ABSTRACT
Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.
AUTHOR SUMMARY Malaria parasites cause disease after invading human red blood cells, implying that a vaccine that interrupts this process could play a significant role in malaria control. Multiple Plasmodium parasite species can cause malaria in humans, and most malaria outside Africa is caused by Plasmodium vivax. There is currently no effective vaccine against the blood stage of any malaria parasite, and progress in P. vivax vaccine development has been particularly hampered because this parasite species cannot be cultured for prolonged periods of time in the lab. We explored whether a related species, P. knowlesi, which can be propagated in human red blood cells in vitro, can be used to screen for potential P. vivax vaccine targets. We raised antibodies against selected P. vivax proteins and testedtheir ability to recognize and prevent P. knowlesi parasites from invading human red blood cells, thereby identifying multiple novel vaccine candidates.
INTRODUCTION
Malaria remains a major global health challenge, with an estimated 228 million cases and >400,000 deaths in 2018 (1). While there are five Plasmodium species that can cause malaria in humans, the majority of clinical cases are caused by P. falciparum and P. vivax. P. falciparum causes almost all malaria cases in Africa, but P. vivax is the dominant cause of malaria in the Americas, and causes a similar number of cases as P. falciparum in South-east Asia (1)⍰. As well as having different global distributions, the two species are also very different biologically, which has significant implications for control. P. vivax, along with P. ovale, can form hypnozoites during its liver stage, which are quiescent forms of the parasite that remain dormant from weeks to years in the liver, re-emerging upon stimulation to cause a relapse of malaria. Hypnozoites can therefore act as a continuous source of infection even in the absence of active transmission. This hurdle is made more significant by the fact that primaquine and tafenoquine, the only drugs used to treat hypnozoites, are frequently contraindicated due to their toxicity in patients with glucose-6-phosphate deficiency, a common polymorphism in regions of the world where P. vivax is most prevalent (2,3)⍰. In addition, sexual stage development in P. vivax is much more rapid than in P. falciparum (4,5), meaning that even with rapid treatment with antimalarials, onwards transmission can still occur. These features limit the effectiveness of current chemotherapeutic interventions, making the search for an effective vaccine even more important for P. vivax.
The complex life-cycle of Plasmodium vivax parasites present multiple potential intervention strategies, including preventing transmission to the mosquito, targeting the liver stage to prevent disease and relapse, and targeting blood stages to limit disease and lower the potential of transmission from one infected individual to another. Indeed, vaccine targets across all these various stages of the parasite are under investigation, although in general far fewer antigens have been studied in depth in P. vivax relative to P. falciparum (reviewed in (6,7)). This is particularly the case for blood stage targets, where only a few targets such as P. vivax apical membrane antigen 1 (PvAMA1) (8,9)⍰ and P. vivax merozoite surface protein 1 (PvMSP119) (10,11)⍰ have advanced to pre-clinical study. The furthest advanced P. vivax vaccines, by far, are based on P. vivax Duffy Binding Protein (PvDBP), the only blood stage target that has reached clinical Phase Ia trials (12); these are PvDBPII-DEKnull (12)⍰ and PvDBPII (11–15)⍰⍰⍰. This is in stark contrast to P. falciparum, where multiple targets in different stages have been tested in Phase Ia (Reviewed in (6)⍰), and the RTS,S pre-erythrocytic vaccine has advanced beyond Phase III to pilot testing across three countries in Africa (16). More P. vivax targets clearly need to be screened if vaccine development for this species is to advance.
It was previously believed that P. vivax was completely dependent on the interaction between PvDBP and its receptor, Duffy Antigen Receptor for Chemokine (DARC) (17–20) to invade human erythrocytes. However, it has recently been shown that P. vivax is also able to infect individuals who are Duffy negative, so express little or no DARC on the surface of their erythrocytes (21–24). While the invasion of Duffy negative erythrocytes could still rely on PvDBP (25,26), the sole focus on PvDBP as a vaccine candidate clearly needs to be reassessed and additional targets evaluated, either as potential substitutes for PvDBP, or to be used in combination with it. As noted above, erythrocyte invasion is a very complex process, and while the process is much less well-understood in P. vivax than it is in P. falciparum (27)⍰, other P. vivax ligands such as reticulocyte-binding protein 2 (RBP2b) (28), GPI-anchored micronemal antigen (GAMA) (29)⍰, and erythrocyte binding protein 2 (ebp2) (30) have all been shown or proposed to be involved, enabling the identification of possible combinatorial vaccines (31)⍰.
In this study we took a reverse vaccinology approach to identify new P. vivax vaccine targets, building on previous work where we expressed a panel of 37 full-length recombinant P. vivax vaccine targets predicted to be involved in erythrocyte invasion (32)⍰. Polyclonal antibodies were generated against 8 of these proteins, and the antibodies were tested for their ability to inhibit merozoite invasion. P. vivax preferentially invades immature erythrocytes (33)⍰ which are difficult to obtain (34–37), which has limited the development of continuous culture of P. vivax in vitro, despite herculean efforts (38). As a first-stage screen we therefore performed invasion inhibition assays using P. knowlesi, a close phylogenetic relative of P. vivax (39,40) that has been adapted to in vitro cell culture in human erythrocytes (41,42). We also took advantage of the amenability of P. knowlesi to genetic manipulation to explore the function of some of the target genes, and to swap P. knowlesi genes for their P. vivax orthologues to establish whether this would affect antibody inhibition. Together, this work prioritises new targets for P. vivax vaccine development, and presents additional evidence that P. knowlesi can be used as a readily manipulatable in vitro model for P. vivax.
RESULTS
Generation of polyclonal antibodies against new P. vivax vaccine candidates
We have previously expressed a pilot library of 37 P. vivax proteins that were either shown to localise to merozoite organelles with a role in invasion, or were predicted to do so based on the localisation of their respective P. falciparum homologues (32). In all cases, the full-length extracellular domains of these proteins were expressed using a mammalian protein expression system. This approach, which increases the likelihood of correct folding of disulphide-linked extracellular domains, has been used extensively for P. falciparum invasion-associated proteins (43) to generate antigens capable of inducing potent invasion-inhibitory antibodies, including for the major P. falciparum blood-stage vaccine target PfRh5 (31,44)⍰. Comparing immunoreactivity of native or heat-denatured epitopes and testing for protein-protein interactions indicated that the produced P. vivax library was also likely to consist of largely functional proteins (32)⍰. To test whether this library could also be used to generate inhibitory antibodies, rabbit polyclonal antibodies were raised against eight targets, selected to represent a range of predicted subcellular localizations and including PvDBP as a positive control (Table 1). In all cases, antibodies were raised against the complete recombinant ectodomain of the candidates. As outlined in the Methods, 1mg of antigen was used to immunize each rabbit, and total IgG purified from serum using Protein A affinity chromatography.
