Immune responses against a single CD8+-T-cell epitope induced by virus vector vaccination can successfully control Trypanosoma cruzi infection

Abstract

In order to develop CD8+-T-cell-mediated immunotherapy against intracellular infectious agents, vaccination using recombinant virus vectors has become a promising strategy. In this study, we generated recombinant adenoviral and vaccinia virus vectors expressing a single CD8+-T-cell epitope, ANYNFTLV, which is derived from a Trypanosoma cruzi antigen. Immunogenicity of these two recombinant virus vectors was confirmed by the detection of ANYNFTLV-specific CD8+ T cells in the spleens of immunized mice. Priming/boosting immunization using combinations of these two recombinant virus vectors revealed that the adenovirus vector was efficient for priming and the vaccinia virus vector was effective for boosting the CD8+-T-cell responses. Moreover, we also demonstrated that the ANYNFTLV-specific CD8+-T-cell responses were further augmented by coadministration of recombinant vaccinia virus vector expressing the receptor activator of NFkappaB (RANK) ligand as an adjuvant. By priming with the adenovirus vector expressing ANYNFTLV and boosting with the vaccinia virus vectors expressing ANYNFTLV and RANK ligand, the immunized mice were efficiently protected from subsequent challenge with lethal doses of T. cruzi. These results indicated, for the first time, that the induction of immune responses against a single CD8+-T-cell epitope derived from an intrinsic T. cruzi antigen was sufficient to control lethal T. cruzi infection.

FIG. 1.

Generation of recombinant virus vectors. A. Primary structure of T. cruzi trans-sialidase surface antigen (TSSA) and an H-2K^b-restricted CD8⁺-T-cell epitope, ANYNFTLV. The gene encoding for TSSA was first identified to be present among the clusters of genes encoding for enzymes involved in de novo pyrimidine biosynthesis in the genome of T. cruzi Tulahuen strain (11). The report (11) showed a schematic representation of the 25 kb segment containing not only five genes that encode all six enzymes of de novo pyrimidine biosynthesis but also an additional gene, orf, which was described only as surface protein of T. cruzi (DNA accession number: AB010287). We designated the surface protein as TSSA, since its amino acid sequence was highly homologous to the T. cruzi trans-sialidase superfamily protein. The T. cruzi trans-sialidase usually consists of four parts; i.e., signal peptide, catalytic domain, C-terminal repeats, and hydrophobic region for GPI anchor. TSSA, however, consists of only two parts, signal peptide and catalytic domain. An H-2K^b-restricted CD8⁺-T-cell epitope, ANYNFTLV (536-543), was identified on TSSA (19). B. A minigene encoding the MANYNFTLV peptide was inserted either into pAxCAwt, a transfer vector for replication-deficient adenovirus, or into pMCO3, a transfer vector for highly attenuated vaccinia virus (MVA). Detailed procedures for generating recombinant viruses are described in the Materials and Methods. CAG, modified chicken β-actin promoter with the cytomegalovirus immediate-early enhancer (32); poly A, poly(A) addition signal; IRES, internal ribosome entry site; EGFP, enhanced green fluorescent protein; E/L, synthetic early/late MVA promoter; 7.5, MVA P7.5 promoter; GUS, gene encoding Escherichia coli β-glucuronidase.

FIG. 2.

Immunogenicity of recombinant adenovirus expressing ANYNFTLV. C57BL/6 mice were administered with either Ad-MANY or Ad-GFP intramuscularly at three different doses (A, B) or intraperitoneally at two different doses (C, D). The mice were sacrificed 14 days after the immunization, and their spleens were removed. A half of splenocytes from individual mice were cultured with irradiated EL-4 cells pulsed with ANYNFTLV peptide for one week. The freshly isolated splenocytes (A, C) or the 1-week cultured splenocytes (B, D) were subjected to the ELISPOT assay for IFN-γ-producing cells in response to ANYNFTLV peptide-pulsed EL-4 cells. The number of IFN-γ-secreting cells/10⁶ cells was counted 24 h later. The number of IFN-γ-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-γ-secreting cells that appeared against peptide-pulsed EL-4. Data represent the mean ± standard deviation of three mice in each group. The data are representative one of three independent experiments.

FIG. 3.

Immunogenicity of recombinant MVA expressing ANYNFTLV. C57BL/6 mice were administered intramuscularly (A, B) or intraperitoneally (C, D) with two different doses of either MVA-MANY or MVA-p3. The mice were sacrificed 11 days after the immunization, and their spleens were removed. A half of splenocytes from individual mice were cultured with irradiated EL-4 cells pulsed with ANYNFTLV peptide for 1 week. The freshly isolated splenocytes (A, C) or the one-week cultured splenocytes (B, D) were subjected to the ELISPOT assay for IFN-γ-producing cells in response to ANYNFTLV peptide-pulsed EL-4 cells. The number of IFN-γ-secreting cells/10⁶ cells was counted 24 h later. The number of IFN-γ-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-γ-secreting cells that appeared against peptide-pulsed EL-4. Data represent the mean ± standard deviation of three mice in each group. The data are representative one of three independent experiments.

FIG. 4.

