Plasmodium berghei circumvents immune responses induced by merozoite surface protein 1- and apical membrane antigen 1-based vaccines

Abstract

Background: Two current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP1(19)) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic.

Methodology and results: In this study, we assessed the protective efficacies of a series of MSP1(19)- and AMA1-based vaccines using the P. berghei rodent malarial parasite and its transgenic models. Immunization of mice with a baculoviral-based vaccine (BBV) expressing P. falciparum MSP1(19) induced high titers of PfMSP1(19)-specific antibodies that strongly reacted with P. falciparum blood-stage parasites. However, no protection was achieved following lethal challenge with transgenic P. berghei expressing PfMSP1(19) in place of native PbMSP1(19). Similarly, neither P. berghei MSP1(19)- nor AMA1-BBV was effective against P. berghei. In contrast, immunization with P. yoelii MSP1(19)- and AMA1-BBVs provided 100% and 40% protection, respectively, against P. yoelii lethal challenge. Mice that naturally acquired sterile immunity against P. berghei became cross-resistant to P. yoelii, but not vice versa.

Conclusion: This is the first study to address blood-stage vaccine efficacies using both P. berghei and P. yoelii models at the same time. P. berghei completely circumvents immune responses induced by MSP1(19)- and AMA1-based vaccines, suggesting that P. berghei possesses additional molecules and/or mechanisms that circumvent the host's immune responses to MSP1(19) and AMA1, which are lacking in P. yoelii. Although it is not known whether P. falciparum shares these escape mechanisms with P. berghei, P. berghei and its transgenic models may have potential as useful tools for identifying and evaluating new blood-stage vaccine candidate antigens for P. falciparum.

Figure 1. Construction and expression analysis of MSP1₁₉-BBVs.

(A) Schematic diagram of three MSP1₁₉-BBV genomes. MSP1₁₉ was expressed as a MSP1₁₉-gp64 fusion protein under the control of the polyhedron promoter. Numbers indicate the amino acid positions of MSP1₁₉-gp64 fusion protein and endogenous gp64 protein. pPolh, polyhedrin promoter; SP, the gp64 signal sequence; FLAG, the FLAG epitope tag; pgp64, gp64 promoter. (B) Western blot analysis of MSP1₁₉-BBVs. AcNPV-PfMSP1₁₉surf (lanes 1, 2, 3 and 10), AcNPV-PbMSP1₁₉surf (lanes 4, 5, 6 and 11) and AcNPV-PyMSP1₁₉surf (lanes 7, 8, 9 and 12) were treated with the loading buffer with 5% 2-ME (lanes 1, 4, 7, 10, 11 and 12), 0.5% 2-ME (lanes 2, 5 and 8) or without 2-ME (lanes 3, 6 and 9) and examined using the 5.2 mAb (lanes 1–3), P. berghei-hyperimmune serum (lanes 4–6), P. yoelii-hyperimmune serum (lanes 7–9) and anti-gp64 mAb (lanes 10–12). Positions of MSP1₁₉-gp64 fusion protein and endogenous gp64 are shown at the right panel of lanes 10–12. (C–H) Immunofluorescence patterns of sera obtained from mice immunized with three MSP1₁₉-BBVs on paraformaldehyde fixed erythrocyte smears infected with P. falciparum (C–D), P. berghei (E–F) and P. yoelii (G–H). The smears were incubated with serum obtained from an individual mouse immunized either with AcNPV-PfMSP1₁₉surf (C), AcNPV-PbMSP1₁₉surf (E) or AcNPV-PyMSP1₁₉surf (G), and antibody binding was detected with secondary FITC-labeled antibody. Cell nuclei were visualized by DAPI staining on the corresponding smears (D, F and H). Scale bar, 10 µm.

Figure 2. Kinetics of PfMSP1₁₉-specific antibody titers and parasitemia during the course of infection.

Groups of mice were non-immunized or immunized either i.m. or i.n. with AcNPV-PfMSP1₁₉surf, and then challenged i.v. with 10³ Pb-PfM19 pRBC. Parasitemia was monitored daily 4 days after challenge and sera were collected periodically post-challenge to measure antibody titers. The bar chart indicates PfMSP1₁₉-specific antibody titers on the left vertical axis. The line graph indicates the course of parasitemia (%) on the right vertical axis. (+), death.

Figure 3. Construction and expression analysis of AMA1-BBVs.

(A) Schematic diagram of four AMA1-BBV genomes. AMA1 was expressed as an AMA1-gp64 fusion protein under the control of the polyhedron promoter. Numbers indicate the amino acid positions of AMA1-gp64 fusion protein and endogenous gp64 protein. pPolh, polyhedrin promoter; SP, the gp64 signal sequence; FLAG, the FLAG epitope tag; pgp64, gp64 promoter. (B) Western blot analysis of AMA1-BBVs. AcNPV-PyAMA1-D123surf (lane 1), AcNPV-PyAMA1-D3surf (lane 2), AcNPV-PbAMA1-D123surf (lane 3) and AcNPV-PbAMA1-D3surf (lane 4) were treated with the loading buffer containing 1% 2-ME and examined using P. yoelii-hyperimmune serum (lanes 1 and 2), or P. berghei-hyperimmune serum (lanes 3 and 4). (C–J) Immunofluorescence patterns of sera obtained from mice immunized with four AMA1-BBVs on methanol-acetone fixed smears of erythrocytes infected with P. yoelii (C and E) and P. berghei (G and I). The smears were incubated with serum obtained from an individual mouse immunized either with AcNPV-PyAMA1-D123surf (C), AcNPV-PyAMA1-D3surf (E), AcNPV-PbAMA1-D123surf (G) or AcNPV-PbAMA1-D3surf (I), and antibody binding was detected with a secondary FITC-labeled antibody. Cell nuclei were visualized by DAPI staining on the corresponding smears (D, F, H and J). Scale bar, 10 µm.