144) was used to co-immunize C57 BL/6 mice. The titer of anti-HBcIgG was detected by ELISA and the antigen-specific CD8(+) T cells (CD8(+)IFN-gamma(+) T cells) were detected by intracellular cytokine staining at different time points.

Results: After co-immunization by pIL-15 and pHBc144 DNA vaccine the antigen-specific CD8(+) cells of mice increased gradually, the first peak of immune response appeared 14 d later, then the number of antigen-specific CD8(+) Ts cells decreased gradually and maintained at a steady level in 3 mo. After boosting, the number of antigen-specific CD8(+) T cells reached the second peak 10 d later with a double of the 1st peak, then the number of antigen-specific CD8(+) T cells decreased slowly. IL-15 as a gene adjuvant had no significant effect on humoral immune responses induced by hepatitis B virus core gene DNA vaccine, but increased the memory antigen-specific CD8(+) T cells induced by hepatitis B virus core gene DNA vaccine.

Conclusion: DNA vaccine constructed by HBc Ag 1-144 amino acid induces effective cell immunity, and cytokine plasmid-delivered IL-15 enhances the longevity of CD8(+) T cells.

Figure 1

Immunization schedule of pHBc144 and pIL-15 plasmid.

Figure 2

Electrophoretogram of PCR products. The human IL-15 coding sequence (GenBank U14407) was RT-PCR amplified from human PBL mRNA stimulated with LPS.

Figure 3

SDS-PAGE and Western blotting of recombinant polypeptides. M: polypeptide molecular marker; lane 1: BL21; lane 2: pET32c ; lane 3: pET-15 uninduced ; lane 4: pET-15 induced.

Figure 4

IL-15 bioactivity in supernatants of cells transfected with pIL-15 expression plasmid. COS cells were transfected with pIL-15 (▲) expression constructs or vector pcDNA3 (×), and the supernatants were collected 72 h after transfection. The supernatants were tested by CTLL-2 bioassay to measure IL-15 bioactivity. IL-2 (5 × 10³ U/ mL) was used as a positive control.

Figure 5

Kinetics of antigen-specific CD8⁺ T-cell response in spleen (A) and PBMCs (B) following DNA immunization. Mice were immunized with pHBc144 plasmid DNA or vector DNA intramuscularly. At various time points, splenocytes and PBMCs were harvested, stimulated for 5 h with specific peptide, double stained with anti-CD8a-PE and anti-IFN-γ-APC. A total of 1 × 10⁵ events were acquired by a flow cytometer. The indicated values on the lines of the figure (plotted on a log₁₀ scale) reflect the percentage of peptide-specific CD8⁺ T-cells to total CD8⁺ T-cells in the spleens or PBMCs (about three mice/group at each time point). The cut-off value was 0.1%.

Figure 6

Percentages of HBc specific CD8⁺ T cells in the spleen after primary (A) and secondary immunization (B) and in PBMCs after primary (C) and secondary immunization (D) detected by ICCs directly ex vivo at various time points. C57BL/6 mice were immunized with pHBc144 DNA or pIL-15 DNA and boosted at d 30 later. The splenocytes and harvested at various time points. Then the cells were cultured for 5 h in the absence or presence of the HBcAg peptide and double stained with anti-CD8a-PE and anti-IFN-γ-APC. CD8⁺IFN-γ⁺cells are enclosed in ovals, and the numbers in the upper right quadrants represent the percentage of CD8⁺IFN-γ⁺ cells to total CD8⁺ T cells. The data from each group at each time point represent mean ± SD (n = 3). Naïve mice were used as negative controls. The cut-off value was 0.1%.