PTRAMP, CSS and Ripr form a conserved complex required for merozoite invasion of Plasmodium species into erythrocytes

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: major_revision

Abstract
Invasion of erythrocytes by members of the Plasmodium genus is an essential step of the parasite lifecycle, orchestrated by numerous host-parasite interactions. In P. falciparum Rh5, with PfCyRPA, PfRipr, PfCSS, and PfPTRAMP, forms the essential PCRCR complex which binds basigin on the erythrocyte surface. Rh5 is restricted to P. falciparum and its close relatives; however, PTRAMP, CSS and Ripr orthologs are present across the Plasmodium genus. We investigated PTRAMP, CSS and Ripr orthologs from three species to elucidate common features of the complex. Like P. falciparum, PTRAMP and CSS form a disulfide-linked heterodimer in both P. vivax and P. knowlesi with all three species forming a complex with Ripr by binding its C-terminal region, termed the PTRAMP-CSS-Ripr (PCR) complex. Cross-reactive antibodies targeting the PCR complex differentially inhibit merozoite invasion. The crystal structure of a cross-reactive antibody reveals an inhibitory epitope on the C-terminal tail of PvRipr. Cryo-EM visualization of the P. knowlesi PCR complex confirms predicted models and demonstrates a core invasion scaffold in Plasmodium spp. with implications for vaccines targeting multiple species of malaria-causing parasites.

Introduction
There are more than 200 species of Plasmodium that infect a diverse range of hosts including primates, rodents, reptiles and birds. At least six species, including Plasmodium falciparum, P. vivax and P. knowlesi, have the ability to infect humans. P. falciparum is the most lethal species to infect humans, while P. vivax is the most widespread globally1,2. P. knowlesi is confined mainly to regions of Southeast Asia and is transmitted from macaques by zoonotic infection3,4. The Plasmodium genus can be divided into three main subgenera or clades that include Laverania (includes P. falciparum), Plasmodium (includes most human infective species such as P. vivax and P. knowlesi) and Vinckeia (primarily rodent infective species).

The three clades of the Plasmodium genus represent distinct evolutionary branches distinguished by geographic distribution and severity of disease; however, they can also exhibit distinct host cell selectivity. P. vivax has a strict preference for invasion of reticulocytes in the blood whereas P. falciparum can invade both reticulocytes and the more mature normocytes5. While the core machinery for invasion of reticulocytes and normocytes by P. vivax and P. falciparum, such as the parasite actomyosin motor, is conserved, there are distinct ligand-receptor interactions that provide selectivity for host cell invasion (reviewed in ref. 6). In P. falciparum, many of these ligands are dispensable for invasion7. The notable exception is reticulocyte-binding protein homolog 5 (Rh5)8,9 which is an essential P. falciparum ligand that binds to the receptor basigin on human erythrocytes10. Rh5 can play a role in host tropism through polymorphisms in the protein and differential binding to basigin of other non-human primates8,11.

In P. falciparum Rh5 functions in a complex of five proteins that include CyRPA (Cysteine Rich Protective Antigen)12, Ripr (Rh5 interacting protein)13, PTRAMP (Plasmodium thrombospondin-related apical merozoite protein)14,15, and CSS (cysteine-rich, small, secreted)16 that has been termed the PCRCR complex17–19. PTRAMP and CSS form a disulfide-linked heterodimer that tethers the PCRCR complex to the merozoite membrane via the transmembrane domain of PTRAMP18. All proteins in the PCRCR complex are functionally essential for P. falciparum merozoite invasion of human erythrocytes and, crucially, antibodies and nanobodies that bind to individual proteins can inhibit merozoite invasion18,20–25. Conditional gene knockouts of each PCRCR protein in P. falciparum display the same phenotype, whereby the merozoites can interact with the erythrocyte surface and produce the strong deformation of the host membrane typical during normal invasion; however, the merozoite fails to internalize18,26. The PCRCR complex has been hypothesized to capture and anchor the increased membrane surface contact formed between the merozoite and erythrocyte membrane that is created during strong deformation driven by the merozoite’s actomyosin motor18. This facilitates the establishment of the moving junction and is followed by the downstream events of invasion and ultimately the internalization of the merozoite into the erythrocyte27,28.

Despite being essential for P. falciparum invasion, Rh5 orthologs are absent in species outside of the Laverania subgenus29,30. Consequently, utilization of basigin as a host receptor for invasion is not universal, as demonstrated for both P. knowlesi and P. vivax16, suggesting that other parasite ligand-receptor interactions facilitate host cell attachment in other Plasmodium spp. Non-Laverania species do, however, possess orthologs of other components of the P. falciparum PCRCR complex. P. knowlesi orthologs of PTRAMP, CSS and Ripr have been shown to be essential for merozoite invasion using conditional gene knockouts16 and more recently a high-resolution transposon mutagenesis screen for growth31,32. It has been suggested that PkPTRAMP, PkCSS and PkRipr form a complex and that PkPTRAMP provides the means for erythrocyte binding16. The PkCyRPA ortholog was also identified, and while it has been shown to be essential for parasite growth, it does not appear to be part of this complex in P. knowlesi16.

Here, we hypothesized that PTRAMP, CSS and Ripr form a common basis for invasion complexes across Plasmodium spp. We leveraged recent insights into the PCRCR complex to characterize these proteins in several species of Plasmodium to elucidate the conserved features of merozoite invasion complexes. Our findings revealed a conserved PCR trimeric complex common to all Plasmodium clades that forms a core invasion scaffold. Additionally, cross-reactive antibodies targeting PTRAMP, CSS, and Ripr were identified that differentially inhibit merozoite invasion of erythrocytes, including an antibody targeting Ripr that inhibited both P. knowlesi and P. falciparum growth. Identification of a conserved molecular scaffold presents an attractive approach for the development of vaccines targeting multiple species of malaria-causing parasites.

Results
PTRAMP, CSS and Ripr are conserved in all clades of Plasmodium
We first confirmed that the proteins constituting the P. falciparum PCRCR complex are conserved in other Plasmodium spp. by searching for orthologs and found that PTRAMP, CSS and Ripr were present in all subgenera (Fig. 1a). This conservation contrasts with Rh5 which is only present in P. falciparum and other Laverania species. Whilst CyRPA is relatively conserved, none of the species within the Vinckeia subgenus possess a CyRPA ortholog. This suggests PTRAMP, CSS and Ripr (PCR) form a conserved three-membered complex present across all Plasmodium spp.

AlphaFold3 was used to predict the structure of the PCR complex of different Plasmodium spp. to understand the assembly of the three proteins (Fig. 1b, Supplementary Fig. 1)33,34. This revealed a common architecture of the PCR complex in which PTRAMP and CSS together form a platform that is bound by the C-terminal end of Ripr. The PfCSS crystal structure aligned with the AlphaFold3 prediction of PfPCR with an RMSD of 0.711 Å, suggesting that no conformational changes are required within PfCSS to facilitate PfRipr binding (Fig. 1b).

The engagement of Ripr with both PTRAMP and CSS in the AlphaFold 3 predictions was similar in all Plasmodium spp. but was inconsistent with existing biophysical data for P. falciparum which had suggested that PfCSS alone was sufficient for PfRipr binding18. However, this interaction was low affinity with the KD of CSS binding to Ripr in the low micromolar range18,19. In contrast, the PCR model predicted significant interaction of both PTRAMP and CSS with Ripr for P. falciparum, P. vivax and P. knowlesi. PfPTRAMP, PfCSS and PfRipr all contain multiple predicted N-linked glycan motifs and whilst there is very limited glycosylation in P. falciparum these proteins were expressed in insect cells and therefore are predicted to undergo extensive glycosylation (Fig. 1c)35. To further investigate PfPTRAMP-PfCSS (PfPC) - PfRipr binding, we expressed recombinant glycosylation-modified variants that either completely lacked glycans or had significantly reduced glycosylation (Fig. 1c, d).

Using biolayer interferometry (BLI), we determined that non-glycosylated forms of PfRipr and PfPC interacted with a KD of 158.5 nM (Fig. 1e), representing a ~10-fold stronger interaction compared to previous measurements (1–4 μM)18,19 and equivalent to the affinity measured between Rh5 and CyRPA (179 nM)36. The enhanced binding affinity of non-glycosylated proteins suggests that N-linked glycans on PfRipr and PfPC, added during heterologous expression, had interfered with their interaction interface in previous studies. Despite removal of a majority of the PfPTRAMP glycans monomeric PfPTRAMP showed no binding to PfRipr (Supplementary Fig. 2). These results suggest that the complete PfPC heterodimer is necessary for high-affinity PfRipr binding.

The P. vivax orthologs of PTRAMP and CSS form a disulfide-linked heterodimer
To investigate whether the disulfide-linked PC heterodimer is conserved as the basis for Ripr binding across Plasmodium species, as predicted by AlphaFold334, we co-expressed P. vivax PTRAMP and CSS orthologs in mammalian cells. The resulting PvPC heterodimer could be separated into its component monomers through reduction of the intermolecular disulfide bond (Fig. 2a). Nanobodies were raised against the purified PvPC heterodimer to enable further structural and biophysical characterization (Supplementary Fig. 3). Crystallization of the PvPC heterodimer was achieved by truncating the predicted disordered N-terminal repeat region of PvCSS and adding nanobody D7 (Fig. 2b, Supplementary Tables 1–3). The resulting structure confirmed that PvCSS adopts the previously characterized two-domain degenerate 6-Cys fold seen in PfCSS (Fig. 2c, Supplementary Fig. 4)18. No electron density was observed for either the growth-factor domain (GFD) or the thrombospondin repeat (TSR) domain of PvPTRAMP, despite space being available within the crystal lattice (Supplementary Fig. 4c). This suggests that the majority of PvPTRAMP was insufficiently stabilized within the crystal lattice to produce coherent diffraction. Nevertheless, clear electron density extended from PvCSS cysteine 122, the predicted site of disulfide formation with PvPTRAMP (Fig. 2d). Modeling of PvPTRAMP residues 42 to 53 revealed the structural basis for PvPC heterodimerization (Fig. 2e, f). Specifically, PvPTRAMP forms an interrupted β-strand that extends across both β-sheets of the CSS D1 domain, establishing multiple backbone interactions (Fig. 2c, f, Supplementary Table 2).

Alignment of available PTRAMP and CSS sequences showed that the two cysteines involved in heterodimerization are conserved across most species (Supplementary Figs. 5, 6). One exception is Plasmodium inui, which has tyrosine and serine substitutions in PTRAMP and CSS, respectively (Supplementary Figs. 5, 6). Nevertheless, AlphaFold33 modeling predicts a similar interface between PTRAMP and CSS in this region (Supplementary Fig. 1).

PvRipr binds the PvPC heterodimer to form a high affinity complex
Biophysical analysis revealed that PvPC binds to PvRipr with high affinity (KD = 28.8 ± 3.9 nM) (Fig. 3a, Supplementary Fig. 7a, b). While monomeric PvPTRAMP was sufficient for binding, it showed approximately 10-fold weaker affinity (KD = 292.5 ± 25.6 nM) (Fig. 3b). No interaction was detected between PvRipr and PvCSS (Fig. 3c), and none of the PvPCR components bound to PvCyRPA at the tested concentrations (Fig. 3d). Mass photometry analysis confirmed the formation of a stable PvPCR complex, with PvPC and PvRipr each displaying monodisperse peaks when analyzed individually (Supplementary Fig. 7c). Formation of the PvPCR complex, following incubation of PvPC and PvRipr, was evidenced by the emergence of a higher molecular weight peak corresponding to a mass of 207 ± 29 kDa which is consistent with a 1:1:1 complex of PTRAMP:CSS:Ripr (Fig. 3e). The formation of a stable complex was further validated by the co-elution of PvPC and PvRipr in size-exclusion chromatography (Supplementary Fig. 7d).

