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. 2020 Jun 19;14(6):e0008323.
doi: 10.1371/journal.pntd.0008323. eCollection 2020 Jun.

Cross-species reactivity of antibodies against Plasmodium vivax blood-stage antigens to Plasmodium knowlesi

Fauzi Muh  1 , Namhyeok Kim  1 , Myat Htut Nyunt  2 , Egy Rahman Firdaus  1 , Jin-Hee Han  1 , Mohammad Rafiul Hoque  1 , Seong-Kyun Lee  1 , Ji-Hoon Park  1 , Robert W Moon  3 , Yee Ling Lau  4 , Osamu Kaneko  5 , Eun-Taek Han  1
Affiliations

Cross-species reactivity of antibodies against Plasmodium vivax blood-stage antigens to Plasmodium knowlesi

Fauzi Muh et al. PLoS Negl Trop Dis. .

Abstract

Malaria is caused by multiple different species of protozoan parasites, and interventions in the pre-elimination phase can lead to drastic changes in the proportion of each species causing malaria. In endemic areas, cross-reactivity may play an important role in the protection and blocking transmission. Thus, successful control of one species could lead to an increase in other parasite species. A few studies have reported cross-reactivity producing cross-immunity, but the extent of cross-reactive, particularly between closely related species, is poorly understood. P. vivax and P. knowlesi are particularly closely related species causing malaria infections in SE Asia, and whilst P. vivax cases are in decline, zoonotic P. knowlesi infections are rising in some areas. In this study, the cross-species reactivity and growth inhibition activity of P. vivax blood-stage antigen-specific antibodies against P. knowlesi parasites were investigated. Bioinformatics analysis, immunofluorescence assay, western blotting, protein microarray, and growth inhibition assay were performed to investigate the cross-reactivity. P. vivax blood-stage antigen-specific antibodies recognized the molecules located on the surface or released from apical organelles of P. knowlesi merozoites. Recombinant P. vivax and P. knowlesi proteins were also recognized by P. knowlesi- and P. vivax-infected patient antibodies, respectively. Immunoglobulin G against P. vivax antigens from both immune animals and human malaria patients inhibited the erythrocyte invasion by P. knowlesi. This study demonstrates that there is extensive cross-reactivity between antibodies against P. vivax to P. knowlesi in the blood stage, and these antibodies can potently inhibit in vitro invasion, highlighting the potential cross-protective immunity in endemic areas.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Cross-species reactivity of antibodies against P. vivax merozoite surface proteins to P. knowlesi parasites by immunofluorescence assay.
Localization of a panel of antibodies for P. vivax surface proteins (green) in P. knowlesi co-stained with anti-PkMSP1-19 (red) as a surface marker. DIC, differential interference contrast; DAPI, 4’,6-diaminidino-2-phenylindole (blue). All parasites shown are segmented schizonts (24–28 hours post-invasion). Bars indicate 5 μm.
Fig 2
Fig 2. Cross-species reactivity of antibodies against P. vivax apical organelles proteins to P. knowlesi parasites by immunofluorescence assay.
(A) Localization of a panel of antibodies for P. vivax micronemal proteins (green) in P. knowlesi co-stained with anti-PkDBPα (red) as a microneme marker and DAPI as nuclear marker (blue). (B) Localization of a panel of antibodies for P. vivax rhoptry proteins (green) in P. knowlesi co-stained with anti-PkRhopH2 (red) as a rhoptry marker and DAPI as nuclear marker (blue). All parasites shown are segmented schizonts (24–28 hours post-invasion). Bars indicate 5 μm.
Fig 3
Fig 3. Cross-species reactivity of antibodies against P. vivax parasitophorous vacuole membrane molecules and negative control antibodies to P. knowlesi parasites by immunofluorescence assay.
(A) Localization of antibodies for P. vivax parasitophorous vacuole proteins (green) in P. knowlesi co-stained with anti-PkMSP1-19 as a merozoite surface marker. (B) Localization of PBS-immunized (NI) and HisGST antibodies to P. knowlesi. All parasites shown are segmented schizonts (24–28 hours post-invasion). Bars indicate 5 μm.
Fig 4
Fig 4. Cross-species activity of antibodies against P. vivax antigens to inhibit erythrocyte invasion by P. knowlesi parasites.
Graph showing inhibition activity (%) of antibodies against P. vivax antigens to erythrocyte invasion by P. knowlesi A1-H.1 (2 mg/mL rabbit IgG). PkDBPα, PkAMA1 rabbit polyclonal IgG, and 2C3 monoclonal antibody served as control. NI, PBS-immunized rabbit IgG; DG, dense granules; 2C3, Anti-Fy6 monoclonal antibody (25 μg/mL). Graphs show the mean and error bars denote ±1 SD of duplicate test wells in two independent experiments by using one-way ANOVA with Dunnett’s multiple comparison test of means of antibody inhibition rate with mean of control anti-HisGST. ns, no significant difference, p>0.05.
Fig 5
Fig 5. Growth inhibition activity of antigen-specific IgG from P. vivax-infected patients to P. knowlesi parasites.
(A) Different migration on SDS-PAGE of reduced and non-reduced recombinant proteins immobilized on agarose beads. Proteins were successfully refolded as shown with Coomassie Brilliant Blue in different migration patterns with and without DTT treatment. BSA was served as control. Proteins were then used for immobilization with CNBr-bead for antigen-specific antibody purification from P. vivax-infected patients serum. (B) Western blot analysis of recombinant PvRBP1a, PvRhopH2, and Pv41 proteins with anti-His-tag antibody. (C) Growth inhibition activity of IgGs specific to P. vivax antigens to P. knowlesi. Different concentration of antigen-specific antibodies from human(Pv41, PvRhopH2, PvRBP1a) ranging from 0.1, 0.2 and 0.5 mg/mL were used. IC50 of Pv41 antigen-specific human antibodies was higher than PvRhopH2 antigen-specific human antibodies. αNaive indicates IgG purified healthy individual who have never experienced malaria infection.
Fig 6
Fig 6. Cross-species reactivity of sera from P. knowlesi- and P. vivax-malaria clinical patients against P. vivax and/or P. knowlesi proteins.
(A) IgG responses of pooled P. vivax-infected patient serum from the Republic of Korea (ROK) and knowlesi-infected patient serum (Malaysia) with P. vivax recombinant proteins after subtraction with pooled healthy serum tier in 1:25 dilution. (B) IgG responses of pooled P. vivax-infected patient serum from the Republic of Korea (ROK) and P. knowlesi-infected patient serum (Malaysia) with P. vivax recombinant proteins.
Fig 7
Fig 7. P. knowlesi blood-stage cross-reactivity with individual patient serum.
The human IgG response of PkMSP1-42, PkMSA180-N, and PkAMA1. Individual with outlier reactivity was indicated in black dot. The prevalence of antibody response was compared to the patients (K, knowlesi; V, vivax) and healthy (H) using the Mann-Whitney test. *** = p<0.001.
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