Plasmid-encoded interleukin-15 receptor alpha enhances specific immune responses induced by a DNA vaccine in vivo

Abstract

Plasmid-encoded DNA vaccines appear to be a safe and effective method for delivering antigen; however, the immunogenicity of such vaccines is often suboptimal. Cytokine adjuvants including interleukin (IL)-12, RANTES, granulocyte-macrophage colony-stimulating factor, IL-15, and others have been used to augment the immune response against DNA vaccines. In particular, IL-15 binds to a unique high-affinity receptor, IL-15R alpha; is trans-presented to CD8(+) T cells expressing the common betagamma chain; and has been shown to play a role in the generation, maintenance, and proliferation of antigen-specific CD8(+) T cells. In this study, we took the unique approach of using both a cytokine and its receptor as an adjuvant in an HIV-1 vaccine strategy. To study IL-15R alpha expression, a unique monoclonal antibody (KK1.23) was generated to confirm receptor expression in vitro. Coimmunization of IL-15 and IL-15R alpha plasmids with HIV-1 antigenic plasmids in mice enhanced the antigen-specific immune response 2-fold over IL-15 immunoadjuvant alone. Furthermore, plasmid-encoded IL-15R alpha augments immune responses in the absence of IL-15, suggesting its role as a novel adjuvant. Moreover, pIL-15R alpha enhanced the cellular, but not the humoral, immune response as measured by antigen-specific IgG antibody. This is the first report describing that IL-15R alpha itself can act as an adjuvant by enhancing an antigen-specific T cell response. Uniquely, pIL-15 and pIL-15R alpha adjuvants combined, but not the receptor alpha chain alone, may be useful as a strategy for generating and maintaining memory CD8(+) T cells in a DNA vaccine.

FIG. 1.

The generation of an interleukin-15 receptor α (IL-15Rα) monoclonal antibody. (A) Coomassie staining of the recombinant human IL-15Rα protein in 2-fold dilutions from 12.0 to 0.16 μg of protein. (B) A commercial anti-human IL-15Rα antibody can detect recombinant IL-15Rα protein in a direct ELISA. (C) Immunization schedule for anti-human IL-15Rα monoclonal antibody generation in BALB/c mice. (D) Hybridoma supernatants from clone KK1.23 have high titers of antibody against recombinant IL-15Rα protein by ELISA. (E) Purified monoclonal antibody KK1.23 binds recombinant IL-15Rα in ELISA and specifically as shown by Western blot analysis. CFA, complete Freund's adjuvant; IFA, incomplete Freund's adjuvant; IV, intravenous.

FIG. 2.

Construction and expression of the IL-15Rα DNA plasmid. (A) Human IL-15Rα cDNA was inserted into a pVAX1 expression vector. Plasmid IL-15Rα expresses the appropriate size protein (∼30 kDa) as detected by radioactive in vitro translation with the R&D (B) or the KK1.23 monoclonal (C) anti-human IL-15Rα antibody. (D) Monoclonal KK1.23 antibody (IgG1 isotype) does not bind nontransfected HeLa cells (original magnification, × 20). (E) pTRACER-IL-15Rα-transfected cells (green) stained with a mouse IgG1 isotype control (original magnification, × 20). (F and G) The KK1.23 anti-human IL-15Rα antibody (red) binds pIL-15Rα-pTRACER-transfected cells. Original magnification: (F) × 20; (G) × 60.

FIG. 3.

The combination of pIL-15 and pIL-15Rα augments immune responses compared with either plasmid delivered alone. (A) Immunization schedule for groups of BALB/c mice that were injected with DNA formulations containing combinations of vector, antigenic plasmid (HIV-1 Gag and Pol), pIL-15, and/or pIL-15Rα. The combination of pIL-15/pIL-15Rα was given either in the same leg or split between two different legs (pIL-15 in one, pIL-15Rα in another). Intramuscular immunizations were given with electroporation three times, and mice were killed 1 week after the final boost. (B and C) To determine cellular responses, splenocytes from immunized mice were used in an IFN-γ ELISpot assay. Splenocytes were stimulated overnight with medium (negative control), concanavalin A (positive control), or antigenic peptide (HIV-1 Gag and Pol pools) and IFN-γ spot-forming units were counted.

FIG. 4.

IL-15Rα DNA plasmid is immunogenic in a dose-dependent manner without pIL-15. (A) BALB/c mice were immunized as shown in Fig. 3A with DNA formulations containing combinations of vector, 5 μg of antigenic plasmid (HIV-1 Gag and Pol), and increasing doses of IL-15Rα (7.5, 10, or 15 μg). IFN-γ ELISpot was carried out on splenocytes stimulated with R10 (negative control), concanavalin A (positive control), and (A) HIV-1 Gag peptide pools or (B) HIV-1 Pol peptide pools.

FIG. 5.

pIL-15Rα augments IFN-γ secretion primarily by CD8⁺ T cells. (A) The contribution of IFN-γ secretion by CD8⁺ T cells was measured by ex vivo depletion of CD8⁺ T cells from the splenocytes of immunized mice, using Miltenyi beads. The total antigenic response is shown for whole splenocytes (solid columns) and CD8⁺ cell-depleted splenocytes (gray columns) and was measured by IFN-γ ELISpot. (B) Antibody titers against HIV-1 Gag p24 antigenic protein were determined from serum samples of BALB/c mice immunized with the same constructs and timeline as described in Materials and Methods. Dilutions of sera taken at the time of death were run on an ELISA plate coated with p24 and detected with an HRP-conjugated anti-mouse IgG antibody to measure levels of antigen-specific IgG. Background responses in diluent wells only were subtracted from the sample optical density values before graphing.

FIG. 6.

The combination of pIL-15 and pIL-15Rα does not enhance memory responses. Mice were immunized three times and rested for 30 weeks before being killed. (A) IFN-γ secretion was measured from splenocytes of immunized mice by IFN-γ ELISpot. (B) Intracellular cytokine staining was also used to determine the memory response from immunized mice. Splenocytes from immunized mice were stimulated for 5 hr with medium, PMA/ionomycin, or the dominant and subdominant HIV-1 Gag and Pol antigenic peptides in the presence of brefeldin A. (C) Sera was taken from immunized mice and run in an HIV-1 Gag p24 ELISA. Dilutions of sera were analyzed for antigen-specific IgG. Background responses in diluent wells only were subtracted from the sample optical density values before graphing.

FIG. 7.

pIL-15Rα can act as an adjuvant in the absence of endogenous IL-15. To explore the mechanism of IL-15Rα as an adjuvant, we looked at the ability of human IL-15Rα to bind murine IL-15. (A) Radiolabeled human IL-15Rα binds mouse IL-15 and is coimmunoprecipitated with anti-mouse IL-15 antibody. Immunizations were also repeated in control C57BL/6 (B) and IL-15 knockout (C) mice, according to the schedule in Fig. 3A, and IFN-γ ELISpots were performed on splenocytes.