The C-terminus of Ripr is sufficient for PTRAMP-CSS binding in multiple species of Plasmodium
Previous studies have demonstrated that a three-membered PTRAMP-CSS-Ripr complex is involved in P. knowlesi invasion16. We confirmed that PkPTRAMP and PkCSS form a disulfide-linked heterodimer analogous to those observed in P. falciparum and P. vivax (Fig. 4a) and found that the PkPC heterodimer bound PkRipr with high affinity (KD = 0.6 ± 0.1 nM) (Fig. 4b, Supplementary Fig. 8a, b). Monomeric PkPTRAMP, but not monomeric PkCSS, was sufficient for this interaction (Fig. 4c, d). Like its P. vivax orthologs, PkPCR formed a stable complex as demonstrated by mass photometry, with a molecular weight of 206 ± 13 kDa consistent with a 1:1:1 stoichiometry (Fig. 4e, Supplementary Fig. 8).

We performed a comparative biophysical analysis of PC-Ripr binding to identify the minimal regions of Ripr required for complex formation. Several truncations in the Ripr tail region were generated for P. falciparum, P. vivax, and P. knowlesi proteins and assessed for their ability to bind their cognate PC heterodimer (Supplementary Fig. 9)19. The tail region of Ripr, which encompasses epidermal growth factor (EGF)-like domains 5–10 and the C-terminal domain (CTD), was sufficient for heterodimer binding in all three species with no observable impact on affinity (Fig. 4f, Supplementary Fig. 9)19. A shorter construct containing only EGFs 9 and 10 plus the CTD also retained the ability to bind the heterodimer (Fig. 4f). Interestingly, complete removal of all EGF domains, leaving only the CTD, abolished binding for P. falciparum proteins but not for P. vivax or P. knowlesi (Fig. 4f). These results demonstrate that heterodimer binding requires only a discrete region of Ripr, with some species-specific differences in the minimal binding requirements.

Anti-PCR antibodies are cross-reactive and differentially inhibit Plasmodium spp. invasion
Plasma samples from P. falciparum37, P. vivax38 and P. knowlesi39,40 infected individuals from Thailand (Tha Song Yang) and Malaysia (Sabah) were assessed to determine the extent of patient antibody response to the components of the PCR/PCRCR invasion complexes (Supplementary Fig. 10). IgG antibodies were assessed one week after clinical presentation and compared with malaria-naïve negative controls (IgG temporal kinetics from clinical presentation, one week, and one month post infection is shown in Supplementary Fig. 10). Significant IgG antibody reactivity was detected for P. falciparum patients against the PCRCR complex components except for PfCyRPA (Fig. 5a). Antibodies from P. vivax patients showed reactivity to the PvPCR components PvPC, PvCSS and PvRipr, but not PvPTRAMP (Fig. 5a). Similarly, antibodies from P. knowlesi patients showed reactivity to PkPC, PkCSS and PkRipr but not PkPTRAMP. Overall, there was a consistently low response to monomeric PTRAMP compared to other antigens and a consistently high response to monomeric CSS suggesting that the antibody response to the PC heterodimer is predominantly against CSS. High antibody responses were also observed for Ripr from all species and is consistent with Ripr being immunodominant as reported previously41. Furthermore, antibody responses to CSS and Ripr were broadly cross-reactive, with the antigens of all three species cross-reacting with sera from individuals independent of infective species (Supplementary Fig. 10).

We sought to investigate the potential of antibodies and nanobodies to inhibit growth of multiple Plasmodium species, given the serological cross-reactivity observed. Monoclonal antibodies (mAbs) and nanobodies generated against PvPC and mAbs against PvRipr were evaluated for cross-reactivity with their P. falciparum and P. knowlesi orthologs (Supplementary Figs. 11–13). All tested antibodies bound to PkPC and PkRipr, with four out of seven showing cross-reactivity across all three species (Fig. 5b, Supplementary Figs. 11, 12)42. All anti-PvPC nanobodies were cross-reactive with PkPC, however, this cross-reactivity was much lower against PfPC with only one out of eight binding PfPC (Supplementary Fig. 13). Epitope binning revealed that both 2D9 and 4E2, which bind PvPC, compete for Ripr binding. Of all the Ripr binding antibodies, only 4H10, which binds the tail of Ripr excluding EGF 6–8, competes for PvPC binding. Notably, anti-Ripr mAbs 5B3 and 5B4 both recognize EGF 6–8, a region previously associated with inhibitory activity in P. falciparum (Supplementary Fig. 12d)41,43. Growth inhibition assays (GIAs) were performed against P. knowlesi to assess the inhibitory potential of anti-PvPC nanobodies and anti-PvPCR antibodies. Initial screening revealed that two anti-Ripr antibodies (5B3 and 5B4) and both anti-PC antibodies (2D9 and 4E2) inhibited parasite growth at 0.5 mg/mL (Fig. 5c). This inhibition was dose-dependent, with 5B3 and 5B4 showing half-maximal effective concentrations (EC50) of 74 µg/mL and 520 µg/mL respectively, while 2D9 and 4E2 exhibited an EC50 of 635 µg/mL and 892 µg/mL respectively (Fig. 5d). The cross-reactive mAb 5B3 also inhibited P. falciparum growth, albeit with a significantly higher EC50 of approximately 2 mg/mL (Fig. 5e). In contrast, neither 4E2 nor 2D9 inhibited P. falciparum growth, despite showing moderate inhibition in P. knowlesi. This reduced inhibitory effect may be attributed to varying affinities of these antibodies for different Ripr orthologs (Supplementary Fig. 12e). The mAb 4H10 showed no inhibitory activity in P. knowlesi or P. falciparum18.

Following screening of the antibodies in P. knowlesi and P. falciparum GIAs (Fig. 5c–e, Supplementary Fig. 14), we assessed their potential inhibitory effect on P. vivax merozoite invasion and parasite growth in ex vivo GIAs. Assays performed on Cambodian P. vivax parasites revealed no inhibitory effect for any of the tested antibodies (Fig. 5f). To validate the P. vivax results, we evaluated a subset of these antibodies for their ability to inhibit growth in the closely related species P. cynomolgi44. The data from these assays were consistent with the P. vivax findings, confirming that none of the antibodies could inhibit parasite growth in either of these two species (Fig. 5g). These results demonstrate that while antibodies against the PCR complex may exhibit cross-reactivity across recombinant PCR complexes from multiple species of Plasmodium, this cross-reactivity does not necessarily correlate with growth inhibitory capacity. As these antibodies were raised against the P. vivax protein, these results either suggest minor functional differences between the complexes of P. falciparum and P. knowlesi compared to P. vivax and P. cynomolgi or that the PCR components are less critical for invasion of P. vivax and P. cynomolgi.

The anti-PvRipr antibody 5B3 targets a conserved epitope on EGF 7 and 8 of Ripr
To understand the molecular basis of the cross-reactivity exhibited by the inhibitory antibody 5B3, we determined the crystal structure of the antigen-binding fragment (Fab) of 5B3 in complex with PvRipr EGF7–8 (Fig. 6, Supplementary Table 4). Both EGF7 and EGF8 adopt canonical EGF-like folds, comprising two antiparallel β-sheets and three surface-exposed loops. The domains are connected via a short EGF7 linker (residues Phe801–Asn803)45–47. 5B3 primarily engages the β-sheets, B-loop, and C-loop of EGF7, with additional contacts to the EGF7 linker and the 310-helix of EGF8 (Fig. 6a). Sequence alignment of Ripr EGF7–8 domains from P. vivax, P. knowlesi, P. falciparum and P. cynomolgi orthologs revealed 71% sequence similarity. 79% of the total surface area on PvRipr EGF7-8 buried by antibody 5B3 is conserved across species, providing a structural explanation for its cross-species reactivity. A small number of polymorphic residues, such as Glu786 and Asn802, contribute to hydrogen bonds or salt bridges but make only moderate contributions to the total buried surface area (BSA) (Fig. 6b, Supplementary Table 5). The antibody-antigen interface is dominated by heavy chain contacts, with interactions mediated by all three heavy complementarity-determining regions (HCDRs) and framework region 3 (HFR3). In contrast, the light chain contributes only a single contacting residue, Trp91, from LCDR3. Most interactions involve conserved epitope residues (Fig. 6c, Supplementary Table 5), consistent with 5B3’s cross-reactivity. Among the few polymorphic contact residues Glu786 in PvRipr forms both a salt bridge and a hydrogen bond with H-Arg73, as well as an additional hydrogen bond with H-Ser28 (Fig. 6d). Substitution with valine in PfRipr would abolish those specific interactions, yet all van der Waals interactions with HCDR1 and HFR3 are predicted to be preserved. Similarly, Asn802 interacts with multiple tyrosine residues in HCDR1 and HCDR3. Structural modeling suggests that the Asn802Glu substitution in PfRipr is well tolerated owing to the similar side chain size, with sufficient spatial accommodation and the potential to form additional hydrogen bonds within the tyrosine-rich environment, without disrupting existing interactions (Fig. 6e). Together, these data reveal that 5B3 engages a highly conserved epitope across Ripr orthologs. While a limited number of polymorphic Ripr residues contribute to the interface, they do not abrogate binding, supporting 5B3’s broad reactivity against Ripr from multiple Plasmodium species.

Cryo-EM analysis of PkPCR supports AlphaFold predictions
To provide more confidence in the predicted models of the PCR complexes, and to understand how inhibitory antibodies may function, we carried out cryo-electron microscopy (cryo-EM) experiments on the PkPCR complex. Cryo-EM analysis of the PkPCRtail complex revealed an overall shape consistent with the AlphaFold prediction (Fig. 7a, Supplementary Fig. 15). The addition of 5B3 Fab allowed unambiguous assignment of the orientation of the two-dimensional (2D) classes (Fig. 7a). Furthermore, comparison of Fab bound and unbound classes showed no discernible differences in the PCR complex which confirmed that 5B3 binds to the tail region of Ripr without interfering with complex formation (Fig. 5b, Supplementary Figs. 12, 15). This suggests that parasite inhibition by 5B3 likely has a direct effect on Ripr function during invasion rather than on the complex.

The cryo-EM data, combined with the PvPC crystal structure, strongly support the predicted PCR complex structure (Fig. 7b). In this complex, the PTRAMP-CSS heterodimer is formed by an intermolecular disulfide bond. This heterodimer engages Ripr via two interfaces: PTRAMP clinching the CTD of Ripr, and the D2 domain of CSS acting with EGF9 of Ripr (Fig. 7b). The PTRAMP-Ripr interface is predicted to involve several complementary electrostatic interactions between the CTD of Ripr with both the GFD and TSR of PTRAMP. The CSS-Ripr interface is predicted to be predominantly hydrophobic in nature, with the two β-strands of EGF9 packing against the α-helix of CSS and its neighboring β-strand. In total, the predicted interface formed between PkRipr and the PkPC heterodimer has a BSA of approximately 2,000 Å2. The remaining mass of Ripr likely extends below the PCR complex, where it may interact with other invasion proteins.

