Antigen export during liver infection of the malaria parasite augments protective immunity

Abstract

Protective immunity against preerythrocytic malaria parasite infection is difficult to achieve. Intracellular Plasmodium parasites likely minimize antigen presentation by surface-expressed major histocompatibility complex class I (MHC-I) molecules on infected cells, yet they actively remodel their host cells by export of parasite factors. Whether exported liver-stage proteins constitute better candidates for MHC-I antigen presentation to CD8(+) T lymphocytes remains unknown. Here, we systematically characterized the contribution of protein export to the magnitude of antigen-specific T-cell responses against Plasmodium berghei liver-stage parasites in C57BL/6 mice. We generated transgenic sporozoites that secrete a truncated ovalbumin (OVA) surrogate antigen only in the presence of an amino-terminal protein export element. Immunization with live attenuated transgenic sporozoites revealed that antigen export was not critical for CD8(+) T-cell priming but enhanced CD8(+) T-cell proliferation in the liver. Upon transfer of antigen-specific CD8(+) T cells, liver-stage parasites secreting the target protein were eliminated more efficiently. We conclude that Plasmodium parasites strictly control protein export during liver infection to minimize immune recognition. Strategies that enhance the discharge of parasite proteins into infected hepatocytes could improve the efficacy of candidate preerythrocytic malaria vaccines. Importance: Vaccine development against Plasmodium parasites remains a priority in malaria research. The most advanced malaria subunit vaccine candidates contain Plasmodium surface proteins with important roles for parasite vital functions. A fundamental question is whether recognition by effector CD8(+) T cells is restricted to sporozoite surface antigens or extends to parasite proteins that are synthesized during the extensive parasite expansion phase in the liver. Using a surrogate model antigen, we found that a cytoplasmic antigen is able to induce robust protective CD8(+) T-cell responses, but protein export further enhances immunogenicity and protection. Our results show that a cytoplasmic localization does not exclude a protein's candidacy for malaria subunit vaccines and that protein secretion can enhance protective immunity.

FIG 1

Transgenic OVA and expOVA Plasmodium sporozoites and liver-stage parasites express and export the surrogate antigen ovalbumin (OVA). (A) Schematic of transgenic P. berghei parasite lines that express a truncated version of the surrogate antigen OVA. The H-2^b-restricted CD8⁺ T-cell epitope (SIINFEKL) and CD4⁺ epitope (ISQAVAAHAEINEA) are highlighted in red and blue, respectively, in the schematic. Antigen expression is controlled by the preerythrocytic-stage-specific UIS4 promoter. The expOVA parasites express OVA fused to an N-terminal protein export element (PEXEL), whereas OVA parasites produce a cytoplasmic version of the antigen. (B) Indirect immunofluorescence microscopy of early liver-stage parasites (18 h after infection). (C) Quantification of fluorescence signal revealed by IFAs of infected Hepa 1-6 cells at 24 h postinjection. Bars and whiskers show means ± standard deviations. Results are representative of two independent experiments (n ≥ 8). (D, E) Late liver-stage parasites (48 h after infection) (D) and P. berghei sporozoites (E). Parasites were stained with a polyclonal anti-OVA antibody (red) and the nuclear dye Hoechst 33342 (blue). In addition, liver-stage parasites were stained with an anti-P. berghei Hsp70 antibody (green). (B, D) To visualize the liver-stage tubulovesicular network (LSTVN), early liver-stage parasites were also stained with an anti-PbUIS4 antiserum. Scale bars: 5 µm (B, D); 2 µm (E).

FIG 2

CD8⁺ T-cell responses induced by intraparasitic and exported antigen are cytolytic in vivo. (A) Schematic diagram of methodology. Mice received 3 × 10⁵ OT-1 cells and were either left untreated or immunized by i.v. injection of 10,000 irradiated wild-type (WT), expOVA, or OVA sporozoites. Six days later, target cells were prepared by pulsing syngeneic splenocytes with the SIINFEKL or no peptide prior to labeling with CFSE and transfer to mice (1 × 10⁷ pulsed cells/mouse each). After 18 h, spleens of recipient mice were harvested and analyzed by CFSE fluorescence. (B) Representative histogram plots showing the fate of target cells in naive mice (top left), mice immunized with irradiated WT sporozoites (top right), and mice immunized with expOVA (bottom left) or OVA (bottom right) sporozoites. (C) Quantification of in vivo cytolytic activity. Bars and whiskers show means ± standard deviations. *, P < 0.05 (Kruskal-Wallis test). Results are representative of one of two experiments with three mice per group per experiment.

FIG 3

Antigen export enhances CD8⁺ T-cell proliferation in vivo. C57BL/6 CD45.1 mice received OT-1 cells as indicated in panel C; the cells were obtained from CD45.2 donor mice and labeled with CFSE prior to transfer. After 18 h, mice received 10,000 irradiated WT, expOVA, or OVA sporozoites. Three days later, spleens and livers were harvested from recipient mice and cells stained with CD8, CD3, CD45.1, and CD45.2 antibodies. (A) Representative histogram plots showing the gating strategy for CD45.2⁺ CD8⁺ T cells. SSC, side scatter; FSC, forward scatter. (B) Representative histogram plots showing proliferation of hepatic CD45.2⁺ CD8⁺ T cells from mice immunized with WT (left), expOVA (center), or OVA (right) sporozoites. (C) T-cell proliferation from livers and spleens of sporozoite-immunized mice (n = 3 each). Shown is the percentage of the CFSE population of OT-1 CD8⁺ CD45.2 T cells originating from the liver (left) or spleen (right). The amount of cells transferred is indicated. *, P < 0.05 (Kruskal-Wallis test).

FIG 4

IFN-γ production in CD8⁺ T cells recognizing OVA and Plasmodium berghei sporozoite-derived antigens. (A) Mice were immunized weekly with two doses of 10,000 irradiated P. berghei sporozoites. Seven days after the last immunization, cells from livers and spleens were tested for their capacity to produce IFN-γ after restimulation in vitro with SIINFEKL peptide. As controls, splenic and hepatic cells were left unstimulated or stimulated with a sporozoite-specific peptide, TRAP_130–138 (PbTrap₁₃₀). Representative flow cytometry plots show IFN-γ production by CD8⁺ T cells in spleens and livers of mice. (B) Percentages of IFN-γ⁺ CD8⁺ T cells are shown as means ± standard deviations. Results are representative of two independent experiments (n = 3 or 4 mice). *, P < 0.05; **, P < 0.01 (Mann-Whitney test).

FIG 5

Immunization with expOVA sporozoites enhances protection against reinfection. (A) Quantification of parasite liver loads in immunized mice that received OT-1 or OT-2 cells. C57BL/6 mice received 2 × 10⁵ OT-I (CD8) T cells (OT-1) or OT-II (CD4) T cells (OT-2). Next, mice were immunized once with 10,000 irradiated WT (black), expOVA (red), or OVA (green) sporozoites. Control mice were immunized once without prior T-cell transfer. Twelve days after the last immunization, animals were challenged by i.v. injection of 10,000 sporozoites of the corresponding genotype. After 42 h, livers were removed and parasite loads were quantified by real-time PCR. Bars and whiskers show means ± standard deviations. *, P < 0.05; **, P < 0.01 (Mann-Whitney test).