Anti-Plasmodium vivax antibodies are able to recognise orthologues in P. knowlesi
Given the inherent difficulty in acquiring P. vivax ex vivo isolates for testing, we explored the use of P. knowlesi, which has recently been adapted to continuous in vitro cell culture in human erythrocytes (41,42) as a model for P. vivax vaccine candidate screening. P. knowlesi, which falls into the same clade of simian parasites as P. vivax (39,40) naturally infects the Kra cynomolgus macaque (Macaca fascicularis) but causes severe zoonotic malaria in Southeast Asia (45), falls into the same clade of simian parasites as P. vivax (39,40). While this phylogenetic relationship is reflected in a higher of conservation between the P. vivax and P. knowlesi genomes than the P. vivax and P. falciparum genomes (39), the degree of conservation varies between at the individual gene level. Sequence alignment between our P. vivax candidate proteins and their orthologues in P. knowlesi showed a range of sequence similarities (Table 1), from a pairwise identity score of 51% for PvDBP and its closest P. knowlesi orthologue PkDBPα, to higher identity scores for several targets (PvGAMA, Pv12, PvARP, PvCyRPA and Pv41), reaching 80% in the case of Pv41. In contrast, a lower degree of conservation was found for the merozoite surface proteins PvMSP3.10 and PvMSP7.1, both members of multigene families which are known to be highly polymorphic within and between Plasmodium species.
To explore whether variable degrees of homology would limit our ability to test specific targets in P. knowlesi, we first determined whether antibodies raised against P. vivax (Pv) targets can recognise their P. knowlesi (Pk) orthologues in immunoblots using P. knowlesi schizont-stage protein lysates (Figure 1).
Antibodies raised against Pv12, PvARP, Pv41, PvMSP7.1 and PvDBP produced a single immuno-reactive band, while several bands were detected with antibodies against PvGAMA, PvMSP3.10 and PvCyRPA, suggesting either post-translational modifications or proteolytic processing events. While multiple factors could affect signal strength, including expression level in schizont stages, there was a correlation between % Pv/Pk identity and the strength of the immunoblot signal, PvCyRPA being the exception with a weak detection signal despite 68% identity. Antibodies against Pv12, PvGAMA, PvMSP3.10, PvCyRPA all detected proteins around their expected molecular weight based on estimates from the corresponding orthologous protein in P. knowlesi. In contrast, anti-PvARP, Pv41 and PvMSP7.1 detected proteins larger than the expected molecular weight suggesting that they might migrate more slowly, which is not uncommon in extracellular proteins. Anti-PvDBP detected a protein half the expected size suggesting either that PkDBPα (the closest P. knowlesi orthologue of PvDBP) is highly processed, or that anti-PvDBP antibodies cross-react with multiple PkDBP proteins. Overall however, immunoblotting showed that the majority of anti-P. vivax antibodies recognised P. knowlesi proteins.
Anti-P. vivax antibodies are able to localise orthologous target proteins to P. knowlesi invasion organelles
To further explore the use of P. knowlesi as a model for P. vivax reverse vaccinology studies, we tested the antibodies in indirect immunofluorescence assays using mature P. knowlesi schizonts (Figure 2). Out of the 8 polyclonals, only anti-PvMSP3.10 (which shows the lowest percentage of identity between Pv and Pk) did not produce a specific signal (Figure 2). Anti-PvGAMA, PvCyRPA, PvDBP and PvARP all labelled punctate foci within the merozoites, while anti-Pv12, Pv41 and PvMSP7.1 all appeared to label the entire merozoite surface. No staining was observed with pre-immune antiserum (Figure S1), confirming that the labelling was antigen-specific.
To establish the specific location of each antigen, anti-P. vivax antibodies were used in co-localization experiments with antibodies specific to proteins of known cellular locations i.e. AMA1, MSP1-19 and GAP45 located in microneme, merozoite surface and inner membrane complex (IMC), respectively (see Methods for antibody sources). Anti-Pv12, Pv41 and PvMSP7.1 all showed a clear co-localization with anti-MSP1 (Figure 3A, Figure S2A) suggesting that their orthologous targets are located on the merozoite surface. Anti-PvGAMA and, to a lesser extent, anti-PvCyRPA and anti-PvDBP co-localised with anti-AMA1, suggesting that their orthologous targets are located in apical secretory organelles such as the micronemes (Figure 3B, Figure S3). Anti-ARP appeared to be apically located but did not co-localise with any known markers that we tested (Figure 3B and Figure S3), such that its exact location remains to be determined. To confirm that antibodies against Pv12, Pv41 and PvMSP7.1 were labelling the merozoite surface and not the IMC, which produce similar staining patterns in late schizonts, co-staining with the IMC marker anti-GAP45 was calso arried out in early schizonts, as the IMC and merozoite surface are easier to distinguish earlier in the cell cycle. In all cases there was no co-localisation with anti-GAP45 in early schizonts (Figure S2B), confirming a merozoite surface, not an IMC, location. In all cases the co-localization of the anti-P.vivax antibody targets as determined by immunolocolisation were identical to those predicted based on their P. falciparum homologues.