While PfPC lacks the ability to bind erythrocytes directly, it enhances Rh5 binding when incorporated into the PCRCR complex18. Previous studies have shown that PkPTRAMP can bind erythrocytes; however, these experiments were performed with monomeric PkPTRAMP and not heterodimeric PkPC16. We performed flow-cytometry based erythrocyte binding assays to assess whether PkPC or the PkPCR complex bound to erythrocytes. Neither PkPC nor PkPCR showed significant binding to erythrocytes relative to the positive control, PfRh5 (Fig. 7c, Supplementary Fig. 16a). Considering that P. vivax invades reticulocytes exclusively, we extended our investigation to include reticulocyte binding assays. Similarly, we observed no binding of PvPC or PkPC to reticulocytes (Fig. 7d, Supplementary Fig. 16b). Collectively, our analysis found no evidence of erythrocyte or reticulocyte binding by PkPC, PvPC, or the PkPCR complex.

We therefore hypothesize that PTRAMP, CSS and Ripr form a core invasion scaffold in Plasmodium parasites. This scaffold provides the basis for the assembly of species-specific complexes that are adapted for binding a diverse set of host erythrocyte receptors (Fig. 7e). In P. falciparum this complex incorporates CyRPA and Rh5 which facilitate invasion via the essential interaction with basigin. The equivalent proteins in P. vivax and P. knowlesi that are responsible for erythrocyte binding are yet to be identified. If findings from P. falciparum are applicable to these species, these interactions will be an essential step in merozoite invasion.

Methods
Recombinant protein expression
All gene sequences used were retrieved from the VEuPathDB (accessed through www.plasmodb.org)51 from reference strains (3D7 for P. falciparum, PvP01 for P. vivax (with the exception of PvRBP2b for which the Sal-1 sequence was used), and strain H for P. knowlesi). All genes were synthesized by Genscript (Singapore) unless otherwise stated. Expi293F cells (Life Technologies) as above.

P. falciparum
Recombinant PfRh5, PfCyRPA, PfPTRAMP, PfPC and PfRipr constructs were produced as described previously, with some changes made to the synthetic gene constructs used for expression18. PfPTRAMP, comprising residues 31 to 307, was subcloned into the pAcGP67a vector with a C-terminal C-tag. Four potential N-linked glycosylation sites were removed, at positions Asn112, Asn149 and Asn155 by mutation to Gln, and at position Asn195 by mutation of Thr197 to Ala, to produce PfPTRAMP_31-307_4x. To produce a large amount of pure monomeric PfPTRAMP, another construct was made that contains all the same mutations and also contains Cys60 Ser mutation to prevent disulfide formation, termed PfPTRAMP_31-307_4xC60S. PfCSS was subcloned into the pAcGP67a vector with a C-terminal FLAG-tag preceded by a TEV protease cleavage site. This construct has all six potential N-linked glycosylation sites removed at positions Asn74, Asn88, Asn192, Asn234, Asn261 and Asn283 by mutation of Ser76, Thr90, Ser194, Thr236 and Thr263 to Ala and Asn283 to Gln, to produce PfCSS_21-290_6×18. The previous PfRipr construct18 was altered with the following mutations: Thr966Ala-Ser1023Ala yielding the construct PfRipr_20-1086_2xA. PfRipr tail (aa 717-1086), PfRiprEGF 9,10,CTD (aa 899-1086) and PfRiprCTD (aa 981-1086), were all synthesized by Genscript and purified in an identical fashion to PfRipr.

P. vivax
The pvptramp gene (PVP01_1436800, aa 21-297) excluding the transmembrane and cytoplasmic domains was subcloned into a modified pTRIEX2 vector that contains an N-terminal Small Ubiquitin-like Modifier (SUMO)-Flag tag followed by a Tobacco Etch Virus (TEV) protease cleavage site (from here on termed SFT). Potential N-glycosylation sites were assessed and one site, Asn115, was removed by mutation of Ser117 to Ala. This yielded SFT_PvPTRAMP_21-297_S117A. This construct was then further cloned to incorporate a C-terminal Avitag, yielding SFT_PvPTRAMP_21-297_S117A_Avi. Both constructs were expressed in Human Embryonic Kidney (HEK) Expi293F cells (Life Technologies) as secreted soluble proteins. Transient transfection was carried out as per the manufacturer’s protocol and the culture medium harvested 5–6 days post-transfection. The proteins were purified via multiple rounds of binding and eluting using Anti-Flag M2 Affinity Gel (Merck) and 100 μg/mL of Flag peptide (Genscript) in HBS (20 mM HEPES pH 7.2, 150 mM NaCl). The eluted fractions were pooled and incubated with TEV protease (1 mg of TEV for every 10 mg of protein) overnight at 4°C. His-tagged TEV was removed by applying the protein solution to nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (Qiagen) and collecting the flow through. The flow through was then concentrated on a 10,000 dalton (Da) Molecular Weight Cut-Off (MWCO) Amicon Ultra-15 Centrifugal Filter (Merck) and applied to an S75 Increase 10/300 column (Cytiva) connected to an AktaPure (Cytiva) to separate the TEV-cleaved PvPTRAMP from the SUMO-Flag tag. Peak fractions were assessed for purity via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and pure fractions were pooled and concentrated. Glycerol was added (10% v/v final) to the concentrated protein and then aliquoted and flash frozen in liquid nitrogen (LN2).

The full-length pvcss gene (PVP01_1344100, aa 22-381) was subcloned into a modified pTRIEX2 vector with a C-terminal Flag-tag preceded by a TEV protease cleavage site (from here on termed TF). N-glycosylation sites were predicted and three potential sites, Asn114, Asn180, Asn352, were mutated via three mutations: Ser116Ala, Thr182Ala and Ser354Ala. This yielded PvCSS_22-381_S116A_T182A_S354A_TF. This construct was further cloned to remove a predicted N-terminal repeat region to aid in crystallization. This yielded the construct PvCSS_115-381_S116A_T182A_S354A_TF. These constructs were expressed as soluble secreted proteins in HEK Expi293F cells (Life Technologies) as above.

To generate disulfide-linked PvPTRAMP-PvCSS (PvPC), PvPTRAMP and PvCSS were co-expressed in HEK Expi293F cells (Life Technologies) at a ratio of 50:50 PvPTRAMP:PvCSS for full length CSS and a ratio of 40:60 PvPTRAMP:PvCSS for PvCSS_115. The expression and purification were carried out as above with size-exclusion chromatography performed using either an S200 Increase 10/300 GL (Cytiva) or S200 16/600 HiLoad (Cytiva).

The full-length pvripr gene (PVP01_0816800, aa 22-1074) was subcloned into pAcGP67a with a C-terminal 6xHis-tag yielding PvRipr_22-1074_His and expressed in Sf21 cells using the flashBAC ULTRA baculovirus system (Oxford Expression Technologies). After initial transfection, the P1 virus was amplified and titrated several times to produce a P3 virus. This P3 virus was used for large scale expression. The proteins were expressed as soluble secreted proteins. The culture medium was harvested 3 days after the addition of P3 virus. The media was concentrated via tangential-flow filtration with a 3000 Da MWCO (Merck) to reduce the volume ~10-15 fold. This resultant concentrate was then dialyzed into TBS (20 mM Tris pH 8.5, 150 mM NaCl) overnight at 4°C. Imidazole was then added to a final concentration of 10 mM and the culture media passed over Ni-NTA Agarose resin (Qiagen), washed with TBS+ 20 mM imidazole, and eluted in TBS+ 500 mM imidazole. The eluted protein was then concentrated on a 30,000 Da MWCO Amicon Ultra-15 Centrifugal Filter (Merck) and applied to an S200 Increase 10/300 GL column (Cytiva) connected to an Akta Pure (Cytiva). Peak fractions were pooled, concentrated and flash frozen as above. PvRipr truncations (PvRipr tail (aa 665-1074), PvRiprEGF9,10,CTD (aa 844-1074), PvRiprCTD (aa 972-1074)) were synthesized via the Genscript mutagenesis service. The truncated constructs were subcloned into the pAcGP67a vector and purified in an identical manner to the full-length construct.

PvRiprEGF 6-8 (aa 717-843) was synthesized and subcloned into pET28a yielding a construct with an N-terminal 6xHis tag followed by a TEV site. Expression was carried out in Escherichia coli (E. coli) strain SHuffle® T7 (New England Biolabs) grown in terrific Broth with 40 μg/mL of kanamycin. One litre of culture was grown in incubators at 37°C and shaking at 180 revolutions per minute (rpm) until an optical density at 600 nm (OD600) of around 1.0 was reached. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) (Astral) was then added to a final concentration of 1 mM, and protein expression was continued at 16°C for 16–18 h. Cells were then harvested via centrifugation and the pellet resuspended in TBS pH 8.5, and with cOmplete ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche). The resuspended cells were then sonicated, and the cellular extract clarified by centrifugation at 30,000 × g for 30 minutes at 4°C. Imidazole was added to the clarified supernatant to a final concentration of 10 mM and then passed over pre-equilibrated Ni-NTA resin, washed with TBS pH 8.5+ 20 mM imidazole, and then eluted in TBS pH 8.5 containing 500 mM imidazole. The eluted protein was then concentrated on a 10,000 MWCO Amicon Ultra-15 Centrifugal Filter (Merck) and injected onto an S200 16/600 HiLoad (Cytiva) equilibrated in TBS pH 8.5. Peak fractions were then concentrated, supplemented with glycerol to a final concentration of 10% (v/v) and flash frozen in liquid nitrogen.

PvRiprEGF 7-8 (764-843) was synthesized and subcloned into pcDNA34CK vector with the addition of a N-terminal 6xHis tag followed by a TEV site. Two predicted N-glycosylation sites, Asn767 and Asn780, were removed with the following mutations: Asn767Gln, Asn780Gln. This yielded the construct PvEGF7-8N767Q,N780Q. The protein was expressed in HEK Expi293F cells and purified in a similar manner to PvPTRAMP above. The proteins were purified via binding and eluting using a 5 mL HisTrap Excel column (Cytiva) in PBS with 500 mM imidazole, followed by size-exclusion chromatography using an S200 Increase 10/300 column (Cytiva) connected to an Akta Pure (Cytiva) in TBS pH 8.0. Peak fractions were pooled and incubated with TEV protease overnight at 4°C. His-tagged TEV was removed by applying the protein solution to Ni-NTA Agarose resin and collecting the flow through. Protein was assessed for purity via SDS-PAGE. The flow through was then buffer exchanged into TBS pH 8.0 and concentrated on a 3000 Da MWCO Amicon Ultra-4 Centrifugal Filter (Merck). Glycerol was added (10% v/v final) to the concentrated protein and then aliquoted and flash frozen in liquid N2.

The pvcyrpa gene (PVP01_0532400, aa 24-362) was subcloned into pTRIEX2-TF vector which yielded PvCyRPA_22-362_TF. Two predicted N-glycoslyation sites, Asn78 and Asn282, were removed with the following mutations: Thr80Ala, Thr284Ala. The protein was expressed in HEK Expi293F cells and purified in an identical manner to PvPTRAMP above.

PvRBP2b (PVX_094255, aa 161-1454) was purified as described previously52.

P. knowlesi
The pkptramp gene (PKNH_1437600, aa 21-297) excluding the transmembrane and cytoplasmic domains was subcloned into a modified pTRIEX2-SFT as was done for PvPTRAMP. Potential N-glycosylation sites were assessed and two sites, Asn115 and Asn261, were removed by mutation of Ser117 and Ser263 to Ala. This yielded SFT_PkPTRAMP_21-297_S117A_S263A. A PkPTRAMP construct expressing a C-terminal Avitag for biotinylation was made using PCR and restriction digests to yield SFT_PkPTRAMP_21-297_S117A_S263A-Avi. Expression of PkPTRAMP and PkPTRAMP-Avi was carried out in an identical manner to PvPTRAMP described above.

