The dual action of human antibodies specific to Plasmodium falciparum PfRH5 and PfCyRPA: Blocking invasion and inactivating extracellular merozoites

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the current leading blood-stage malaria vaccine candidate. PfRH5 functions as part of the pentameric PCRCR complex containing PTRAMP, CSS, PfCyRPA and PfRIPR, all of which are essential for infection of human red blood cells (RBCs). To trigger RBC invasion, PfRH5 engages with RBC protein basigin in a step termed the RH5-basigin binding stage. Although we know increasingly more about how antibodies specific for PfRH5 can block invasion, much less is known about how antibodies recognizing other members of the PCRCR complex can inhibit invasion. To address this, we performed live cell imaging using monoclonal antibodies (mAbs) which bind PfRH5 and PfCyRPA. We measured the degree and timing of the invasion inhibition, the stage at which it occurred, as well as subsequent events. We show that parasite invasion is blocked by individual mAbs, and the degree of inhibition is enhanced when combining a mAb specific for PfRH5 with one binding PfCyRPA. In addition to directly establishing the invasion-blocking capacity of the mAbs, we identified a secondary action of certain mAbs on extracellular parasites that had not yet invaded where the mAbs appeared to inactivate the parasites by triggering a developmental pathway normally only seen after successful invasion. These findings suggest that epitopes within the PfCyRPA-PfRH5 sub-complex that elicit these dual responses may be more effective immunogens than neighboring epitopes by both blocking parasites from invading and rapidly inactivating extracellular parasites. These two protective mechanisms, prevention of invasion and inactivation of uninvaded parasites, resulting from antibody to a single epitope indicate a possible route to the development of more effective vaccines.


Introduction
Globally, malaria remains a serious problem with 247 million cases of malaria worldwide, and 619,000 deaths in 2021 [1], with the majority of disease burden and death due to Plasmodium falciparum (Pf).Despite a growing arsenal of antimalarial drugs, it is unlikely that drugs alone will be enough to eradicate malaria and a highly effective malaria vaccine is needed, that would continue to provide protection when medicines are not available.Antibodies are known to play a key role in protection from malaria [2].Low-level antibody responses to parasite invasion protein PfRH5, can be naturally acquired following many years of malaria exposure and associate with clinical immunity and inhibit parasite growth in-vitro [3].High levels of PfRH5 vaccine-induced antibodies can block merozoite invasion of erythrocytes [4], and are associated with delayed time to detection of parasitemia in a controlled human malaria infection trial in vaccinated individuals [5].PfRH5 forms a chain-like PCRCR complex with P. falciparum Cysteine-Rich Protective Antigen (PfCyRPA), PfRH5-interacting protein (PfRIPR), P. falciparum cysteine-rich small secreted protein (PfCSS) and P. falciparum Plasmodium thrombospondin-related apical merozoite protein (PfPTRAMP) [6][7][8], which has been shown to localize at the tight junction between the merozoite and erythrocyte immediately prior to invasion [6,9].PfRH5, PfCyRPA, PfRIPR, PfCSS and PfPTRAMP are required for merozoite invasion of erythrocytes with PfRH5, PfCyRPA, PfRIPR each able to stimulate the production of cross-strain neutralizing antibodies [6,[9][10][11][12][13][14][15].Of the five PCRCR components, PfRH5, PfCyRPA and PfRIPR are the most well characterized and are compelling malaria vaccine candidates with experimental vaccination of non-human primates and humans with PfRH5 producing potent neutralizing antibodies [16,17].Understanding the effect of antibodies targeting the PCRCR complex antigens, both in isolation and combination, is now critical to effective next-generation blood-stage vaccine design.Importantly, the amount of antibody required for protection may be greatly reduced by exploiting synergistic interactions between antibodies.