Screening anti-P.vivax antibodies for inhibitory activity in P. knowlesi identifies novel invasion-blocking candidates
Having established that anti-P. vivax antibodies could be used to specifically detect homologues in P. knowlesi, we explored whether the same antibodies could inhibit P. knowlesi erythrocyte invasion or intra-erythrocytic development. Serial two-fold dilutions of purified total IgG were prepared starting from 10 mg/ml, and incubated with synchronized ring-stage P. knowlesi parasites for 24 hours. Assays were carried out in two biological replicates each with three technical replicates, where invasion and growth inhibition were measured by flow cytometry using Far-red Cell Trace staining of erythrocyte and SYBR green staining of parasite DNA. Invasion was quantified as the percentage of erythrocytes that were both SYBR green and Far-red Cell Trace positive as compared to only control treated erythrocytes, while growth was quantified as the percentage of cells that were only SYBR green positive as compared to control treated erythrocytes. As shown in Figure 4 and Table 2, compared to the positive and negative controls for inhibition (heparin and rabbit IgG respectively), inhibitory activity fell into two broad groups: inhibitory (top panel, anti-Pv12, Pv41, PvGAMA and PvDBP which gave IC50 values of 4.17, 11.24, 6.64 and 4.54 mg/ml respectively) and not inhibitory (bottom, anti-PvARP, PvCyRPA, PvMSP7.1, PvMSP3.10). The low level of inhibition observed with anti-PvMSP7.1 and PvMSP3.10 could be due to the low degree of homology between Pv and Pk homologs, and the lack of inhibition observed with the anti-PvMSP3.10 was consistent with the absence of cross-reactivity with PkMSP3 homologues in immunofluorescence assays. Strong inhibition with anti-PvDBP, the only P. vivax blood stage vaccine target in the advanced stage of vaccine development, was confirmatory and comparable to other studies (46)⍰. Antibodies to two other targets, Pv12 and PvGAMA, had broadly similar IC50s to anti-PvDBP, while antibodies to Pv41, which interacts with Pv12 (32), also had strong inhibition. All three targets were therefore worthy of further investigation.
Gene editing in P. knowlesi establishes that Pk41 and PkGAMA are not essential for blood-stage growth
Gene essentiality is one potential prioritisation factor in ranking vaccine candidates, as targeting the product of a gene that is absolutely required for parasite development is by definition more likely to yield growth inhibitory activity. Given that antibodies against Pv12, Pv41, and PvGAMA all inhibited P. knowlesi growth, we used genome editing to determine whether the orthologous genes in P. knowlesi could be knocked out. We also targeted PkARP as a positive control, as anti-PvARP antibodies had no inhibitory effects on P. knowlesi (Figure 4), while constructs targeting PkDBPα were included as a negative control, as this gene has previously been shown to be essential (47)⍰. Gene targeting was carried out using a CRISPR-Cas9 two-vector approach, with one vector containing Cas9 and guide RNA expression cassettes as well as the selection marker, while the other one was a donor template for repair consisting of eGFP flanked by 5’ and 3’ untranslated regions of each respective gene (Figure S4A). Thus, after drug selection based on the selection marker in the guide vector and not in the donor vector, integration of the construct would both eliminate the endogenous gene, and result in eGFP expression. Transfection of P. knowlesi was followed by selection with 100 nM pyrimethamine for 6 days to select for Cas9 expression, and cultures were maintained for up to 3 weeks. Transfections were repeated at least twice for each pair of constructs.
Parasites were recovered from all transfections. Genomic DNA was extracted from recovered lines, and used for genotyping to establish whether integration had occurred. Only parasites transfected with Pk41 and PkGAMA knockout constructs gave bands of the size expected if gene deletion had occurred (Figure S4B). Whole genome sequencing analysis confirmed this result, showing no reads mapping to the deleted regions of the wildtype (WT) P. knowlesi genome (Figure S5 and S6), indicating that integration of the knock-out construct had occurred. By contrast, genotyping of parasites transfected with Pk12, PkARP and PkDBPα constructs did not differ from WT cultures. No WT band was amplified for Pk41 and PkGAMA knockout lines, whereas WT parasite controls yielded bands of the expected size (Figure S4B). Pk41 and PkGAMA therefore appear to be non-essential for P. knowlesi growth, whereas Pk12, PkARP and PkDBPα were not able to be disrupted using this approach. For PkARP this was unexpected, given that anti-PvARP antibodies had no effect on growth or invasion and the three different guide RNAs used for this gene were effective in targeting this locus in subsequent experiments, shown below.
To confirm that Pk41 and PkGAMA expression was absent in the knockout lines, fluorescence and immunofluorescence assays were performed. Both knockout lines expressed eGFP (Figure 5A and B), while localisation assays with anti-Pv41 and anti-PvGAMA gave no specific signal (Figure 5C), showing only background staining in clear contrast to WT parasites (Figures 3 and 4). This confirms that these parasites were not expressing Pk41 and PkGAMA, and therefore that Pk41 and PkGAMA are redundant for intra-erythrocytic growth, despite the fact that anti-P41 and anti-GAMA antibodies were shown to inhibit parasite growth (Figure 4). Testing the antibodies in growth assays using the knockout strain showed no detectable inhibition, confirming that the antibodies were specific to their immunogens (Figure 6).
Allele replacement of P. knowlesi genes with P. vivax orthologues increases the inhibitory effect of anti-P. vivax antibodies
A true test of the inhibitory effectiveness of the anti-P. vivax antibodies would be in the context of the proteins that they were raised against, but P. vivax culture and invasion assays are not available for routine use. To test an alternative approach, we sought to replace P. knowlesi target genes with their P. vivax orthologues, generating chimeric P. knowlesi strains expressing P. vivax proteins. Replacement constructs were created in which the Pv12, Pv41, PvGAMA and PvARP open reading frames were flanked by the 5’ and 3’ UTRs of their P. knowlesi counterparts, and these were transfected in combination with the same Cas9/gRNA vectors used in the knockout studies, in order to replace Pk12, Pk41, PkGAMA and PvARP with Pv12, Pv41, PvGAMA and PvARP respectively (Figure S7). After selection of transfected parasites with 100 nM pyrimethamine and expansion of the resulting parasites lines, genomic DNA was extracted for genotyping. All lines gave bands of the expected size (Figure S7) indicating that integration of these replacement constructs had occurred at the expected locus, and no WT bands were detected. Whole genome sequencing analysis confirmed that no reads mapped at the targeted region when comparing with Pk reference genome (Figures S8-11).
Localisation assays with anti-Pv12, PvARP, Pv41 and anti-PvGAMA antibodies all gave specific signals in the replacement lines (Figure 7) with anti-Pv12 and Pv41 (Figure 7A and 7C) indicating merozoite surface localisation, while anti-PvGAMA and PvARP (Figure 7B and 7D) appeared as punctate signals, just like signals in wildtype P. knowlesi parasites (Figures 2 and 3). These chimeric parasites are therefore viable and able to correctly express and localize Pv12, PvARP, Pv41 and PvGAMA. The chimeric P. knowlesi strains had similar growth rates as the WT strains (Figure S12), indicating that the P. vivax genes can substitute for the function of their P. knowlesi counterparts, emphasizing the phylogenetic relationship between the two parasites. To test whether replacing the P. knowlesi genes with their P. vivax counterparts increased the inhibitory activity of anti-P. vivax antibodies, we tested for growth and invasion inhibition, comparing WT and chimeric replacement lines. In all cases inhibition was increased when using the chimeric lines (Figure 8), indicating that while P. knowlesi is a useful model as a first screen for P. vivax reverse vaccinology studies, sequence differences between P. vivax antigens and their P. knowlesi orthologues can lead to underestimation of the inhibitory effect when only wildtype P. knowlesi parasites are used.