The pkcss gene (PKNH_1353400, aa 22-362) was subcloned into pTriEX2-TF. Five potential N-glycosylation sites, Asn96, Asn161, Asn175, Asn243 and Asn333 were removed via five mutations: Ser98Ala, Thr163Ala, S177Ala, S245A and S335A. This yielded PkCSS_22-362_S98A_T163A_S177A_S245A_S335A_TF. Expression of PkCSS was carried out in an identical manner to PvCSS above.

Expression of the PkPC heterodimer was carried out in an identical manner as described for PvPC. The ratio of PkPTRAMP:PkCSS deoxyribonucleic acid (DNA) used was 60:40 when PkPTRAMP-SFT and PkCSS-TF were being used, and 50:50 when PkPTRAMP-Avi was being used.

The pkripr gene (PKNH_0817000, aa 22-1096) was subcloned into pAcGP67a with a C-terminal 6xHis-tag yielding PkRipr_22-1096_His, as per the PvRipr construct. Truncations of the full-length construct were made by Genscript using the mutagenesis service, to produce PkRipr tail (aa 669-1096), PkRiprEGF9,10,CTD (aa 848-1096) and PkRiprCTD (aa 994-1096). All PkRipr constructs were expressed and purified in an identical manner to the equivalent PvRipr constructs.

For all constructs containing an Avitag, in vitro biotinylation was carried out as previously described53.

Antibodies and nanobodies
One alpaca was subcutaneously immunized six times 14 days apart with 130 μg (800 μg total) of recombinant PvPC. GERBU FAMA (GERBU Biotechnik GmbH, Heidelberg, Germany) was used as an adjuvant. Whole blood was collected three days after the last immunization for the preparation of lymphocytes. Nanobody library construction was carried out according to established methods54. Briefly, alpaca lymphocyte mRNA was extracted and amplified by reverse transcription PCR (RT-PCR) with nanobody-encoding, gene-specific primers. This produced a library of nanobody cDNA sequences that contained approximately 108 sequences. The sequences that were cloned into the pMES4 phagemid vector were amplified in E. coli TG1 strain and subsequently infected with M13KO7 helper phage for downstream recombinant phage expression.

Handling of the alpaca for scientific purposes was approved by Agriculture Victoria, Wildlife and Small Institutions Animal Ethics Committee, project approval No. 26-17.

Biopanning was performed over two rounds with 1 μg of immobilized antigen as previously described54. Ninety-four positive clones were taken for further screening via enzyme-linked immunosorbent assay (ELISA). Clones showing positive binding by ELISA (n = 93) were sequenced. Of these, 71% were full length Variable Heavy domain of Heavy chain (VHH) (n = 66).

Nanobodies were expressed in the periplasm of E. coli WK6 cells as described previously55. Briefly, bacteria (250 mL) were grown in Terrific Broth at 37°C to an OD600 of 0.7. The cultures were then induced with 1 mM IPTG (Astral) and grown overnight at 28°C. Cells were harvested and resuspended in PBS containing 20% sucrose and 20 mM imidazole to rupture the periplasm. EDTA was added to a final concentration of 5 mM, and the cells were incubated on ice. MgCl2 was then added to a final concentration of 10 mM, and the periplasmic extract was harvested via centrifugation. The nanobodies were purified via standard Ni-NTA purification methods.

Monoclonal antibodies were raised in mice as per the Walter and Eliza Hall Animal Ethics Committee approved procedures. All monoclonal antibodies were produced by the WEHI Antibody Facility. Mice were injected with 80–180 μg of protein three times and then boosted once with 30–60 μg. After cloning of hybridomas, the supernatants were tested via ELISA and BLI. Based on these results, several hybridomas for each antigen were selected for further scale up of purified immunoglobulin G (IgG).

The plasmids of chimeric anti-PvRipr 5B3 Fab were synthesized by subcloning the variable domains of light and heavy chain of antibody 5B3 into pcDNA34CK and pcDNA34CH1 vectors respectively. The Fab was expressed in HEK Expi293F cells in a similar manner to PvPTRAMP above, with culture medium harvested 6 days post-transfection. The Fab was purified from the culture supernatant via binding and eluting using a 1 mL HiTrap MabSelect VL column (Cytiva) connected to an Akta Pure, in 50 mM sodium citrate buffer pH 2.5. The elution fractions are neutralized using 1 M Tris buffer pH 9.0, and the Fab was assessed for purity via SDS-PAGE. The elution fractions were pooled, concentrated and buffer exchanged into PBS on a 10,000 Da MWCO Amicon Ultra-15 Centrifugal Filter (Merck). The purified Fab was stored at 4°C.

Structure prediction
Prediction of PCR complexes from multiple Plasmodium species was done using the AlphaFold3 server34.

Biolayer interferometry (BLI)
Biolayer interferometry (BLI) experiments were carried out on an Octet Red96e (Sartorius) at 25°C. For kinetics analysis ligands were immobilized onto either anti-penta-His (His1K), streptavidin (SAX or SAX2) or Ni-NTA (NTA) biosensors (Sartorius) depending on the affinity tag present on the protein (His-tag or biotinylated Avitag). Ligands were diluted to 10–40 μg/mL in 1x kinetics buffer (PBS, pH 7.4, 0.1% (w/v) bovine serum albumin (BSA), 0.02% (v/v) Tween-20) prior to immobilization. Biosensors were initially dipped in kinetics buffer for 30–60 s to establish a baseline signal, and then dipped into wells containing the ligand, followed by another 30–60 s baseline. After the second baseline step, the ligands were then dipped into wells containing two-fold dilution series of analyte. Association was measured for 120 s and then the biosensors were dipped into kinetics buffer to measure the dissociation for another 120 s. Data were analysed using Sartorius Data Analysis software 11.0. Kinetic curves were fitted using a 1:1 binding model.

Competition studies for anti-PvPC nanobodies were performed using Ni-NTA (NTA) biosensors (Sartorius) with His-tagged nanobodies as the ligand (diluted to 5 μg/mL in kinetics buffer). After a 30 s baseline step, the biosensors were dipped into wells containing an irrelevant nanobody that does not bind to PvPC to quench the biosensor and ensure no free sites are present for the downstream steps. Following a second baseline step, the biosensors were dipped into PvPC diluted to 500 nM in kinetics buffer. After loading of PvPC onto the biosensors, a final baseline step was performed before the biosensors were dipped into either secondary nanobody (at 10 μg/mL diluted in kinetics buffer) or PvRipr (at 200 nM diluted in kinetics buffer). Data were analysed using Sartorius’ Data Analysis software 11.0 and the epitope bins were assessed by normalization and manual curation.

Antibody kinetics were determined similarly to the above methods. Anti-Mouse IgG Fc Capture (AMC) biosensors (Sartorius) were used to immobilize mouse monoclonal antibodies at a concentration of 5–20 μg/mL in kinetics buffer. Antibody competition studies were carried out in a similar manner to the nanobodies, however anti-pentaHis (His1K) biosensors were used.

Protein crystallization
PvPC_115 was purified as above. PvPC and nanobody D7 were co-complexed with the nanobody at 3x molar excess. The free nanobody was separated from the PvPC-nanobody complex by size-exclusion chromatography on an S200 Increase GL 10/300 (Cytiva) in HBS. Peak fractions were pooled and concentrated to ~5–6 mg/mL and setup in coarse screen sitting drop crystal trays at the Monash Macromolecular Crystallization Facility. Needle-like crystals formed after ~9 days in 0.2 M ammonium sulfate ((NH4)2SO4) and 20% (w/v) polyethylene glycol (PEG) 3350. Further in-house optimization of conditions yielded large crystals in 0.2 M (NH4)2SO4 and 16% (w/v) PEG-3350. Crystals were looped in mother liquor containing 10% (v/v) glycerol and flash frozen in liquid nitrogen. Diffraction data were collected with the MX2 beamline at the Australian Synchrotron (Clayton, Australia) at 100 K (λ = 0.9537 Å). Statistics are in Supplementary table 1.

PvEGF7-8N767Q,N780Q and 5B3 Fab were purified as above. PvEGF7-8N767Q,N780Q and 5B3 Fab were co-complexed with PvEGF7-8_N767Q_N780Q at 2x molar excess. Free PvEGF7-8_N767Q_N780Q was separated from the co-complex by size-exclusion chromatography on an S200 Increase GL 10/300 (Cytiva) in HBS pH 7.5. Fractions were assessed for purity and co-complexation via SDS-PAGE. Peak fractions were pooled and concentrated to 5 mg/mL and setup in coarse screen sitting drop crystal trays at the Bio21 Macromolecular Crystallization Facility. Thin-plate crystals formed after ~4 days in 0.2 M (NH4)2SO4 and 25% w/v PEG 3350. Further in-house optimization of conditions yielded singular rod-like crystals in 0.2 M (NH4)2SO4, 20% PEG-3,350 and 0.1 M sodium acetate buffer pH 4.4. Crystals were looped in mother liquor containing 15% (v/v) glycerol and flash frozen in LN2. Diffraction data were collected with the MX2 beamline at the Australian Synchrotron (Clayton, Australia) at 100 K (λ = 0.9537 Å). Statistics are in Supplementary table 4.

Structure determination and model building
Both diffraction data were processed with the XDS package56 before being scaled and merged using Aimless57 in the CCP4 suite58. The program Matthews59 was used to estimate the number of molecules in the asymmetric unit.

For the structure of PvPC-D7 an AlphaFold233 model of PvCSS constituting residues 115-381 was used as a search model for molecular replacement using Phaser60. After 3 copies of PvCSS were fitted, additional searches were performed with a nanobody structure. To ensure the best fit possible, a BLASTp (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) search was performed with nanobody D7 to find the structure with the highest sequence similarity for molecular replacement searches. This search yielded a nanobody (PDB: 7N0R), which was used as a search model with the complementarity determining region 3 (CDR3) sequence removed61. The structure was then iteratively refined in Phenix62 and assessed and modified with Coot63. Clear density extended from the unpaired cysteine in PvCSS, C122, that was not accounted for by either PvCSS or nanobody D7. Due to the crystals being setup with the PvPC heterodimer, it was reasonable to conclude that this density belonged to PvPTRAMP. PvPTRAMP residues 41-53 were built into the electron density de novo independently for each of the three molecules in the asymmetric unit. Refinement and model statistics are described in Supplementary Table 1. For analysis of the contacts formed between PvPTRAMP and PvCSS, and PvCSS and D7, the program Contact (part of CCP4 suite)58 was used in conjunction with PISA server (https://www.ebi.ac.uk/pdbe/pisa/) and are summarized in Supplementary Tables 2 and 3. Nanobodies were renumbered according to the Kabat numbering system, as determined by ANARCI63.

For the structure of PvRipr EGF7-8 in complex with 5B3 Fab, 1 molecule was estimated in the asymmetric unit. The AlphaFold2 prediction of PvRipr EGF7-8 and the crystal structure of an HIV-2 neutralizing Fab fragment (PDB: 3NZ8) with the CDRs deleted, were used as models for molecular replacement using Phaser. Alternating rounds of structure building and refinement were carried out using Coot and Phenix. Refinement and model statistics are found in Supplementary Table 4. Interactions between PvRipr EGF7-8 and 5B3 were analyzed using PISA and CCP4 Contact and are summarized in Supplementary Table 5. 5B3 was renumbered according to the Kabat numbering system, as determined by ANARCI.

Multiple sequence alignment
Multiple sequence alignments were computed using ESPript3.064.

Mass photometry
Mass photometry experiments were carried out on a TwoMP mass photometer (Refeyn). Each well was focused after the addition of 10 μL of filtered PBS. Once focused, 10 μL of protein at either 50 nM (P. knowlesi) or 100 nM (P. vivax) was added, mixed, and events were recorded for one minute using AcquireMP (Refeyn). Raw data processing was done in DiscoverMP (Refeyn) and the data exported and presented using Prism v9 (GraphPad). In-house recombinant mouse mixed lineage kinase-like (MLKL), human glutamine synthetase and human catalase were used for the construction of a calibration curve.

