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. 2018 Apr 10;8(1):5781.
doi: 10.1038/s41598-018-23728-1.

Cross-species analysis of apical asparagine-rich protein of Plasmodium vivax and Plasmodium knowlesi

Affiliations

Cross-species analysis of apical asparagine-rich protein of Plasmodium vivax and Plasmodium knowlesi

Fauzi Muh et al. Sci Rep. .

Abstract

The Plasmodium falciparum apical asparagine (Asn)-rich protein (AARP) is one of malarial proteins, and it has been studied as a candidate of malaria subunit vaccine. Basic characterization of PvAARP has been performed with a focus on its immunogenicity and localization. In this study, we further analyzed the immunogenicity of PvAARP, focusing on the longevity of the antibody response, cross-species immunity and invasion inhibitory activity by using the primate malaria parasite Plasmodium knowlesi. We found that vivax malaria patient sera retained anti-PvAARP antibodies for at least one year without re-infection. Recombinant PvAARP protein was strongly recognized by knowlesi malaria patients. Antibody raised against the P. vivax and P. knowlesi AARP N-termini reacted with the apical side of the P. knowlesi merozoites and inhibited erythrocyte invasion by P. knowlesi in a concentration-dependent manner, thereby suggesting a cross-species nature of anti-PvAARP antibody against PkAARP. These results can be explained by B cell epitopes predicted in conserved surface-exposed regions of the AARP N-terminus in both species. The long-lived anti-PvAARP antibody response, cross-reactivity, and invasion inhibitory activity of anti-PvAARP support a critical role of AARP during the erythrocyte invasion and suggest that PvAARP induces long-lived cross-species protective immunity against P. vivax and P. knowlesi.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic structure, B-cell epitope prediction and sequence diversity of PvAARP and PkAARP. (a) Schematic structures of PvAARP (285 amino acids [aa]) and PkAARP (239 aa). Asparagine (Asn)- and proline (Pro)-rich regions are indicated in yellow and blue, respectively. Regions used to generate PvAARP N-terminus (rPvAARP-N, aa 21−112), PkAARP N-terminus (rPkAARP-N, aa 21−112) and full-length PvAARP (rPvAARP-FL) and PkAARP (rPkAARP-FL) recombinant proteins are indicated under each structure. Four predicted B-cell epitopes (#1−#4) are highlighted with arrows and aa sequences. (b and c) SP, signal peptide. B-cell epitope prediction of PvAARP-N (b) and PkAARP-N (c). Yellow areas above the threshold (red line) were predicted to be part of the B-cell epitope, and green areas were unlikely to be a part of the B-cell epitope. (d) Sliding window plot of the nucleotide diversity of the pkaarp gene encoding the N-terminus using 29 pkaarp sequences with a window size of 60 and a step size of 3.
Figure 2
Figure 2
SDS-PAGE and western blot analysis of PvAARP and PkAARP. (a) Crude recombinant proteins of full-length PvAARP and PkAARP [rPvAARP-FL and rPkAARP-FL] were expressed with the WGCF expression system. (b) Recombinant PvAARP-N [Pv, 0.5 μg] and PkAARP-N [Pk, 0.5 μg] proteins were expressed in E. coli and purified to a single band [arrowhead]. (c) Specific band have been detected with anti-glutathione S-transferase antibody [Gst], mouse serum [M] and rabbit immunized [R] with PvAARP-N or PkAARP-N. (d) P. knowlesi A1-H.1 parasite lysate was recognized with anti-PvAARP-N [α-Pv] and anti-PkAARP-N [α-Pk] antibody, nor non-immunized rabbit (α-NI). The full length of SDS-PAGE and western blots are presented in the Supplementary Fig. S2. (e) Immunofluorescence assay of anti-PvAARP-N and PkAARP-N with P. knowlesi A1-H.1. pRBC, parasitized-red blood cells; uRBC, uninfected-red blood cells; DAPI, 4’,6-diaminidino-2-phenylindole. Bars indicate 5 μm.
Figure 3
Figure 3
(a) IgG responses of pooled vivax patient serum [V], pooled knowlesi patient serum [K] or pooled healthy individual serum [H] to the full-length PvAARP-FL [rPvAARP-FL] or PkAARP [rPkAARP-FL] recombinant proteins. (b) IgG responses of 32 sets of archived vivax malaria patient sera to rPvAARP-FL. The IgG response is represented by normalized mean fluorescence intensity [MFI]: MFI of the test sample/[MFI + 2 standard deviations].
Figure 4
Figure 4
Invasion inhibition activity of anti-PvAARP-N and anti-PkAARP-N against P. knowlesi A1-H.1 into human erythrocytes. Purified IgG [0.25, 0.5, 1.0, 2.0 and 4.0 mg/mL] from rPvAARP-N and rPkAARP-N-immunized, non-immunized [NI], and His-GST-immunized rabbit, as well as anti-DARC [2C3] monoclonal antibodies [25 μg/mL] were examined for their inhibitory activity against erythrocyte invasion by P. knowlesi A1-H.1 into human erythrocytes. ns, not significant different. p > 0.05; single asterisk, p < 0.05; double asterisks, p < 0.01; triple asterisks, p < 0.001.

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