DISCUSSION
To date only a limited number of Plasmodium vivax blood stage vaccine candidates have been investigated (reviewed in (6,7,48,49)). This is in large part because it is currently not possible to continuously culture P. vivax blood stages in vitro, which rules out many biological assays. We have explored whether P. knowlesi, which has a close phylogenetic relationship with P. vivax (39,40) and has been adapted to in vitro culture in human erythrocytes (41,50), could be used to screen for P. vivax blood-stage vaccine candidates, as it can for drug-resistance candidates (51). Such an approach has proven viable to explore the most advanced P. vivax blood-stage vaccine candidate, PvDBP (46). In this case we sought to apply the P. knowlesi model to systematically screen for new blood-stage antigens, using a panel of polyclonal antibodies generated against candidates from a previously published library of P. vivax schizont expressed proteins (32). A similar approach has recently been applied to a panel of P. vivax blood stage targets, although functional testing did not include knockout and gene replacement strategies (52). We focused on seven P. vivax targets: two merozoite surface proteins (PvMSP7.1 and PvMSP3.10); two 6-Cysteine domain proteins (Pv12 and Pv41) and three proteins not belonging to other families (PvGAMA, PvCyRPA and PvARP), with PvDBP included as a positive control.
There was a broad correlation between the ability of anti-P. vivax antibodies to specifically recognise their P. knowlesi orthologues in immunoblot and immunofluorescence assays and the percent identity within the P. vivax/P. knowlesi antigen pairs. However, it is worth noting that a single dominant protein band was identified in 5/8 cases, and clear intracellular localisations defined in 7/8 cases, despite levels of identity as low as 50%, suggesting the system has broader utility than homology levels alone might indicate. This high level of cross-reactivity may be in part be due to our strategy of raising polyclonal antibodies against full-length protein ectodomains, whereas many antigen studies focus only on smaller sub-domains, which limits the chances that cross-reactive responses will be generated. There is also some evidence that the eukaryotic expression system we use increases the likelihood of generating antibodies against folded, functional domains (43), which are more likely to be of utility in assays such as immunofluorescence or growth inhibition, where conformation-dependent epitopes are more important. Using these antibodies in growth inhibition assays revealed robust dose-dependent inhibition of P. knowlesi growth by anti-Pv12, Pv41 and PvGAMA antibodies, in some cases on a similar level to anti-PvDBP.
Pv12 and Pv41 are members of a family of 6-cysteine domain proteins, other members of which are under investigation as transmission-blocking vaccine targets in P. falciparum (53–55). The P. falciparum orthologues of Pv12 and Pv41 form a heterodimer and are localised on the merozoite surface (56). We have previously shown that Pv12 and Pv41 are also able to heterodimerize (32), and here we show that their P. knowlesi orthologues also colocolise to the merozoite surface, suggesting key elements of the function of these two proteins are conserved across Plasmodium species. However, there are also elements that are different. Immunoepidemiology studies show that antibody responses to Pv12 and Pv41 are commonly induced by exposure to P. vivax infection (32,57–59), and have been associated with protection against severe P. vivax malaria, in keeping with the inhibitory activity of anti-Pv12 and Pv41 antibodies shown here. By contrast, in P. falciparum, anti-Pf12 and Pf41 antibodies have no inhibitory effects on parasite growth in vitro, and the genes can be deleted, suggesting a level of functional redundancy in this species (56). Previous immunofluorescence studies of Pv12 in P. vivax suggest that it localises to the rhoptries, rather than merozoite surface as in our experiments (60)⍰, although whether this apparent difference is due to differences in the stage of the parasite during erythrocytic schizogony used in the assays is not known.
We localised PkARP to the apical region of the merozoite, suggesting a possible location in the rhoptries like the PfARP homologue in P. falciparum (60), but in contrast to previous studies suggesting a merozoite surface location in P. vivax and P. knowlesi (52,61); again, experimental differences in the stage of parasites used might explain the different observations. PkGAMA also localised to apical organelles, replicating previous observations of a micronemal location in P. vivax (29) and P. falciparum (62,63). We were unable to inhibit P. knowlesi growth using anti-PvARP antibodies, contradicting what has been shown in P. knowlesi (64) and P. falciparum (60). However, when we replaced PkARP with PvARP, there was a reversal of the activity of anti-PvARP, suggesting that PkARP may lack key inhibitory epitopes recognised by our polyclonals, which were raised against PvARP. Anti-PvGAMA had invasion and growth inhibitory effects on P. knowlesi in both WT and chimeric lines, replicating the observations of anti-PfGAMA effects on P. falciparum (63), suggesting a conserved role of this protein in invasion across species. Immunoepidemiology studies also show that antibody responses to PvARP and PvGAMA are commonly induced by exposure to P. vivax infection (14,32,57–58,65), and have been associated with protection against severe P. vivax malaria.
How do these relatively new candidates compare to the much more well-studied vaccine candidate PvDBP? Clearly PvDBP has been the subject of decades of work, meaning that there are multiple lines of evidence supporting its candidacy. The limitations of this target are also well known, specifically the challenge of strain-specific antibody responses, which may be able to be overcome with epitope engineering (28,66–68). One question in weighing up the candidacy of any vaccine antigen is whether the gene that encodes it is essential for parasite growth, as targeting a non-essential gene would seem likely to select for parasites that do not rely on the gene product, and so are able to escape the vaccine.
According to the current model, PvDBP is essential for invasion, as P. vivax primarily invades reticulocytes via the interaction between PvDBP and its host receptor, Duffy Antigen Receptor for Chemokine (DARC) (17–20). However, it has now clearly been demonstrated that P. vivax is also able to infect individuals who are Duffy negative, lacking DARC expression on their red blood cell surface (21,22). This could indicate that P. vivax is able to utilize other ligands for invasion such as PvRBP2b (28) and erythrocyte binding protein 2 (ebp2) (30), although it is also possible that PvDBP is still involved in the invasion of Duffy negative cells (26). It is also worth noting that the genetic essentiality of PvDBP for parasite growth has never been able to be directly tested, as P. vivax cannot be cultured and therefore cannot be genetically manipulated. Studies in P. knowlesi, which has three homologues of PvDBP, suggest that at least one is required for invasion of human erythrocytes (47), but whether this is true of PvDBP remains to be proven unequivocally.