Human samples
Human plasma samples were utilized from three cohorts of Plasmodium infected patients along with three cohorts of malaria-naïve negative controls. Patients infected with P. vivax were recruited from Tha Song Yang, Thailand, during the year 2014, as previously described38, with a subset of 34 included in the current study. Patients infected with P. falciparum37 and P. knowlesi39,40 were recruited from Sabah, Malaysia, during the years 2010–2018 and 2012–2014, respectively. P. falciparum (n = 31) and P. knowlesi (n = 33) infected patients were included in the current study. Plasma samples were assayed from time of clinical presentation, 1 week later, and 1 month later. 28 malaria-naïve samples from the Melbourne Volunteer Biospecimen Donor Registry (VBDR) and 29 malaria-naïve samples from the Thai Red Cross (TRC) were utilized to create seropositivity cut-offs. Individuals from the TRC donated blood in Bangkok, a malaria-free region of Thailand, and had not had malaria diagnosed in the year prior nor had they traveled to endemic regions in the prior three years, as previously described65. An additional set of afebrile healthy controls (n = 30) were assayed from Sabah, Malaysia; however, these individuals may have had prior Plasmodium infections and were thus not utilized to create the seropositivity cut-off.

Ethical approval for sample use was provided by WEHI Human Research Ethics Committee (14/02), with original study approval in Thailand (Faculty of Tropical Medicine, Mahidol University, MUTM 2014-025-01 and 02) and Malaysia (Menzies School of Health Research, HREC 12-1815, 16-2544, 10-1431, 12-1807). All individuals gave informed consent and/or assent to participate in the studies.

Multiplexed antibody assay
Recombinant Plasmodium proteins were coupled to unique regions of magnetic, fluorescent, Bio-Plex microbeads (Bio-Rad) following the manufacturer’s instructions and as previously described66. Briefly, 200 μL of microbeads were washed then activated for 20 min with sulfo-N-hydrosuccinimide (50 mg/mL) and N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) (50 mg/mL) in monobasic sodium phosphate (pH 6.2). Following further washing, the activated microspheres were resuspended in PBS with 1–6 μg of Plasmodium protein. After overnight incubation, the coupled beads were washed and then stored in PBS-TBN (PBS, 0.1% (w/v) BSA, 0.02% (v/v) TWEEN-20, 0.05% (w/v) sodium azide, pH 7) at 4°C until further use. Microbeads were always kept protected from light.

Plasma samples were diluted in PBT (1X PBS, 1% (w/v) BSA, 0.05% (v/v) Tween-20) at a dilution of 1:100. For P. vivax and P. falciparum antigens, plasma samples from hyper-immune individuals from PNG were used as a positive control. For P. knowlesi antigens, plasma samples from acutely infected P. knowlesi patients were used as the positive control. Both positive control pools were used to create a modified reference standard curve, starting at 1:50 with a 5 point 5-fold serial dilution. Diluted samples (50 μL) all controls and patients) were added to black flat-bottom 96-well plates and mixed with 50 μL of the coupled-antigen bead mixture (0.1 μL of each coupled antigen per well in PBT), then incubated for 30 minutes. The plate was washed and then 100 μL of 1:100 phycoerythrin (PE)-conjugated anti-human secondary antibody (Jackson Immunoresearch) was added and incubated for a further 15 min. Plates were washed then resuspended in PBT before being read on a MAGPIX instrument. Median fluorescent intensity was converted to arbitrary relative antibody units (RAU) using the standard curves, to adjust for plate-plate variation65.

Statistical analysis
An antigen-specific seropositivity cut-off was set as the mean of the negative controls (VBDR + TRC) plus two times the standard deviation. Data are presented as the fold change of the mean peak week 1 antibody response relative to the seropositivity cut-off. RAU values of samples and control cohorts are shown in Supplementary Fig. 10.

Growth inhibition assays
P. knowlesi growth inhibition assays were undertaken using P. knowlesi YH1 parasites over 2 cycles of growth (~64 h) using standard conditions67. Antibodies were initially screened at 0.5 mg/mL for inhibitory activity before 2-fold serial dilution dose response curves were undertaken to define potency for inhibitory antibodies. Parasitemia was determined using flow cytometry (BD Acurri) after staining with 10 mg/mL of ethidium bromide, with data analysed using FlowJo software (BD Life Sciences). P. knowlesi growth in the presence of antibodies was compared to that of untreated control wells to define growth inhibitory activity. All experiments were performed a minimum of three times with duplicate wells unless stated otherwise.

P. falciparum growth inhibition assays were performed as described previously18.

P. vivax growth was analysed using an ex vivo invasion assay performed as described previously with slight modification68. P. vivax samples were collected in 2023 from infected individuals in Kampong Speu, Western Cambodia. P. vivax infection was determined using rapid diagnostic testing (CareStartTM Malaria Pf/pan rapid diagnostic tests, Accessbio) or microscopy and species-specific PCR to ensure monoinfection. Venous blood was collected in lithium heparin tubes and immediately sent on ice to the Malaria Research Unit at Institute Pasteur, Cambodia. There, erythrocytes were separated from the plasma, and the plasma was discarded. Erythrocytes were then suspended in warm Roswell Park Memorial Institute (RPMI) medium before leukocyte depletion using a non-woven fabric filter. The work presented here was approved by the National Ethics Committee for Health Research in Cambodia (192 NECHR, July 11, 2022). All patients and/or their parents/guardians provided informed written consent for samples to be taken and used for these purposes.

Infected erythrocytes were enriched using a potassium chloride (KCl)-Percoll density gradient and then transferred into culture in supplemented Iscove′s Modified Dulbecco′s Medium (IMDM) (Gibco) (supplemented with 0.5% (w/v) Albumax II (Gibco), 2.5% (v/v) heat-inactivated human serum, 25 mM HEPES (Gibco), 20 μg/mL gentamicin (Sigma) and 0.2 mM hypoxanthine (C-CPro)). The stage of the parasite culture was then assessed via thin blood smear. In the case of a majority ring culture, parasites were allowed to mature through to the schizont stage (~40 h) before starting the experiment. If the culture consisted mainly of trophozoites, the experiment was carried out after 18-20 h. The enriched schizonts were then mixed 1:1 with reticulocytes (previously enriched from cord blood or adult peripheral blood from malaria-naïve donors) and pre-labeled with Celltrace Far Red Dye for quantitation. The cultures were incubated with either 500 μg/mL (anti-tetanus toxin 43038) or either 100 μg/mL or 500 μg/mL (monoclonal antibodies and nanobodies) of biologics in a volume of 50 μL in 384-well plates. Cells were stained with Hoechst 33342 to stain parasite DNA and parasitemia was quantified via flow cytometry, with new infections being defined as Far Red/Hoechst double-positive cells. For quantitation, data were normalized against parasites mock treated with PBS. Observed control invasion rates ranged from 0.46 to 5.3% (median = 0.7%).

P. cynomolgi assays were performed as previously described44. Both P. falciparum and P. knowlesi GIAs were carried out in parallel to P. cynomolgi assays to serve as positive controls for antibody inhibition. P. cynomolgi strain Berok R9 was maintained in rhesus red blood cells (Emory Primate Center) at 2% hematocrit in RPMI 1640 with 10% human O+ serum and gassed (1% O₂, 5% CO₂, 94% N₂) at 37°C. The invasion assay was setup with 0.2% hematocrit and 2–3% schizontemia in 30 μl volumes in 384-well plates using antibodies 2D9, 4H10, 5B3, 5B4, IgG control and heparin (positive control) After 12 h of incubation, the parasite DNA was stained with Vybrant™ DyeCycle™ Violet (Invitrogen), and 100,000 cells were analyzed via a Cytek-Northern Lights flow cytometer. Invasion was measured by the percentage of newly parasitized erythrocytes (CellTrace Far Red+/ Vybrant™ DyeCycle™ Violet+). Inhibition was assessed relative to control wells without antibodies. Data analysis was done using GraphPad Prism v10.

Electron microscopy
All electron microscopy was carried out at the Bio21 Ian Holmes Imaging Centre, University of Melbourne.

For negative staining, purified PkPCRtail + 5B3 Fab (at ~0.1 mg/mL) was applied to formvar and carbon coated, glow discharged copper grids (300 mesh, ProSciTech). Four microlitres of protein was incubated for one minute, then blotted off, washed twice in water, and then stained with 1% (w/v) uranyl acetate for two minutes before being blotted and dried thoroughly. The grids were then imaged on a Tecnai F30 operating at 200 kV. Two-dimensional classification was performed in cryoSPARC (v4.4.1)69.

For cryo-electron microscopy, freshly purified PkPCRtail and PkPCRtail + 5B3 Fab at 0.5–1 mg/mL was applied to glow discharged UltrAuFoil (Quantifoil MicroTools GmbH) grids (300 mesh, R1.2/1.3) or HexAuFoil (Quantifoil MicroTools GmbH) and then blotted for 5 seconds with a blot force of 7 (UltrAuFoil) or 10 (HexAuFoil) before being plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) operating at 4°C and 100% humidity. Grids were screened for good ice quality on an FEI Talos Arctica (Thermo Fisher Scientific) operating at 200 kV. Grids showing sufficient thin and amorphous ice were then transferred to an FEI Titan Krios G4 (Thermo Fisher Scientific) for data collection. Data were collected using an acceleration voltage of 300 kV and a Falcon 4i detector (Thermo Fisher Scientific) using EPU automation software. Data were collected over three sessions, two for PkPCRtail (from two independent grids) and one for PkPCRtail + 5B3. Pixel sizes used for collection were 0.506 Å/pixel with a total dose of 50 e-/Å2 for PkPCRtail and 0.808 Å/pixel with a total dose of 40 e-/Å2 for PkPCRtail + 5B3 and all datasets were collected with a nominal defocus range of −0.5 μm to −2 μm. CryoSPARC (v4.4.1-v4.6.2)69 was used for all data processing. Gain and motion corrected, and contrast transfer function (CTF)-estimated movie stacks were curated to select for good CTF fit and to remove micrographs that showed signs of significant drift, contained obvious frost contamination, or had no visible particles. This resulted in 8730 and 826 movie stacks for session one and two, respectively, for PkPCRtail and 3285 movie stacks for PkPCRtail + 5B3. The curated datasets were then used in multiple rounds of automated picking and 2D class averaging. For PkPCRtail 11,955 particles corresponded to the ‘front view’, and 3067 particles corresponded to the ‘side view’. For PkPCRtail + 5B3 13,478 particles corresponded to the ‘front view’, and 2694 particles corresponded to the ‘side view’. Whilst clear features were visible in this small number of classes, severe orientation bias and the small, flat, and elongated shape of the particles precluded three-dimensional reconstruction.

Reticulocyte enrichment for flow cytometric binding assays
Cord blood was obtained through a material transfer agreement (MTA, ID# M19/110) with the Bone Marrow Donor Institute (BMDI) at the Royal Children’s Hospital in Melbourne, Australia under the human ethics project “14/09, Malaria parasite growth and invasion into reticulocytes” which was approved by the Walter and Eliza Hall Institute Human Research Ethics Committee (HREC). Cord blood was passed through a RC High Efficiency Leucocyte Removal Filter (Haemonetics Australia) and then centrifuged at 2000 xg for 5 min to separate blood from serum. The blood was then washed three times in 1x human tonicity PBS (HTPBS) before being made up to 50% hematocrit. This 50% solution was then layered on top of a 70% (v/v) Percoll cushion (GE Healthcare). Centrifugation for 25 min at 2100 xg separated the mature erythrocytes from the reticulocytes, with the reticulocytes forming a thin band at the interface between buffer and Percoll. Reticulocytes were stored in 1x HTPBS at 4°C.