PLOS PATHOGENS
The dual action of human antibodies specific to Plasmodium falciparum PfRH5 and PfCyRPA Antibodies recognizing the same complex could be expected to synergize through a wide variety of mechanisms, including, but not limited to, causing a conformational change that enhances binding of a second antibody [18], slowing down invasive processes allowing other antibodies more time to bind [4] or by stabilizing antibody binding through lateral interactions [19].The ideal effect of antibodies would be rapid and lead to permanent disabling of the parasites' ability to invade or survive.While antibody binding to a parasite might temporarily prevent invasion, if that parasite is not disabled by this interaction, then the dissociation of that antibody could leave the parasite virulent and able to attempt invasion again.
In this study we use live cell imaging to examine the visible temporal and morphological effects of mAbs binding to different epitopes of PfRH5 and PfCyRPA on invasion, the postinvasion development of young parasites, and the inactivation of uninvaded merozoites.While the ability of PCRCR antigen-specific antibodies to block invasion is known, this is the first report of their capacity to inactivate uninvaded parasites.This work illuminates new mechanisms through which neutralizing antibodies to certain epitopes of PfRH5 and PfCyRPA function and future studies of the cellular and molecular basis of these observations could open the door to strategies for the rational design of a highly effective blood-stage malaria vaccine.

Antibody inhibition of merozoite invasion
To characterize the effects of mAbs specific for PfRH5 and PfCyRPA on invasion of erythrocytes, live cell microscopy was performed on clonal 3D7 parasites using methods that had been previously developed [20].As expected, parasites in the presence of the control EBL 040 mAb (targeting Ebola virus) [4,21] followed the usual progression of invasion with distinct stages marked first by egress of merozoites from the schizont (Fig 1A, S1 Table and S1 Video) [20,22,23], contact of the merozoite with the erythrocyte (mean 41.95 s after egress [SD 35.80]), and deformation of the erythrocyte membrane (mean duration 52.60 s [SD 106.70]).Following this is the short stage where the PCRCR complex acts preceding the formation of the tight junction between the parasite protein AMA1 on the merozoite surface and the parasite-derived RON complex on erythrocyte surface [24][25][26][27].Here we term this pre-tight junction stage as the "RH5-basigin binding stage" (mean 1.76 s [SD 0.53]), and merozoite invasion of the erythrocyte ensues over the next 10 seconds (mean 10.27 s [SD 2.41]).After this, temporary changes to the invaded erythrocyte occur called echinocytosis, and the merozoite differentiates into an intracellular ring (Fig 1A and S1 Video).
Anti-PfRH5 mAbs were isolated from volunteers immunized in the first human Phase Ia PfRH5 vaccine trial [28].Individual mAbs were characterized: clone R5.004 was found to be strongly neutralizing and to bind directly to the basigin-binding site of PfRH5 (S1A and S1B Fig) [4]; whilst clone R5.008 was found to be moderately neutralizing possibly by hindering PfRH5's access to basigin through steric clashes with the RBC membrane or basigin's RBC binding partners PCMA or MCT1 [4,29].It has recently been discovered that PfRH5 must have its pro-sequence cleaved by plasmepsin X before it can engage basigin [30].
We also analyzed three mAbs which bind PfCyRPA and were composed of variable regions from vaccinated chickens and a human IgG1 constant region [19].These three mAbs all bind to the beta propeller blade 1 and 2 regions of PfCyRPA (S1C and S1D Fig) .PfCyRPA-binding mAbs demonstrated modest levels of inhibition [7].However, in growth inhibition activity (GIA) assays, R5.008 and Cy.009 synergized to strongly inhibit invasion and this combination was therefore chosen for further study (Fig 1B).R5.008 was filmed at a concentration causing less inhibition to directly compare results with the R5.008 + Cy.009 combination, while other individual mAbs were filmed at concentrations resulting in an intermediate level of inhibition to assess the stages of invasion that were affected.We also evaluated PfCyRPA fragment antigen binding (Fab) molecules in GIA assays and found the Fabs consistently demonstrated greater GIA than whole IgGs at equivalent molarities (Figs 1C and S1E-S1G).Since this was especially evident for Cy.007, with Fabs being more than ten-fold more inhibitory than whole IgGs, we included this Fab in our live imaging analysis.
Videos of egress and invasion were analyzed in detail for each of the mAbs, Fabs and the control EBL 040 antibody with a median of 11 videos analyzed for each antibody.The concentrations used are indicated in brackets (in μg/mL) following the antibody's name.The number of merozoite-erythrocyte contacts per egress was equal to or greater than that in the EBL 040 control antibody (mean 16.82 s [SD 8.64]), indicating the parasites were healthy (Fig 2A and After egress, merozoites took 42.0 s to contact the erythrocyte they invaded (all times are means).Following an initial contact of 0.9 s, the merozoites deformed their erythrocytes for 52.6 s followed by the quiescent complex stage of 1.8 s, when it is believed the PCRCR complex and tight junction form ("Rh5-basigin stage").Merozoite internalization or invasion takes 10.3 s after which the merozoite starts to spin 16.4 s later.Spinning lasts 116.3 s and once finished, a pseudopod emerges from the merozoite 159.2 s later.57.2 s after this, the merozoite membrane starts to become irregular to form a pre-ring.After 638.0 s, a fully formed complete ring is evident.40.9 s after invasion is complete, the host erythrocyte develops membranous protrusions in a process called echinocytosis.These protrusions increase in their extent reaching a maximum after 44.2 s.This state continues for 395.9 s until the erythrocyte returns to its usual biconcave shape over 305.9 s.Of note, the timing and duration of echinocytosis is highly variable.SD, range and number (N) of events are indicated in S1 Table .(B) In vitro single cycle growth inhibition activity (GIA) assay dilution series of R5.008 mAb alone (pink), or in combination with Cy.009 mAb held at 0.13 mg/mL (blue), against 3D7 parasites.Predicted Bliss additivity is indicated (grey).The solid green line indicates the GIA of mAb Cy.009 held alone at a fixed concentration of 0.13 mg/ mL.Dotted line indicates 50% GIA.(C) Comparison of the GIA of the Cy.007 mAb (purple) with its Fab fragment (light pink) indicating the latter is much more potent.Assay performed as in B.
https://doi.org/10.1371/journal.ppat.1011182.g001).In addition, the times from egress to first merozoite contact with erythrocytes were similar to EBL 040 (mean 41.95 s [SD 35.80]), demonstrating the experimental conditions were consistent for each antibody combination (S2A Fig and S5A Table).With the exception of R5.004- (22) the antibodies did not greatly reduce the degree with which the merozoites deformed their target RBCs which is thought to be a product of merozoite ligands acting upstream of the PCRCR complex and the actomyosin invasion motor and is positively associated with invasion success (S2B Fig and S5B Table) [20].