In the case of the four candidates identified by initial screening with wildtype P. knowlesi, there are clear 1:1 orthologues between P. vivax and P. knowlesi, providing an even stronger rationale than that of Pv/PkDBP to use P. knowlesi genetic tools to explore candidates. We utilised the fact that P. knowlesi can be readily genetically manipulated (46) to explore whether Pv12, Pv41, PvGAMA and PvARP were essential for parasite growth. Pk41 and PkGAMA could be experimentally deleted, while Pk12 and PkARP could not, even though both anti-PvP41 and PvP12 antibodies had invasion and growth inhibitory effects on WT P. knowlesi. The fact that Pk41 and PkGAMA could be deleted without any apparent effect on growth, whereas antibodies that recognise them inhibit growth, seems contradictory. This could be explained if the antibodies raised against Pv41 and PvGAMA recognized multiple targets in P. knowlesi, but these antibodies had no inhibitory activity when incubated with the relevant knockout strains, nor could they detect any protein in immunofluorescence assays in these lines. This strongly suggests that the antibodies are specific, and rules out off-target explanations for the antibody inhibition data. An alternative explanation is that the process of genetic deletion, which takes some weeks to recover modified parasites, provides an opportunity for the parasites to adapt to the loss of a specific gene, for example by up-regulating the expression of other genes. By contrast, growth inhibition assays occur in a single cycle, which the parasites may find it harder to adapt to. Vaccine-induced antibodies would arguably operate in a similar manner, suggesting that while essentiality might be one element used to prioritise new targets, it should definitely not be the only one. Despite this, it would seem reasonable to argue that Pv12 and PvARP should have a higher priority for follow-up than Pv41 and PvGAMA.
This study clearly highlights several advantages of the P. knowlesi system as a model for testing P. vivax blood-stage antigens, as has been suggested in previous drug (51) and vaccine (46,52) studies. A key one is accessibility -access to ex vivo P. vivax samples is limited and samples are precious, whereas we were readily able to perform multiple in vitro assays using P. knowlesi. A second is also the genetic accessibility of the system, where gene deletions and allele replacements, while not precisely routine, are certainly readily achievable. There are however always limitations in using one species as a model for another, and this study reveals some of these. The study relies on antibodies that were generated against P. vivax proteins being able to cross-react with their P. knowlesi orthologues, and while in almost all cases this proved possible, there was some correlation between the strength of cross-reactivity and the % identity between antigen pairs, meaning the P. knowlesi model will almost certainly be more useful for some antigens than others. The genetic tractability of P. knowlesi offers a potential solution to this problem, as we have shown, by allowing the replacement of endogenous P. knowlesi genes with their P. vivax orthologues, implying that antibodies can be tested against the precise sequence they were generated against. This approach relies on the ability of a P. vivax gene to substitute for P. knowlesi gene function, which may not always be the case, but in all four instances tested here, as well as the Pv/PkDBP swaps carried out by others (46), this has not proven a problem. Ultimately however, no model system is perfect, even in vitro culture of P. vivax itself, which after all is only a model for in vivo growth. It would be extremely useful to the P. vivax vaccine field to carry out a head-to-head comparison across all the four currently available functional models - wildtype P. knowlesi, genetically modified P. knowlesi with allele modifications to insert P. vivax genes, P. cynomologi and P. vivax ex vivo assays. The targets identified here, Pv12, Pv41, PvARP and PvGAMA, along with PvDBP, present a perfect opportunity to carry out such a test.
To conclude, using both antibody and genetic approaches, we exploited the phylogenetic relationship between P. knowlesi and P. vivax to explore blood-stage P. vivax vaccine targets. The data suggests a hierarchy of possible targets, with Pv12 and PvARP being the highest priority as they are genetically essential and can be targeted with inhibitory antibodies, Pv41 and PvGAMA in a second tier as they can be inhibited with antibodies but also genetically deleted, while PvMSP7.1, PvMSP3.10 and PvCyRPA would seem to have the lowest priority. We have demonstrated that antibodies against P. vivax vaccine targets are able to recognise proteins in P. knowlesi as well as inhibit its growth and invasion, and that P. knowlesi has many advantages as a rapid and accessible system to screen P. vivax blood stage targets.
These advantages need to be balanced against the limitations described above, and it is always possible that lack of inhibition and in some case lack of localisation in P. knowlesi using anti-P. vivax antibodies is due to the lower level of similarity between P. vivax and P. knowlesi orthologues, or indeed differences in biology between these species. Until a robust P. vivax culture system is established, which despite extensive effort by multiple teams (34–38)⍰ does not seem likely soon, it will be advisable to use multiple models to screen for candidates, and be clear and upfront about the limitations inherent in all of them.
MATERIALS AND METHODS
In vitro parasite culture
Plasmodium knowlesi parasites were maintained as described in (41). Briefly, P. knowlesi strain A1-H.1 was propagated in human O+ erythrocytes (UK NHS Blood and Transplant), in RPMI 1640 supplemented with Albumax (Thermo Fisher Scientific), L-Glutamine (Thermo Fisher Scientific), Horse serum (Thermo Fisher Scientific), Gentamicin (Thermo Fisher Scientific). The cultures were kept at 2% hematocrit, gassed using a mixture of 5% CO2, 5% O2 and 90% Nitrogen, while being monitored three times per week by counting parasitemia using light microscopy with media change or splitting as appropriate.
Synchronization and enrichment of Plasmodium knowlesi
Synchronization was performed by enriching late stage parasites using Histodenz (Sigma Aldrich) as described in (41). Briefly, parasites were resuspended in 5ml complete media and layered on top of 5 ml of 55% Histodenz in complete culture media in a 15 ml tube (Greiner). The mixture was then centrifuged for 3 minutes at room temperature, 1500 g, acceleration 3 and brake 1, resulting in late stage parasites becoming enriched at the interface. For inhibition assays, these parasites were returned to culture, and assays set up after reinvasion had occurred in the subsequent cycle. For protein extracts, immunofluorescence assays and transfections, this was repeated over three consecutive cell cycles to create very tightly synchronized parasites, with schizont samples from a fourth cycle of Histodenz purification used for subsequent analysis.