Flow cytometry-based erythrocyte binding assays
For assays using mature erythrocytes, erythrocytes were washed twice in PBS and then made up to a density of approximately 1×107 cells/mL in PBS+ 1% (w/v) BSA (PBS-BSA). Each sample used 100 μL of this suspension. Erythrocytes were centrifuged, the supernatant was removed, and the cells were resuspended in a solution containing freshly prepared recombinant proteins in PBS-BSA. Individual proteins were prepared at a final concentration of 2 μM (except for PfRh5, which was prepared at 400 nM), and complexes were mixed with an equimolar amount of protein to a final concentration of 2 μM. After a 45-min incubation at room temperature, the samples were centrifuged, washed, and then incubated with primary antibodies, either 5A9 (anti-Rh5), 4E2 (anti-PC) or 5E11 (anti-Ripr). After a 45-min incubation the cells were again centrifuged and then incubated with Alexa-488 anti-mouse fluorescent antibody at a dilution of 1:100. After a 45-min incubation, cells were washed twice in PBS and then resuspended before analysis on an Attune NxT flow cytometer (Thermo Fisher Scientific). For each sample 50,000 events were recorded. The data were then analysed in FlowJoTM v10.7 Software (BD Life Sciences). Antibody background was subtracted from the positive population recorded in the presence of recombinant protein and this background-subtracted value has been plotted in the summary figures.