S2A Table
The number of invasions per egress was reduced in the R5.004- (22)    of schizont egress and the number of erythrocytes available for invasion can vary, a complementary measure of invasion was also used: the percentage of merozoites that invade after contacting an erythrocyte.Using this measure, invasion was reduced for all anti-PfRH5 and -PfCyRPA antibodies compared to the control (mean 19

Antibody effects on early pre-invasion interactions
Pre-invasion consists of the time from first contact between the erythrocyte and merozoite to the start of invasion, with early pre-invasion events including initial contact and erythrocyte deformation (Fig 1A).The antibodies did not greatly change the short period of time from first merozoite contact to the start of erythrocyte deformation, although R5.004- (22) ).Our interest in the duration of deformation stemmed from earlier work where the PfRH5 specific mAb R5.011, was found to increase the pre-invasion period, which primarily consists of RBC deformation, thereby allowing the invasion-neutralizing mAb R5.016 more time to bind PfRH5 and inhibit invasion [4]

Antibody effects on the complex stage; late pre-invasion interactions
The PfRH5-basigin binding stage, the time from the end of deformation to the start of invasion, is the time PCRCR is thought to have its primary function, aiding rhoptry release and tight junction formation [6,9,20].This stage of invasion is tightly regulated, with a mean time of 1.76 s [SD 0.53 s] (Fig 1A and S1 Table) [20].In comparison to control antibody EBL 040, both R5.004-( 22 ).Although there was only a single instance of an invasion under these conditions, the R5.008-