Antibody production and purification
Rabbits were immunized with 1mg of his-tagged P. vivax full-length ectodomain, expressed in HEK239E cells as previously described (32), and purified by nickel affinity chromatography. Immunisations were carried out using Freunds complete/incomplete adjuvant by Cambridge Research Biochemicals. Total IgG was purified using Protein G gravitrap kit (GE healthcare). Eluted total IgG was concentrated by centrifugation at 4°C for 30 mins using 100000MWCO vivaspin (Sartorius). The concentrate was then dialyzed using a dialysis tube (Millipore) overnight at 4°C with 1 litre of RPMI 1640 (Thermo Fisher Scientific), before repeating concentration if necessary. Total IgG concentration was measured bynanodrop (NanoDrop).
Protein extraction and Western blotting
To generate protein extracts, schizont stage parasites enriched from 5-10 mL of culture at 5-10% parasitemia were treated with 0.15% saponin (Sigma Aldrich) and protease inhibitor (Cat. No. 5892970001, Sigma Aldrich) at 1X for 1min on ice to release parasites from their host cell. After pelleting and two rounds of washing in ice cold 1X PBS (Sigma Aldrich) with protease inhibitor 1X, parasites were treated with 1 µl of DNAse I (Thermoscientific) for 30mins at 37°C., before mixing 1:1 with Laemmli sample buffer 2X and incubating for 30mins-1h at 37°C to gently denaturate the sample. The pellet was then frozen down at -80°C until needed. Samples were diluted 0, 1:5 or 1:10 in Laemmli, and 5µl of the diluted samples were loaded onto a 4-12% bis-tris NuPage gel (Thermo Fisher Scientific) and run at 200V for 50min in MOPS buffer (Thermo Fisher Scientific). For electrophoresis, 1 µg/µl of samples was mixed with 2.5 µl NuPage LDS sample buffer (4X) (Thermo Fisher Scientific), 1 µl NuPage Reducing agent (10X) (Thermo Fisher Scientific) and deionized water to 6.5 µl. The mixture was then incubated at 72°C for 10 mins while shaking at 300 RPM. 10 µl of the sample was then resolved on a 4-12% bis-trisNuPage gel (Thermo Fisher Scientific) with 1X MOPS SDS gel buffer (Thermo Fisher Scientific) at 200 V for 50 minutes.
Western blot transfer was carried out in wet conditions at 30V for 60min, and membrane blocked overnight while shaking at 4°C in 5% milk (Marvel) containing 0.077% sodium azide (Sigma Aldrich). Primary antibodies were diluted in 5% milk/PBS/0.1% TWEEN 20 at concentrations as follows: anti-PvGAMA 1:2400; Pvp12, PvMsp7.1 and PvMSP3.10 1:400; Pvp41, PvARP and PvCyRPA 1:800; PvDBP 1:1600. Primary incubation was carried out overnight at 4°C. Blots were then washed three times, each 10mins, in PBST (1X PBS and 0.1% Tween), before incubating with secondary anti-Rabbit HRP (Abcam) at 1:20000 dilution in 5% milk/PBS/0.1% for 45 mins at room temperature. Blots were washed again three times, each 10mins, in PBST before developing with ECL prime Western Blotting detection reagent, (GE Healthcare). Expected molecular weight of the P. vivax candidate proteins and their orthologous proteins in P. knowlesi was predicted using Protein Molecular weight calculator (69) based on the amino acid sequences of the respective protein sequences from PlasmoDB.
Immunofluorescence assays
Cells were synchronized, harvested from culture and enriched using Histodenz as described above based on (41). These were then washed with 1X PBS (Sigma Aldrich) for 5 mins and fixed with 4% paraformaldehyde (Agar scientific)/ 0.0075% glutaraldehyde (Sigma Aldrich) in 1X PBS for 30 minutes at room temperature, followed up with washing in 1x PBS while shaking for 5 mins. Thin smears of fixed cells were made on Poly-L-Slides (Sigma Aldrich) and stored in -80°C freezer until needed. For processing, slides were incubated briefly at room temperature, permeabilized with 0.1% Triton X-100 (Sigma Aldrich) in 1X PBS for 30 mins at room temperature then washed once with 1x PBS while shaking for 5 mins. Blocking was carried out overnight at 4°C in a humidified dark chamber using Blocking Aid Solution (Thermo Fisher Scientific). Primary antibody diluted in Blocking Aid Solution (Anti-PvGAMA 1:1200; Pvp12, PvMsp7.1 and PvMSP3.10 1:200; PvARP 1:400; PvCyRPA and PvDBP 1:800; Pvp41 1:200, PkMSP1-19 1:2000; PfAMA1 1:1000) was then added and incubated overnight in a humidified dark chamber at 4°C followed by washing three times for 5 mins in 1X PBS while shaking. Secondary antibody diluted in Blocking Aid Solution (Alexa Fluor 555 Goat-anti rabbit (Thermo Fisher Scientific) (1:500) for anti-P. vivax rabbit polyclonals and Alexa Fluor 488 Goat-anti rat (Thermo Fisher Scientific) (1:500) for PkMSP1-19 and PfAMA1) was then added and incubated 1 hour in a humidified dark chamber at room temperature followed by washing three times for 5 mins in 1X PBS while shaking. Hoechst 33342 (Thermo Fisher Scientific), for nucleus staining, was diluted 1:3000 in 1x PBS (Sigma Aldrich) then added and incubated for 10 mins in a humidified dark chamber at room temperature with subsequently washing three times for 5 mins in 1X PBS while shaking. The cells were later mounted with Pro-Long Gold mounting solution (Thermo Fisher Scientific), covered with cover-glass (VWR), left to cure for 24 hours in dark and dry chamber at room temperature and eventually sealed with slide sealer (Biotium), before imaging on a Leica DMi8.