For assays involving reticulocyte-enriched cord blood, erythrocytes were made up in 1x HTPBS + 1% (w/v) BSA (HTPBS-BSA) to a density of approximately 1×107 cells/mL. Each sample used 100 μL of this suspension. Reticulocytes were centrifuged (2000 xg for one minute), the HTPBS-BSA removed, and then resuspended in a solution containing recombinant proteins in HTPBS-BSA and incubated at room temperature for 45 min. PvPC and PkPC were used at a final concentration of 2 μM. Samples were centrifuged after which the protein solution removed, and cells were washed once with HTPBS-BSA and then incubated with 4E2 (anti-PC) at a concentration of 0.05 mg/mL or polyclonal sera (anti-PvRBP2b) at a concentration of 12.5 μg/mL. After a 45-min incubation, the cells were again centrifuged, and the antibody solution was removed. Cells were washed once as before and then stained with Alexa-647 (either anti-rabbit or anti-mouse) at a dilution of 1:100. After 45 min the reticulocytes were again washed and incubated with 50 μL of thiazole orange (BD Retic-Count, BD Biosciences) for 30 min. Finally, the reticulocytes were centrifuged, the Retic-Count solution removed, and cells were washed with 1x HTPBS two times before analysis on an Attune NxT flow cytometer (Thermo Fisher Scientific). For each sample 50,000 events were recorded. The data were then analysed in FlowJoTM v10.7 Software (BD Life Sciences). This involved gating reticulocytes and then applying a quadrant gate according to the thiazole orange staining and the background staining of the antibody in combination with the Alexa 647. This antibody background was subtracted from the positive population recorded in the presence of recombinant protein: this background-subtracted value has been plotted in the summary figures. Positive binding is determined by the double positive population in the upper right-hand quadrant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Key results: The manuscript provides an in-depth analysis of the formation and conservation of the heterotrimeric protein complex PC (PTRAMP-CSS) and RIPR in three Plasmodium species (Pf, Pv and Pk). The authors use advanced techniques such as predictive modelling, X-ray crystallography and cryo-EM to elucidate the complex's structural conformation, supported by biochemical and biophysical analyses that confirm protein interactions. While the complex exhibits notable structural conservation across species, species-specific functional and interaction differences are also identified, offering new insights into the molecular dynamics of erythrocyte invasion.\nValidity: The structural and biophysical data interpretation is robust and well-supported by experimental results. However, certain aspects raise concerns regarding the validity of some conclusions:\na. The discrepancy between the high structural conservation and the functional differences observed among species is not adequately addressed. This undermines the conclusion of analogous complex functionality across the three species. The discussion reveals a duality by initially suggesting similarity but also functional divergence. I recommend that the authors explore potential mechanisms that could explain this divergence in more depth.\nb. In inhibition assays, the requirement of high antibody concentrations to observe effects in P. falciparum raises uncertainty about whether inhibition is due to toxicity or a specific effect. Additional controls or a more detailed discussion are necessary.\nc. It is striking that antibody 5B3 exhibits cross-reactive inhibitory capacity in some species but fails to inhibit P. vivax invasion, which warrants further clarification from the authors.\nSignificance: This work addresses a topic of great importance in Plasmodium molecular biology, particularly regarding the mechanisms of cellular invasion, which are critical for the development of therapies and vaccines. The structural characterization of the PTRAMP-CSS-RIPR complex, along with interspecies functional differences, may open new research avenues and therapeutic strategies. Nevertheless, the lack of clarity in some interpretations might temporarily limit the study’s impact.\nData and Methodology: The employed techniques (crystallography, Cryo-EM, BLI, SPR, biochemical assays) are appropriate and of high quality for the study’s aims. The data presentation is clear, yet expansion of certain key experiments is advised:\na. The absence of real-time studies on complex formation limits understanding of the dynamic assembly process, which could be improved by applying BLI or SPR techniques.\nb. It is also important to evaluate whether individual proteins interact directly with reticulocytes or if the complex must form prior to cell binding, as this has functional relevance.\nSuggested Improvements\na. Incorporate dynamic binding assays (real-time BLI or SPR) to clarify the assembly mechanism of the complex.\nb. Conduct additional studies to explain why, despite high structural conservation, functional differences exist among species, possibly investigating interactions with other proteins or the cellular environment.\nc. Explore the individual interaction of proteins with reticulocytes and potential interspecies functional differences.\nd. Clarify the potential toxic effects of high antibody concentrations in inhibition assays using appropriate controls.\nClarity and Context: The manuscript is written clearly and is accessible to specialists in the field. Results are adequately contextualized with previous studies, although some critical points, such as the functional discrepancies among species, require deeper discussion and justification.\nThis manuscript provides valuable insights into a protein complex relevant to Plasmodium molecular biology. With the suggested clarifications and additional experiments, the study could achieve the rigor and impact expected for publication in a high-profile journal. Therefore, I recommend consideration of the manuscript after a major that adequately addresses the points raised in this report.", "reviewer_2": "The manuscript by Seager and colleagues investigate the structural and epitope similarities between the PCR complex from P. Knowlesi, P. falciparum and P. vivax. It is a well written manuscript, that contains a large amount of data, both directly in the manuscript and as supplementary/extended data. The aim to identify cross-species reactive or even growth inhibitory antibody is very interesting and gives insight to both evolutionary adaptations of the different species as well as the possibilities to develop potential interventions/vaccines targeting all three species.\nI find that the data support their conclusion well, the methods are sound and the data is not over-interpretated.\nI though have a few comments and suggestion to corrections:\nLine 137: the reference to figure 1d is a bit misleading. Fig. 1d rather shows, that the PC complex can also be seen with these two new non-glycosylated constructs, which could be written directly in the text. But it would also be good to show the glycosylated proteins in the same gel, so that the size differences caused by the glycosylations can be seen.\nLine 141: The refence to Fig. 1e would be better to have in line 139, just after “KD of 160 nM” since it does not show the referenced data of affinity between CyRPA and RH5.\nRegarding the PvRipr specific antibody, that 5B3, does it bind the PvPCR complex? I cannot see if it has. I think that information would support your discussion on why it does not inhibit the growth of P. vivax.\nFig. 5: When testing the antibodies in titration (f. 5d and e), you need to add the H2 (or another control antibody) in the same concentration span. This is especially relevant in fig. 5e where the concentration of 5B3 is being tested in really high concentrations.", "reviewer_3": "The authors have used AlphaFold3 to predict the structures of the PCR (PTRAMP, CSS and Ripr) complex from Plasmodium falciparum, vivax and knowlesi. Given that the proteins are conserved across Plasmodium species and that AF3 is trained on published structures, it is unsurprising that the AF3 prediction is highly similar for all three. The authors then crystallized the PvPC heterodimer, yielding a structure of PvCSS residues 115-381 and PvPTRAMP residues 42-53. The structure reveals a disulfide bond stabilizing the interaction between CSS and PTRAMP, which is conserved across the three species. Beyond this, the PvCSS structure is highly consistent with previously published PfCSS structures. The authors follow up with biophysical studies with recombinant protein to show that binding affinity of the various members for each other appear to be stronger than reported in previous studies using recombinant protein expressed in heterologous systems that differ from Plasmodium in terms of glycosylation. Finally, the authors show that anti-PCR antibodies are cross-reactive but show varying levels of growth inhibition across Plasmodium species. Overall, the work offers few technical or biological advances beyond published work and may be more suitable for a journal with a more focused readership.\n1. Lines 260-276: The cryoEM 2Ds are sufficient for showing what general region of the complex a Fab or antibody binds to, but there is not enough information in low resolution 2D class averages to “strongly support the predicted PCR complex structure”. Nor is the PvPC crystal structure, which contains only CSS residues 115-382 and PTRAMP residues 42-53, comprehensive enough to “strongly support the predicted PCR complex structure.”\n2. Have the authors attempted to obtain a 3D reconstruction from the 2D class averages shown in Fig. 6a?\n3. The word “model” is used to variously refer to many different things throughout the manuscript, including a) atomic models from experimental crystal structures, b) predicted models from AlphaFold 3, c) models for biological or biophysical models d) cartoon models. This language should be clarified to avoid confusion. For example, the “models” in Fig 6c and on the right hand side of Fig 6b would more accurately be described as “cartoons” or “schematics” to differentiate from the predicted AF3 model on the left hand side of Fig 6b or the actual atomic model in Fig 2c."}, "author_response": "Dear Editor\nMany thanks for sending the reviews of our manuscript ‘PTRAMP, CSS and Ripr form a conserved complex required for merozoite invasion of Plasmodium species into erythrocytes’ by Seager et al. We have addressed the reviewers comments point by point below and made changes as suggested. Additionally, we have added substantially more data including a crystal structure determination of Ripr domains bound to the antibody 5B3.\nWe believe that this has addressed the reviewer’s comments and look forward to your final decision on this manuscript.\nKind regards\nAlan Cowman\nAuthor Response to Reviewers:\nReviewer #1 (Remarks to the Author):\nKey results: The manuscript provides an in-depth analysis of the formation and conservation of the heterotrimeric protein complex PC (PTRAMP-CSS) and RIPR in three Plasmodium species (Pf, Pv and Pk). The authors use advanced techniques such as predictive modelling, X-ray crystallography and cryo-EM to elucidate the complex's structural conformation, supported by biochemical and biophysical analyses that confirm protein interactions. While the complex exhibits notable structural conservation across species, species-specific functional and interaction differences are also identified, offering new insights into the molecular dynamics of erythrocyte invasion.\nValidity: The structural and biophysical data interpretation is robust and well-supported by experimental results. However, certain aspects raise concerns regarding the validity of some conclusions:\na. The discrepancy between the high structural conservation and the functional differences observed among species is not adequately addressed. This undermines the conclusion of analogous complex functionality across the three species. The discussion reveals a duality by initially suggesting similarity but also functional divergence. I recommend that the authors explore potential mechanisms that could explain this divergence in more depth.\nAuthor response: We thank the reviewer for highlighting this important point. We acknowledge that our initial discussion may have not fully addressed the apparent discrepancy between structural conservation and functional differences observed among species. We have now expanded our discussion.\nThe high structural conservation of the PCR complex across Plasmodium species, as predicted by AlphaFold and supported by our structural and biochemical data, indeed suggests a conserved core function. However, our functional studies, particularly the differential inhibition by antibodies across species, reveal important species-specific differences.\nTo address this apparent discrepancy, we propose several potential mechanisms:\n1. Subtle structural differences: While the overall structure is conserved, small differences in amino acid composition could lead to species-specific alterations in surface properties or dynamics of the complex.\n2. Interaction with species-specific partners: The PCR complex likely interacts with other invasion proteins that are not conserved across species (such as PfRh5 in P. falciparum). These interactions could modulate the function or accessibility of the PCR complex in a species-specific manner.\n3. Epitope accessibility: As suggested by our new structural data on the 5B3 antibody binding, the accessibility of certain epitopes may differ between species during the dynamic process of invasion, despite overall structural similarity.\nWe have included a more detailed discussion of these potential mechanisms in the manuscript (lines 379-406). While fully elucidating these differences will require further research, including structural studies of the complete invasion complexes from multiple species, we believe our work provides a crucial foundation for understanding both the conserved and divergent aspects of the PCR complex across Plasmodium species.\nb. In inhibition assays, the requirement of high antibody concentrations to observe effects in P. falciparum raises uncertainty about whether inhibition is due to toxicity or a specific effect. Additional controls or a more detailed discussion are necessary.\nAuthor Response: We have added a non-immune IgG control to the P. falciparum GIAs. The non-immune control data shows no growth defect in the presence of 10 mg/mL IgG, providing confidence that the results we see with the 5B3 mAb are specific. Further, non-inhibitory antibodies 2D9, 4E2 and 4H10 also show no effect on parasite growth at high concentrations. This additional data has been added to Main Figure 5 as follows:\nPanel b updated with new competition for 4E2 and 2D9.\nPanel c updated with new 4E2 inhibition\nPanel d changed to include new reps and 4E2 data.\nPanel e changed to include 4E2 mAb and non-immune IgG titrations up to 10 mg/ml.\nc. It is striking that antibody 5B3 exhibits cross-reactive inhibitory capacity in some species but fails to inhibit P. vivax invasion, which warrants further clarification from the authors.\nAuthor Response: We agree that the ability of 5B3 mAb to inhibit P. falciparum and P. knowlesi growth but not P. vivax or P. cynomolgi is a surprising result. To try to address this, we have solved the crystal structure of the 5B3 Fab in complex with EGF7 and EGF8 of PvRipr and defined the epitope. This additional data is described in a new Figure (Fig. 6). This figure includes a comparison of this epitope between PvRipr, PkRipr, PfRipr and PcRipr to show the strong homology of this region. Additionally, we determined the K of binding of 5B3 for D PkRipr, PfRipr and PvRipr and whilst there are significant differences it does not explain the growth inhibition for Pf and Pk compared to Pv and Pc. These results suggest that subtle structural differences in the PfPCR and PkPCR complex, compared with that of PvPCR and PcPCR, may allow 5B3 access during merozoite invasion in the former species.\nTo fully define the difference that explains this result it would be necessary to solve the structure of the full complex with the Rh5 equivalent receptor binding protein attached for each with 5B3 bound using Cryo-EM. Currently, the Rh5 equivalent protein for Pk, Pv and Pc PCR complexes are not identified. Whilst we are working towards this identification and plan to determine these structures using Cryo-EM this is a major effort that is outside the scope of this manuscript. However, we have added considerable additional data to start to address this question including a full figure that determines how 5B3 binds to Ripr.\nSignificance: This work addresses a topic of great importance in Plasmodium molecular biology, particularly regarding the mechanisms of cellular invasion, which are critical for the development of therapies and vaccines. The structural characterization of the PTRAMP-CSS-RIPR complex, along with interspecies functional differences, may open new research avenues and therapeutic strategies. Nevertheless, the lack of clarity in some interpretations might temporarily limit the study’s impact.\nAuthor Response: We appreciate the reviewer's recognition of the importance of our work.\nTo address the concern about clarity in interpretations and to enhance the study's impact, we have made several substantial additions and clarifications:\n1. Structural characterization: We have solved the crystal structure of the 5B3 Fab in complex with EGF7 and EGF8 of PvRipr, providing detailed structural information about this important epitope (new Main Figure 6). This adds to our understanding of the PTRAMP-CSS-RIPR complex structure across species.\n2. Interspecies functional differences: We include a comparative analysis of the 5B3 epitope across PvRipr, PkRipr, PfRipr and PcRipr demonstrating strong homology.\n3. Based on our new data, we propose that subtle structural differences in the PCR complex between species might affect the accessibility of the 5B3 epitope during merozoite invasion, potentially explaining the observed functional differences.\n4. We have outlined the next steps needed to fully resolve remaining questions, including solving structures of the full complex with the Rh5 equivalent receptor binding protein for each species.\nThese additions provide a more comprehensive and nuanced understanding of the PTRAMP-CSS-RIPR complex across Plasmodium species. We believe this enhanced clarity strengthens the study's impact and opens up new avenues for research.\nData and Methodology: The employed techniques (crystallography, Cryo-EM, BLI, SPR, biochemical assays) are appropriate and of high quality for the study’s aims. The data presentation is clear, yet expansion of certain key experiments is advised:\na. The absence of real-time studies on complex formation limits understanding of the dynamic assembly process, which could be improved by applying BLI or SPR techniques.\nAuthor Response: We agree that real time studies on complex formation can provide insight into the interactions required for complex formation. We have extensively used BLI with respect to PCRCR subunit interaction and it is included (Fig. 1E, Fig. 3 a-d, Fig. 4 b-d, Ext. Fig 3 b, Ext Fig. 4b, Ext Fig. 5 a-c)(Main Figures 1e, 3a-d, 4b-d, and Supplementary Figures 7b, 8b, and 9a-c in the resubmitted manuscript). We have shown how Ripr engages the PTRAMP-CSS heterodimer with high affinity via the C-terminal domain and EGF domains 9 and 10. All BLI experiments were carried out over the same time of 240 seconds, with association being measured over the course of 120 seconds. This shows that complex formation is quite rapid.\nTo further illustrate the dynamic assembly process, we direct the reviewer to Figure 3 and 4, which shows representative BLI sensorgrams for the association of Ripr with the PTRAMP-CSS heterodimer. The rapid association observed (reaching equilibrium within 120 seconds) suggests that complex formation is not likely to be a rate-limiting step in the invasion process. This rapid assembly is consistent with the need for quick deployment of invasion machinery during the brief window of merozoite viability outside the red blood cell.