Table 1.
Plasmodium vivax vaccine candidates.
Fig 1.
Anti-P. vivax polyclonal antibodies recognise proteins in P. knowlesi schizont protein lysates.
Protein extracts from enriched schizont-stage P. knowlesi cultures were separated on SDS-PAGE gels, transferred to nitrocellulose and probed with anti-P. vivax polyclonal antibodies. Obtained size corresponding to the major bands seen with each antibody is indicated, along with the expected size of the corresponding protein in P. knowlesi (upper row) and its orthologue in P. vivax (middle row) in kiloDaltons (kDa). The arrows indicate the major bands for PvMSP3.10, PVMSP7.1 and PvCyRPA. Note that the expected size of PkMSP7.1 of 39kDa is an average of the molecular weight of four PkMSP7 like proteins (i.e. PKNH_1265900, PKNH_1266000, PKNH_1266100, PKNH_1266300) that range from 32–46 kDa.
Fig 2.
Immunolocalisation of P. knowlesi proteins using polyclonal anti-P. vivax antibodies.
Antibodies raised against P. vivax vaccine candidates were used in immunofluorescence assays using enriched schizont-stage P. knowlesi parasites. Rabbit polyclonals were detected using Alexa 555-labeled anti-rabbit secondary antibody, and compared with parasite nuclei as detected with Hoechst 33342. Merge is an overlay of Alexa 555 and Hoeschst. Scale bar is 2 micrometers.
Fig 3.
Colocalisation of P. knowlesi homologs of P. vivax vaccine candidates with antibodies to proteins of known cellular location.
P. knowlesi proteins were localised using rabbit polyclonal antibodies raised against P. vivax vaccine candidates and co-stained with rat antibodies against A) anti-PkMSP1 or B) anti-PfAMA1 for colocalization (origin of antibodies is described in Methods). Alexa Fluor 555 goat-anti rabbit and Alexa Fluor 488 goat-anti rat were used as secondary antibodies, and parasite DNA localised using Hoechst 33342. The first Merge column is an overlay of Alexa 555 and Alexa488 staining, while the second Merge column is an overlay of Alexa 555, Alexa488 and Hoeschst. Scale bar is 2 micrometers.
Fig 4.
Dose-dependent inhibition of wild-type P. knowlesi by polyclonal antibodies to P. vivax vaccine candidates.
P. knowlesi parasites were grown in the presence of a dilution series of anti-P. vivax antibodies, with invasion events into fluorescently labelled red blood cells quantified using dual colour flow cytomtery (method summarised in S4 Fig). The effect of antibodies was calculated as a percentage, comparing invasion in the presence of antibodies to invasion in the absence of antibodies. Heparin and total IgG from unimmunised rabbits were used as positive and negative controls respectively. The data presented includes observations from two independent experiments, each of which had three replicate wells for each condition/antibody concentration. To determine if the inhibition was statistically significant, all treatments were independently compared to the effect of the same concentration of non-immune rabbit IgG using Mann-Whitney-Wilcoxon test: *p = 0.05–0.01; **p<0.01.
Fig 5.
Gene editing can delete PkP41 and PkGAMA in P. knowlesi.
Pkp41 and PkGAMA were replaced with eGFP to generate Pk41KO and PkGAMAKO strains respectively, as described in the Methods. A) Flow cytometry to establish eGFP expression in knockout lines. Enriched knock out and wild-type P. knowlesi cultures at late stages were labelled with SYBR green in 1X PBS and incubated for one hour after which they were quantified by flow cytometry. Events were gated as both SYBR green and GFP negative (lower left—uninfected RBCs), SYBR green only positive (lower right—RBCs infected with parasites not expressing GFP) and both SYBR green and GFP positive (upper right—RBCs infected with parasites not expressing GFP). Both KO lines expressed GFP, confirming integration of the KO cassette. B) eGFP expression in knock-out P. knowlesi strains as compared to wild-type P. knowlesi, imaged using fluorescence microscopy. Parasite nuclei were stained using Hoechst 33342; Merge is an overlay of eGFP and Hoeschst, confirming parasite expression of GFP. Scale bar is 2 micrometers. C) Proteins in knock out and wild-type P. knowlesi strains were localised using P. vivax polyclonal antibodies and Alexa Fluor 555 labeled secondary antibody then imaged using fluorescence microscopy. Localisation in both knock out and wild-type Pv41 and PvGAMA was performed using anti-Pv41 and anti-PvGAMA antibodies respectively; in both cases the KO line produced only diffuse background staining, confirming knockout of the P. knowlesi genes. Parasite nuclei were stained with Hoechst 33342; Merge is an overlay of Alexa 555 and Hoeschst. Scale bar is 2 micrometers.
Fig 6.
Gene editing to replace P. knowlesi target genes with orthologous P. vivax candidate genes.
Pvp12, PvARP, Ppv41 and PvGAMA gene sequences were used to replace Pkp12, PkARP, Pkp41 and PkGAMA in wild-type P. knowlesi to create lines Pk12Rep, PkARPRep, Pk41Rep and PkGAMARep allele replacement lines. Proteins in allele-replacement and wild-type P. knowlesi strains were localised using anti-P. vivax polyclonal antibodies and Alexa Fluor 555 labelled secondary antibody, and imaged using fluorescence microscopy. Parasite nuclei were stained with Hoechst 33342; Merge is an overlay of Alexa 555 and Hoeschst. In all cases localisation was the same in the wild-type and allele-replacement lines. Scale bar is 2 micrometers.
Fig 7.
Inhibition by polyclonal antibodies to P. vivax vaccine candidates is increased in chimeric P. knowlesi strains expressing homologous P. vivax proteins.
Invasion inhibition assays were carried out as detailed in the Materials and Methods sections and summarised in S4 Fig. Chimeric P. knowlesi strains expressing Pvp12 (PkPv12), PvARP (PkPvARP), Pvp41 (PkPv41) and PvGAMA (PkPvGAMA) were incubated with anti-PvP12, anti-PvARP, anti-PvP41 and anti-PvGAMA antibodies respectively and compared to inhibition in wildtype P. knowlesi. This data represents observations from two independent experiments each with three replicate wells per each treatment. To determine if the inhibition was statistically significant, all treatments were independently compared to the same concentration of total IgG from non-immunised rabbits using Mann-Whitney-Wilcoxon test with a p-value threshold of 0.05; the inhibition of wild-type and chimeric parasites was also compared using the same approach. Black asterisks represents p-values obtained from comparison with non-immune rabbit IgG while red asterisks represents p-values obtained from comparison of wild-type and mutant parasites. *p = 0.05–0.01; **p<0.01.