Table 1.
Characterization of P. vivax blood-stage antigen-specific antibodies, colocalization and the degree of similarity of homologous domains.
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.
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.
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.
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.
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.
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.
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.
Table 2.
Seropositivity of IgG responses to top 3 antigens in P. knowlesi and P. vivax malaria patients and healthy individuals.