\nb. It is also important to evaluate whether individual proteins interact directly with reticulocytes or if the complex must form prior to cell binding, as this has functional relevance.\nAuthor response: We agree with the reviewer that understanding how this complex interacts with either erythrocytes and/or reticulocytes is crucial to its role in invasion and that is why we did the experiments originally presented in Extended Data. To this end, we carried out reticulocyte and normocyte binding experiments with both the P. vivax PC heterodimer and the P. knowlesi PC heterodimer, and erythrocyte binding experiments with the same proteins as well as the P. knowlesi PCR complex including the positive controls ie. PfRh5 and PvRBP2b (originally shown in Extended Data figure 10). These experiments showed that these complexes could not bind to reticulocytes or normocytes. It has also been shown previously that the P. falciparum PC heterodimer is unable to bind to erythrocytes without incorporation into the PCRCR complex (Scally et al. Nature Micro 2022). Performing the equivalent experiment with PvPCR was not feasible due to the low yields of recombinant full length PvRipr and the high concentrations required for the assay. We believe these findings support the idea that PTRAMP-CSS-Ripr complex is a scaffold for other species-specific red blood cell binding proteins (such as PfRh5 in P. falciparum). Whilst the binding data to reticulocytes and normocytes is negative, we do believe it is an important piece of data, given that previous experiments with PfPTRAMP (Siddiqui et al Cell Microbiol 2013) and PkPTRAMP (Knuepfer et al. PLOS Pathogens 2019) suggested that PTRAMP was able to bind erythrocytes. We believe that the use of heterodimeric PTRAMP-CSS, rather than monomeric PTRAMP, provides more relevant data on how these proteins interact with host cells.\nTo make these results clearer and more visible we have moved the reticulocyte and normocyte binding assays to the new Figure 7 panel c and d.\nSuggested Improvements\na. Incorporate dynamic binding assays (real-time BLI or SPR) to clarify the assembly mechanism of the complex.\nAuthor response: We have included BLI experiments that address the assembly of this complex. PTRAMP and CSS are present as a heterodimer before binding to the Ripr CTD. This complex (along with other potential interactors) is present within the micronemes before being exposed on the merozoite surface. Further cell biology and/or imaging experiments will hopefully shed further light on this complex, but we believe such experiments are outside the scope of this study.\nb. Conduct additional studies to explain why, despite high structural conservation, functional differences exist among species, possibly investigating interactions with other proteins or the cellular environment.\nAuthor Response: We appreciate the reviewer's suggestion to further investigate the reasons for functional differences despite high structural conservation among species. We have conducted additional studies and analyses to address this important point:\n1. Structural analysis of antibody binding: We have determined the crystal structure of the 5B3 Fab in complex with PvRipr EGF7-8 (new Figure 6). This structure provides insight into the specific epitope recognized by this cross-reactive but differentially inhibitory antibody.\n2. Comparative epitope analysis: We included a comparison of the 5B3 epitope across PvRipr, PkRipr, PfRipr and PcRipr. While this region shows strong homology, we identified subtle differences that may contribute to functional divergence.\n3. Cellular environment considerations: Our reticulocyte and normocyte binding assays (now in Figure 7, panels c and d) suggest that the PCR complex alone is not sufficient for host cell binding in P. vivax and P. knowlesi, unlike the situation in P. falciparum where the PCRCR complex can bind erythrocytes.\nBased on these new data, we propose that functional differences may arise from:\n1. Subtle structural variations affecting epitope accessibility during the dynamic process of invasion.\n2. Differences in how the PCR complex interacts with other, potentially species-specific, invasion proteins.\n3. Variations in the cellular context of invasion, such as the preference for reticulocytes in P. vivax.\nWe acknowledge that fully explaining these functional differences will require additional research, including:\n1. Identification and characterization of species-specific binding partners of the PCR complex.\n2. Structural studies of the complete invasion complexes from multiple species.\n3. Dynamic studies of complex assembly and function during the invasion process.\nWhile these extensive studies are beyond the scope of the current work, our findings provide a crucial foundation for understanding both the conserved and divergent aspects of the PCR complex across Plasmodium species. We have expanded our discussion in the manuscript to address these points and outline future research directions.\nc. Explore the individual interaction of proteins with reticulocytes and potential interspecies functional differences.\nAuthor Response: As noted above, we carried out erythrocyte and reticulocyte binding assays. Both of these experiments showed no binding for PC heterodimers or the PkPCR trimeric complex. As we have shown that both PTRAMP and CSS exist as a disulfide-linked heterodimer, we believe that assessing the binding of monomeric proteins would not reflect how these proteins function in the parasite. Performing the equivalent experiment with PvPCR was not feasible due to the low yields of recombinant full length PvRipr and the high concentrations required for the assay.\nGiven our previous findings that the complete PCRCR complex is required for PTRAMP-CSS erythrocyte binding in P. falciparum, we believe that other, yet unidentified, proteins are required for PCR binding in P. knowlesi and P. vivax.\nThese results have now been incorporated into figure 7 (panels c and d).\nd. Clarify the potential toxic effects of high antibody concentrations in inhibition assays using appropriate controls.\nAuthor Response: We have now added a non-immune IgG control to the P. falciparum GIAs. The new control data shows no growth defect in the presence of 10mg/mL IgG, giving us confidence that the results we see with 5B3 are specific. Further, non-inhibitory antibodies 2D9, 4E2 and 4H10 also show no effect on parasite growth at high concentrations.\nClarity and Context: The manuscript is written clearly and is accessible to specialists in the field. Results are adequately contextualized with previous studies, although some critical points, such as the functional discrepancies among species, require deeper discussion and justification.\nAuthor Response: See comments above.\nThis manuscript provides valuable insights into a protein complex relevant to Plasmodium molecular biology. With the suggested clarifications and additional experiments, the study could achieve the rigor and impact expected for publication in a high-profile journal. Therefore, I recommend consideration of the manuscript after a major that adequately addresses the points raised in this report.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Seager and colleagues investigate the structural and epitope similarities between the PCR complex from P. Knowlesi, P. falciparum and P. vivax. It is a well written manuscript, that contains a large amount of data, both directly in the manuscript and as supplementary/extended data. The aim to identify cross-species reactive or even growth inhibitory antibody is very interesting and gives insight to both evolutionary adaptations of the different species as well as the possibilities to develop potential interventions/vaccines targeting all three species.\nI find that the data support their conclusion well, the methods are sound and the data is not over-interpretated.\nI though have a few comments and suggestion to corrections:\nLine 137: the reference to figure 1d is a bit misleading. Fig. 1d rather shows, that the PC complex can also be seen with these two new non-glycosylated constructs, which could be written directly in the text. But it would also be good to show the glycosylated proteins in the same gel, so that the size differences caused by the glycosylations can be seen.\nAuthor Response: We have now included the glycosylated PfPC gel in figure 1 panel d as requested.\nLine 141: The refence to Fig. 1e would be better to have in line 139, just after “KD of 160 nM” since it does not show the referenced data of affinity between CyRPA and RH5.\nAuthor Response: We have moved the reference as per the reviewer’s request.\nRegarding the PvRipr specific antibody, that 5B3, does it bind the PvPCR complex? I cannot see if it has. I think that information would support your discussion on why it does not inhibit the growth of P. vivax.\nAuthor Response: Yes, 5B3 can directly bind the PvPCR complex and this is shown in Fig 5c and Extended Figure 8c (now Supplementary Figure 12 in the resubmitted manuscript) which show the competition epitope binning results. PvPC is still able to bind PvRipr once 5B3 is bound, indicating that a PvPCR-5B3 complex is possible. We agree if 5B3 could not bind the complex but bound PvRipr this would explain the inhibition of growth studies. We have inserted two sentences in the Discussion that discusses the 5B3 results. Lines 379-381:\n“Interestingly, the Ripr-binding mAb 5B3, raised against PvRipr, exhibited an unexpected pattern of cross-inhibitory activity. It inhibited invasion of P. knowlesi and P. falciparum, but not P. vivax or P. cynomolgi, despite binding to the PvPCR complex.”\nFig. 5: When testing the antibodies in titration (f. 5d and e), you need to add the H2 (or another control antibody) in the same concentration span. This is especially relevant in fig. 5e where the concentration of 5B3 is being tested in really high concentrations.\nAuthor Response: As mentioned above we have now added a non-immune IgG control to the P. falciparum GIAs. The new control data shows no growth defect in the presence of 10 mg/mL IgG, giving us confidence that the results we see with 5B3 are specific. Further, non-inhibitory antibodies 2D9, 4E2 and 4H10 also show no effect on parasite growth at high concentrations.\nReviewer #3 (Remarks to the Author):\nThe authors have used AlphaFold3 to predict the structures of the PCR (PTRAMP, CSS and Ripr) complex from Plasmodium falciparum, vivax and knowlesi. Given that the proteins are conserved across Plasmodium species and that AF3 is trained on published structures, it is unsurprising that the AF3 prediction is highly similar for all three. The authors then crystallized the PvPC heterodimer, yielding a structure of PvCSS residues 115-381 and PvPTRAMP residues 42-53. The structure reveals a disulfide bond stabilizing the interaction between CSS and PTRAMP, which is conserved across the three species. Beyond this, the PvCSS structure is highly consistent with previously published PfCSS structures. The authors follow up with biophysical studies with recombinant protein to show that binding affinity of the various members for each other appear to be stronger than reported in previous studies using recombinant protein expressed in heterologous systems that differ from Plasmodium in terms of glycosylation. Finally, the authors show that anti-PCR antibodies are cross-reactive but show varying levels of growth inhibition across Plasmodium species. Overall, the work offers few technical or biological advances beyond published work and may be more suitable for a journal with a more focused readership.\nAuthors Response: We appreciate the reviewer's summary of our work. However, we believe our study offers significant technical and biological advances beyond published work. We would like to highlight the key findings and emphasize the additional data we have added in response to reviewer comments:\nKey Findings and Advances:\n1. Conservation of PCR complex: We demonstrate that the PTRAMP-CSS-Ripr (PCR) complex is conserved across all Plasmodium species, including those that lack Rh5, suggesting a fundamental role in invasion beyond the P. falciparum-specific PCRCR complex.\n2. Structural insights: Our crystal structure of the PvPC heterodimer provides the first structural evidence of the PTRAMP-CSS interaction outside of P. falciparum, confirming the conservation of this critical disulfide bond.\n3. Improved biophysical characterization: By optimizing protein expression to reduce glycosylation, we demonstrate significantly stronger binding affinities between complex components than previously reported, providing a more accurate representation of these interactions.\n4. Species-specific functional differences: Despite structural conservation, we reveal species-specific differences in antibody-mediated growth inhibition, highlighting important functional divergences in the PCR complex across Plasmodium species.\n5. Cross-reactive antibodies: We identify antibodies that show cross-reactivity and differential inhibition across multiple Plasmodium species, which has significant implications for vaccine development.\nAdditional Data and Analyses:\nIn response to reviewer comments, we have added substantial new data and analyses:\n1. Crystal structure of 5B3 Fab-PvRipr complex: We determined the structure of the inhibitory antibody 5B3 in complex with PvRipr EGF7-8, providing detailed structural information about this important epitope (new Figure 6).\n2. Comparative epitope analysis: We performed a detailed comparison of the 5B3 epitope across PvRipr, PkRipr, PfRipr and PcRipr revealing subtle differences that may contribute to functional divergence.\n3. Additional growth inhibition controls: We added non-immune IgG controls and data for non-inhibitory antibodies at high concentrations to demonstrate the specificity of inhibitory effects.\n4. Improved presentation of binding assays: We moved the reticulocyte and normocyte binding assay results to the main figures (Figure 7c, d) for greater visibility and impact.\nThese findings and additional data provide significant new insights into the conservation, structure, and function of the PCR complex across Plasmodium species. They reveal important species-specific differences that have implications for understanding invasion mechanisms and developing cross-species interventions. We believe this work represents a substantial advance in our understanding of Plasmodium invasion biology and has broad relevance to the field of malaria research.\n1. Lines 260-276: The cryoEM 2Ds are sufficient for showing what general region of the complex a Fab or antibody binds to, but there is not enough information in low resolution 2D class averages to “strongly support the predicted PCR complex structure”. Nor is the PvPC crystal structure, which contains only CSS residues 115-382 and PTRAMP residues 42-53, comprehensive enough to “strongly support the predicted PCR complex structure.”\nAuthor Response: We acknowledge that the 2D class averages from cryo-EM and our partial crystal structure of PvPC alone are not sufficient to definitively prove the entire PCR complex structure. However, we believe that the combination of these structural data with our extensive biochemical and biophysical analyses provides strong support for our proposed model of protein interactions within the complex.\nThe crystal structure of PvCSS with PvPTRAMP residues 42-53 confirms the presence and location of the intermolecular disulfide bond predicted by AlphaFold. This key interaction forms the basis of the PC heterodimer, which our biochemical data show is crucial for high-affinity Ripr binding.\nOur new crystal structure of the 5B3 Fab with PvRipr EGF7-8 provides additional structural information about a key region of Ripr involved in complex formation. When combined with our binding studies showing that the C-terminal region of Ripr is sufficient for PC binding, this helps to constrain the possible arrangements of Ripr within the complex.\nThe cryo-EM 2D class averages, while low resolution, are consistent with our proposed model and provide visual confirmation of the overall shape and dimensions of the complex.\nImportantly, they show the 5B3 Fab binding to the predicted location on Ripr.\nWhile we agree that no single piece of our data conclusively proves the entire complex structure, we believe that the consistency across multiple independent lines of evidence - structural, biochemical, and biophysical - provides strong support for our proposed model of the PCR complex.\n2. Have the authors attempted to obtain a 3D reconstruction from the 2D class averages shown in Fig. 6a?\nAuthor response: We thank the reviewer for the suggestion. Indeed, we tried extensively to generate a reasonable 3D reconstruction for the PCR complex. Due to the orientation bias, we were unable to produce a 3D reconstruction of sufficient quality for publication. Shown below is an ab-initio reconstruction of PkPCR at low resolution, showing that the model fits reasonably well within the density. We were unable to improve the resolution further and the density shown here clearly suffers from anisotropy.\nFront View Side View Top View\nThis figure shows ab-initio cryoEM density (grey) and Alphafold model of PkPCR (purple for PTRAMP, green for CSS and yellow for Ripr)(loops have been omitted for clarity).\n3. The word “model” is used to variously refer to many different things throughout the manuscript, including a) atomic models from experimental crystal structures, b) predicted models from AlphaFold 3, c) models for biological or biophysical models d) cartoon models. This language should be clarified to avoid confusion. For example, the “models” in Fig 6c and on the right hand side of Fig 6b would more accurately be described as “cartoons” or “schematics” to differentiate from the predicted AF3 model on the left hand side of Fig 6b or the actual atomic model in Fig 2c.\nAuthors Response: We thank the reviewer for bringing this to our attention. Where possible we have tried to qualify the use of model by including a specifier (e.g. predicted model). We have revised our use of the model throughout the manuscript to minimize confusion.\nMany thanks for considering our manuscript. The reviewer’s comments have been very helpful and improved the manuscript and we are pleased they are now happy with our additional data and changes. All of th e editorial changes and formatting required have been done and attached.\nWe look forward to your finale decision\n."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Having reviewed the revised manuscript, the final report, and the point-by-point response letter, I am satisfied that the authors have made a clear and substantive effort to address the main concerns raised during peer review. In particular, the revisions strengthen the discussion of the structure–function relationship in the relevant protein complexes across the evaluated species, with a more transparent and coherent interpretation of the observations obtained using the 5B3 antibody. I also appreciate the authors’ commitment to pursuing additional crystallographic experiments aimed at elucidating underlying mechanisms and identifying conserved regions. These efforts will be valuable not only to reinforce the structural basis of the proposed functional interpretations, but also to better define which elements are truly conserved versus species-specific within this parasite system.\nWhile some elements of the discussion necessarily remain interpretative and, at times, hypothesis-generating, they are generally framed appropriately and help delineate important open questions in these highly complex parasites. Importantly, the authors acknowledge that, although the species examined can infect similar host cell types, this does not imply conserved or identical molecular mechanisms, and the revised text reflects this nuance more carefully.\nOverall, I consider that the manuscript has improved meaningfully and now provides a more balanced and rigorous framework for the conclusions.", "reviewer_2": "The authors have addressed all my comments made in the first review and I find the revised manuscript good and the results of significance to the field of malaria vaccine development.", "reviewer_3": "My previous concerns have been addressed. No further comments at this time."}, "author_response": ""}]

Decision letter