Invasion and growth inhibition assays
Two milli-litres of O+ erythrocytes at 2% hematocrit in incomplete-culture media were labelled using 2 µl of a stock of 1 mM Far-red Cell Trace (Thermo Fisher Scientific); control unstained erythrocytes were incubated with 2 µl of Dimethyl-sulphoxide (DMSO; Sigma Aldrich). After 2 hours of incubation at 37°C while shaking, labelled erythrocytes were washed twice using complete media, then the cells were resuspended in complete media to 2% hematocrit in 2 ml final volume. Labelled erythrocytes were mixed with synchronized rings, generated by enriching schizonts as described above then returning them to culture until reinvasion had occurred. The labelled erythrocyte-parasite mix was incubated with serial dilutions of anti-P. vivax antibodies, with all dilutions made using incomplete medium. Incubations were carried out in 96-well plates, with each well containing 20 µl infected erythrocytes, 20 µl stained erythrocytes, Xµl of diluted total IgG (Xµl because the antibodies were in varying stock concentrations therefore requiring different volumes to be added to get the same final concentrating), and 2.2 µl of a mixture of serum, hypoxanthine and gentamicin (at a ratio of 2, 0.18 and 0.009 respectively). Control wells contained 40 µl of erythrocytes only, or 20 µl of infected erythrocytes/20 µl of unstained erythrocytes, or 40 µl of stained erythrocytes only, or 20 µl of infected erythrocytes and 20 µl of stained erythrocytes, to control for gating in the flow cytometry. Triplicates of each combination were incubated in a 96 well plate for 24 hours in a gassed chamber. To quantify parasite invasion or growth, samples were centrifuged for 3 mins at 450g (acceleration 9, brake 3) at room temperature, supernatant was removed and samples were labelled with SYBR green I nucleic acid dye (Thermo Fisher Scientific) for 1 hour at 37°C while shaking at 52 rpm. After two washes with 100 µl 1 X PBS (Sigma Aldrich), samples were resuspended in 100 µl of 1 X PBS (Sigma Aldrich) and parasites quantified using FACS (Cytoflex, Becton and coulter) as previously described (70). Data was analyzed using FlowJo (FlowJo)then using Excel (Microsoft office), invasion was calculated as the percentage of erythrocytes that were both SYBR green and Far-red Cell Trace positive as compared to only DMSO treated erythrocytes while growth was calculated as the percentage of cells that were only SYBR green positive as compared to only DMSO treated erythrocytes. The results were then plotted using the following R packages; ggplot2 (71)⍰, ggpubr (72), cowplot (73), magrittr (74), readxl (75) and dplyr (76) in R-Studio (R-Studio Inc). IC50 was determined using Probit/logit regression using Excel (Microsoft office).
Genetic modification
Gene repair construct design and assembly
Constructs, guide RNAs and primers (supplementary table S1,2,4,5,6) were designed with (77) and (78). Synthetic DNA codon optimization was performed using gblocks® Gene Fragments (IDT) (PvGAMA_regions1 & 3, PvARP_region1) and GeneArt Gene Synthesis (Thermo Fischer Scientific) (Pv12, Pv41, PvARP_region3). PvGAMA_regions2 and PvARP_regions3 were amplified from expression constructs previously generated by (32)⍰. Other fragments were amplified from P. knowlesi genomic DNA, purified from saponin-lysed P. knowlesi infected erythrocytes using DNA blood kit (Qiagen) according to the manufacturer’s protocol. Gene editing donor vectors were assembled in PUC19 using Gibson assembly according to the manufacturer’s protocol (NEB), using PCR products amplified using KAPA HiFi HotStart ReadyMixPCR Kit (KAPABiosystems) and purified using gel isolation kits (Macherey and Nagel). Primers (Supplementary Tables S1 and S2) and PCR programs (Supplementary Table S3: KAPA2M for all of constructs except KAP121M for final amplification of Pkgama replacement insert) are listed in the Supplementary Material.
Assembly of Cas9/gRNA vectors
The cloning vector (TGL96) was digested using BtgZI (NEB), purified using a gel purification kit (Macherey and Nagel), and treated with shrimp alkaline phosphatase (NEB) to dephosphorylate vector ends. Guide RNAs (Supplementary Table S5) were reconstituted by mixing 1 µl of 100 µM stocks of the forward and reverse strands for each guide with 1 µl of 10x ligation buffer (NEB), 0.5 µl T4 polynucleotide kinase (NEB) and 65 µl nuclease free PCR water. Annealing was carried out by incubating at 37°C for 30 min, then increasing to 94°C for 5 min before cooling at 25°C at a ramp speed of 5°C per-min. Annealed primers were then diluted to 1 µl in 200 µl and ligated (NEB) to the digestedand dephosphorylated Cas9 vector.
Vectors were transformed into chemically competent E. coli according to the manufacturer’s protocol (NEB), and grown overnight. Resulting colonies were screened by colony PCR using GoTaq Green PCR master mix (Promega), with 1 µl of Pk5’ UTR forward and Pk3’UTR reverse primers for each respective construct. Positive colonies were expanded and DNA purified using a miniprep purification kit (Macherey and Nagel) and sequenced to confirm construct integrity (GATC/Eurofins). Sequencing data was analyzed using Benchling (77) and Seqman Pro (DNA star Navigator); positive constructs were expanded and purified for transfection using a maxiprep purification kit (Macherey and Nagel).
Transfection
Transfection was performed largely as described in (41). Late stage P. knowlesi parasites were enriched using Histodenz as described above. In each transfection cuvette (Lonza), 10 µl of schizonts was mixed with 100 µl of P3 solution (Lonza) containing 30 µg each of the relevant donor and guide vectors. Transfections were carried out using program FP158 (Amaxa Nucleofector, Lonza), and the contents were then immediately transferred into a 2 ml sterile eppendorf tube containing 500 µl of complete culture media mixed with 190 µl uninfected erythrocytes. The transfection mix was incubated at 37°C while shaking at 800 rpm in a thermomixer for 30mins, before being transferred into a 6 well plate, gassed and incubated at 37°C for one parasite life cycle. After this selection was applied with 100 nM pyrimethamine (Santa Cruz Biotechnology Inc). For three days, the cultures were monitored by smearing and selection done by changing the media and replacing with fresh media containing 100 nM pyrimethamine (Santa Cruz Biotechnology Inc). On day 4 post transfection the cultures were diluted 1/3 in 5ml fresh media containing 100 nM pyrimethamine and 100 µl erythrocytes. The cultures were then maintained and monitored after every 2nd cycle by smearing/parasitemia counting, with media changed or cultures split as appropriate. Samples where parasites re-appeared were expanded in a total volume of 50 ml with erythrocytes at 2% hematocrit until parasitemia was greater than 5%. DNA was then isolated using a DNA Blood kit (Qiagen) and analysed using gene-specific primers (Supplementary Table S6) and PCR program KAPA18C (Supplementary Table S3). Cultures that contained only modified parasites were phenotyped without cloning, while those that genotyping showed had both modified and WT genotyping bands were cloned by limiting dilution and plaque cloning in flat-bottomed 96-well plates. Wells containing single plaques were identified using an EVOS microscope (4x objective, transmitted light), expanded, and DNA isolated and genotyped as described above as well as whole genome sequenced WGS analysis was then performed on the Welcome Sanger Cluster using bowtie2 (79), samtools (80). Visualisation was perfomedusing Integrative Genomics Viewer (81–83) as described in Pevner (84)⍰.