Immune responses induced by prime/boost immunization using Ad-MANY and MVA-MANY. A. C57BL/6 mice were primed intramuscularly with 5 × 10⁷ PFU of Ad-MANY. Twelve days later, the mice were boosted intramuscularly with 5 × 10⁷ PFU of Ad-MANY or Ad-GFP, or intraperitoneally with 5 × 10⁷ PFU of MVA-MANY or MVA-p3. The mice were sacrificed 14 days after the boost immunization, and their spleens were removed. The freshly isolated splenocytes were subjected to the ELISPOT assay for IFN-γ-producing cells in response to ANYNFTLV peptide-pulsed EL-4 cells. The number of IFN-γ-secreting cells× 10⁶ cells was counted 24 h later. The number of IFN-γ-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-γ-secreting cells that appeared against peptide-pulsed EL-4. Data represent the mean ± standard deviation of three mice in each group. B. Some groups of the prime/boosted mice (n = 4) were infected intramuscular with 10,000 T. cruzi blood-form trypomastigotes at 10 days after the boost immunization. The number of parasites in 5 μl of peripheral blood (parasitemia) was counted at 11, 14, and 20 days postinfection. *, P < 0.05 compared to unimmunized mice by the unpaired Student's t test. Statistical analysis at 20 days postinfection was not achievable due to the death of all unimmunized mice. C. Survival was monitored daily. The survival of Ad-MANY/MVA-MANY-immunized mice was significantly different (P < 0.05 by the unpaired Mann-Whitney U test) from that of unimmunized mice. The data are representative one of two independent experiments.

FIG. 5.

Induction of ANYNFTLV-specific CD8⁺ T cells by prime/boost immunization with high doses of Ad-MANY and MVA-MANY. C57BL/6 mice (n = 3) were first primed with different doses (5 × 10⁸, 5 × 10⁷, or 5 × 10⁶ PFU) of Ad-MANY or 5 × 10⁸ PFU of Ad-GFP, and boosted 14 days later with different doses (5 × 10⁸, 5 × 10⁷, or 5 × 10⁶ PFU) of MVA-MANY or 5 × 10⁸ PFU of MVA-p3. The mice were sacrificed 10 days after the boost immunization, and their spleens were removed. The freshly isolated splenocytes were subjected to the ELISPOT assay for IFN-γ-producing cells in response to ANYNFTLV peptide-pulsed EL-4 cells. The number of IFN-γ-secreting cells× 10⁶ cells was counted 24 h later. The number of IFN-γ-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-γ-secreting cells that appeared against peptide-pulsed EL-4. Data represent the mean ± standard deviation of three mice in each group. *, P < 0.05 determined by the Dunnett's two-tailed t test. The data are representative one of three independent experiments.

FIG. 6.

Adjuvant effect of recombinant MVA expressing murine RANKL. (A) BHK-21 cells were infected with either MVA-RANKL or MVA-p3 and then were stained with biotinylated anti-RANKL monoclonal antibody followed by PE-labeled streptavidin. The bold histograms indicate the staining with anti-RANKL monoclonal antibody and the thin histograms indicate the staining with isotype-matched control rat immunoglobulin G. (B) C57BL/6 mice (n = 3) were first primed with 5 × 10⁸ PFU of Ad-MANY and then boosted 11 days later with 5 × 10⁸ PFU of MVA-MANY or MVA-p3 and 5 × 10⁷ PFU of MVA-RANKL or MVA-p3. The mice were sacrificed 10 days after the boost immunization, and their spleens were removed. The freshly isolated splenocytes were subjected to the ELISPOT assay for IFN-γ-producing cells in response to ANYNFTLV peptide-pulsed EL-4 cells. The number of IFN-γ-secreting cells/10⁶ cells was counted 24 h later. The number of IFN-γ-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-γ-secreting cells that appeared against peptide-pulsed EL-4. Data represent the mean ± standard deviation of three mice in each group. *, P < 0.05 by the Dunnett's two-tailed t test. NS, not significantly different. The data are representative one of two independent experiments.

FIG. 7.

Prime/boost immunization with Ad-MANY/MVA-MANY + MVA-RANKL can control lethal T. cruzi infection. C57BL/6 mice (n = 4) were first primed with 5 × 10⁸ PFU of Ad-MANY or Ad-GFP, and then boosted with 5 × 10⁸ PFU of MVA-MANY or MVA-p3 and 5 × 10⁷ PFU of MVA-RANKL or MVA-p3 11 days later. The mice were infected intraperitoneally with 10,000 (A, B) or 2,000 (C, D) Tulahuen strain of T. cruzi blood-form trypomastigotes 14 days after the boost immunization. The number of parasites in 5 μl of peripheral blood (parasitemia) was counted periodically, and the data represent the mean ± standard deviation of four mice in each group (A, C). Survival was monitored daily (B, D). *, P < 0.05 compared to the unimmunized mice by the Dunnett's two-tailed t test (A, C). The longer survival of Ad-MANY/MVA-MANY + MVA-RANKL group was significantly different (P < 0.05 by the unpaired Mann-Whitney U test) from that of Ad-GFP/MVA-p3 plus MVA-p3 group of mice (B). The survival of other groups was not significantly different from that of the Ad-GFP/MVA-p3 plus MVA-p3 group of mice (B, D).