Persistence of protective immunity to malaria induced by DNA priming and poxvirus boosting: characterization of effector and memory CD8(+)-T-cell populations

Abstract

The persistence of immunity to malaria induced in mice by a heterologous DNA priming and poxvirus boosting regimen was characterized. Mice were immunized by priming with DNA vaccine plasmids encoding the Plasmodium yoelii circumsporozoite protein (PyCSP) and murine granulocyte-macrophage colony-stimulating factor and boosting with recombinant vaccinia encoding PyCSP. BALB/c mice immunized with either high-dose (100 microg of p PyCSP plus 30 microg of pGM-CSF) or low-dose (1 microg of p PyCSP plus 1 microg of pGM-CSF DNA) priming were protected against challenge with 50 P. yoelii sporozoites. Protection 2 weeks after immunization was 70 to 100%, persisted at this level for at least 20 weeks, and declined to 30 to 40% by 28 weeks. Eight of eight mice protected at 20 weeks were still protected when rechallenged at 40 weeks. The antigen (Ag)-specific effector CD8(+)-T-cell population present 2 weeks after boosting had ex vivo Ag-specific cytolytic activity, expressed both gamma interferon (IFN-gamma) and tumor necrosis factor alpha, and constituted 12 to 20% of splenic CD8(+) T cells. In contrast, the memory CD8(+)-Ag-specific-cell population at 28 weeks lacked cytolytic activity and constituted only 6% of splenic CD8(+) T cells, but at the single-cell level it produced significantly higher levels of IFN-gamma than the effectors. High levels of Ag- or parasite-specific antibodies present 2 weeks after boosting had declined three- to sevenfold by 28 weeks. Low-dose priming was similarly immunogenic and as protective as high-dose priming against a 50-, but not a 250-, sporozoite challenge. These results demonstrate that a heterologous priming and boosting vaccination can provide lasting protection against malaria in this model system.

FIG. 1.

Duration of protection. Mice were immunized and challenged as described in Materials and Methods. Shown is the percentage of sterile protection observed in groups of 10 mice challenged at the indicated times after vaccination with the DG-V (high-dose DNA) (•) or dg-V (low-dose DNA) (○) regimen. Nonimmunized controls (□) are also shown. Error bars represent 95% confidence intervals around the proportions of protected mice.

FIG. 2.

CTL responses. (a) Restimulated CTL assay. Spleen cells from 3 mice from either the high (DG-V)- or low (dg-V)-dose DNA group obtained at 2 or 28 weeks postboost were restimulated in vitro for 7 days with peptide PyCSP(280-295) and used as effectors in a chromium release CTL assay with peptide PyCSP(280-288)-labeled targets as described in Materials and Methods. The percent lysis was determined according to the following formula: (experimental release − medium control release)/(maximum release − medium control release) × 100. The percent specific lysis was calculated by subtracting the percent lysis of the control targets from the percent lysis of the specific peptide. Three mice per group were assayed individually, and data represent the means and standard deviations of percent specific lysis at the indicated effector-to-target-cell (E:T) ratios. (b) Ex vivo CTL assay. Freshly isolated spleen cells from 10 mice per group obtained at 2 or 30 weeks postboost were assayed individually and used as effectors as described in Materials and Methods. Data represent the means and standard deviations of percent specific lysis at the indicated effector-to-target-cell ratios.

FIG. 3.

Phenotype of IFN-γ-producing cells. Spleen cells from 3 mice per group obtained at 2 or 28 weeks postboost were cultured with PyCSP(280-288) peptide-pulsed P815 cells for 16 h, stained with antibodies to CD8, C62L, CD45RB, CD69, and CD44, and stained intracellularly with antibodies to IFN-γ. The phenotypes of IFN-γ-producing cells on gated CD8⁺ T cells were determined individually for each of the 3 mice per group. Representative data from one mouse are shown. PE, phycoerythrin; FITC, fluorescein isothiocyanate.

FIG. 4.

Antibody responses. Shown are titers for EIA with recombinant PyCSP or synthetic peptide (QGPGAP)₄ or IFA against air-dried P. yoelii sporozoites as described in Materials and Methods. For the EIAs, results are reported as 0.5 optical density units (the reciprocal of the serum dilution at which the mean optical density reading is 0.5); for IFA, results are reported as the endpoint of the serum dilution at which immunofluorescence could be detected. Geometric mean titers and standard deviations from groups of 10 mice are shown.