Ethics
Human O+ erythrocytes were purchased from NHS Blood and Transplant, Cambridge, UK, and all samples were anonymised. The work complied with all relevant ethical regulations for work with human participants. The use of erythrocytes from human donors for P. falciparum culture and binding studies was approved by the NHS Cambridgeshire 4 Research Ethics Committee (REC reference 15/EE/0253) and the Wellcome Sanger Institute Human Materials and Data Management Committee.
Supporting information
S1 Supplementary figure 1. Preimmune antibody controls for immunolocalization. Localisation of PkGAMA, PkDBP, PkARP, PkCyRPA, PkMSP7.1, Pk41 and Pk12 using pre-immune serum from rabbits immunized with PvGAMA, PvDBP, PvARP, PvCyRPA, PvMSP7.1, Pv41 and Pv12, respectively. Scale bar is 2 micrometers.
S2 Supplementary figure 2. Colocalization of P. knowlesi proteins using polyclonal anti-P. vivax antibodies to P. vivax vaccine candidates with antibodies to proteins of known cellular location. Colocalization of Pk12, PkMSP7.1, Pk41 with A. PkMSP1-19 and B. PkGap45 using antibodies to Pv12, PvMSP7.1, Pv41, PkMSP1 and PfGap45, respectively. Scale bar is 2 micrometers.
S3 Supplementary figure 3. Colocalization of P. knowlesi proteins using polyclonal anti-P. vivax antibodies to P. vivax vaccine candidates with antibodies to proteins of known cellular location. Colocalization of PkGAMA, PkCyRPA, PkDBP, PkARP, Pk12, PkMSP7.1, Pk41 with A) PkMSP-1-19 and B) PkAMA1 using anti-bodies to PvGAMA, PvCyRPA, PvDBP, PvARP, Pv12, PvMSP7.1, Pv41, PkMSP1 and PfAMA1, respectively. Scale bar is 2 micrometers.
S4 Supplementary figure 4. Gene editing strategy to knock out candidate genes. A) General strategy used to knock out Pk12, Pkarp, Pkdbpalpha, Pk41 and Pkgama. Plasmids used are Cas9/gRNA vector and donor vector containing eGFP (GFP) flanked with 5’ and 3’ untranslated region (UTR) for each respective P. knowlesi gene (PkCDS). Primer pairs used for genotyping are P1&P2 and P3&P4, to test for wildtype, P1&P5 and P6&P4 to test for integration of the knockout construct. B) Genotyping of Pk41KO and PkGAMAKO with above primer pairs as compared to WT. On the right side are the obtained molecular weight in kilobase pairs (kb).
S5 Supplementary figure 5. Whole genome sequencing of Pk41 knockout strain. Alignment of p41 knockout strains to P. knowlesi reference genome and the WT strain from which they were generated.
S6 Supplementary figure 6. Whole genome sequencing of PkGAMA knockout strain. Alignment of GAMA knockout strains to P. knowlesi reference genome and the WT strain from which they were generated.
S7 Supplementary figure 7. Gene editing strategy to replace P. knowlesi target genes with orthologous P. vivax candidate genes. A) General strategy used to replace pk12, pkarp, pk41 and pkgama with pv12, pvarp, pv41 and pvgama, respectively. Plasmids used are Cas9/gRNA vector and donor vector containing the P. vivax coding sequence (PvCDS) flanked with 5’ and 3’ untranslated region (UTR) for each respective P. knowlesi gene (PkCDS). Primer pairs used for genotyping are P1&P2 and P3&P4, to test for WT. P1&P5 and P6&P4 to test for integration of the replacement construct. B) Genotyping of pk12, pkarp, pk41 and pkgama allele replacement (Rep) using the above primer pairs as compared to WT. On the right side are the obtained molecular weight in kilobase pairs (kb).
S8 Supplementary figure 8. Whole genome sequencing of Pk41-Pv41 replacement strain. Alignment of p41 allele replacement strains to P. knowlesi reference genome and the WT strain from which they were generated.
S9 Supplementary figure 9. Whole genome sequencing of PkGAMA-PvGAMA replacement strain. Alignment of GAMA allele replacement strains to P. knowlesi i reference genome and the WT strain from which they were generated.
S10 Supplementary figure 10. Whole genome sequencing of PkARP-PvARP replacement strain. Alignment of ARP allele replacement strains to P. knowlesi i reference genome and the WT strain from which they were generated.
S11 Supplementary figure 11. Whole genome sequencing of Pk12-Pv12 replacement strain. Alignment of p12 allele replacement strains to P. knowlesi i reference genome and the WT strain from which they were generated.
S12 Supplementary figure 12. Comparative Growth rate assay between wildtype P. knowlesi and genetically edited P. knowlesi strains. WT (P. knowlesi WT), Gamako (PkGama knock-out clone), Gamarep (PkGama replacement clone), p41ko (Pkp41 knock out clone), p41rep (Pkp41 replacement clone), P12rep (Pkp12 replacement clone), ARPrep (PkARP replacement clone).
ACKNOWLEDGEMENTS
We wish to acknowledge the following for their various contributions: Ellen Knuepfer for the kind donation of Rat anti-PkMSP1-19 serum. Rob Moon and Franziska Mohring for the kind donation of Pk CRISPR-Cas9, guide and donor vectors, and advice on genetic modification, Mehdi Ghorbal for advice on construct design, cloning and genetic manipulation experiments and Allan Muhwezi for advice on cell culture.
Funding was provided by the National Institutes of Health (R01AI137154), European Union (MultiViVax 773073) and the Wellcome Trust (206194/Z/17/Z). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
The views expressed in this article are those of the authors and do not necessarily represent the views of the National Heart, Lung and Blood Institute, the National Institutes of Health, the United States Department of Health and Human Services, or any other government entity.
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