Antibody effects on invasion
Internalization of the parasite, measured from the time the merozoite begins to enter the erythrocyte until it is entirely inside the erythrocyte, typically has little variability and consistently takes about ten seconds (Fig 1A and S1 3.75]) decreased the duration of internalization, the combination of these two antibodies greatly slowed the invasion time (17.74 s), however this combination was so inhibitory there was only a single invasion observed (Fig 3E).When the RH5-basigin binding stage and invasion stages were combined there was an overall increase in the time from the end of deformation to the end of invasion for R5.004-( 22 ).

Antibody effects on echinocytosis
After invasion is completed, the invaded erythrocyte typically becomes spherical with distinctive spikes covering its surface for several minutes before returning to its usual biconcave shape [22,23,31] (Fig 1A).This process is called echinocytosis and is strongly associated with successful invasions [20].We noted that there were significant reductions in the numbers of these events per egress for the R5.008 ).Since PfRH5 likely binds basigin at the end of deformation [20], and PCRCR-complex binding has been linked to echinocytosis [32], we examined the time from the end of deformation to the start of echinocytosis.R5.008- (40)  To further analyze echinocytosis of the newly invaded RBCs, we measured increasing echinocytosis, the time from the initiation of echinocytosis to the point when maximum echinocytosis was reached, the duration of maximum echinocytosis, and decreasing echinocytosis, the time it took for the erythrocyte to recover its usual biconcave shape (Fig 1A and ).Despite echinocytosis being a post invasion phenomenon, the antibodies specific for PfRH5 and PfCyRPA were able to exert an inhibitory/delaying/slowing effect, particularly for Cy.007-(400).Echinocytosis is thought to be triggered by RBC membrane lipid perturbations caused during merozoite invasion and the invasion slowing effects of the antibodies may have increased these effects, thereby extending the period of echinocytosis [20,32].

Overall order and morphology of normal early ring development
We next examined the potential effects of the PfRH5 and PfCyRPA antibodies on the differentiation of merozoites into ring-stage parasites.Current knowledge of new ring development is largely restricted to observations of merozoites spinning or oscillating immediately following invasion in P. knowlesi and P. falciparum [22,32], with the most detailed description of the stages from an electron microscopy study on P. knowlesi [33].Before examining the effects of PfRH5 and PfCyRPA antibodies we first performed live cell imaging on parasites treated with the control EBL 040-(400) and found that the intraerythrocytic merozoites began to spin on average 16.40 s [SD 20.15s] after the completion of invasion (Fig 1A and S11 Video).The spinning lasted an average of 116.30 s [SD 111.20] followed by the growth of a pseudopod like protrusion from the merozoite 159.20 s [SD 297.89] later.Almost a minute later (mean 57.20 s [SD 69.50]), the merozoite lost its rounded shape and became irregular, typically through the growth of the original pseudopod or, less commonly, through the development and growth of secondary pseudopodia.This stage was defined as a preliminary-ring or 'pre-ring' (Fig 1A and S1 Table).Almost 10 minutes after this (mean 638.00 s [SD 434.52]) a 'complete-ring' was formed, defined as the point at which all dense regions on the rings became fluid and mobile and continuously changed shape (Fig 1A and S1 Table).
In the presence of EBL 040-(400), ring differentiation occurred in most merozoites which invaded and became intraerythrocytic, with 97.4% (SD 8.62%] forming a pseudopod-like protrusion (Fig 4A and ).During this time, the parasites remained at the invasion site and were likely tethered to the erythrocyte surface with no occurrences of fission between the parasitophorous vacuole membrane and erythrocyte membrane.
Most uninvaded, extracellular merozoites in the presence of EBL 040 IgG appeared unchanged over the 20-minute observation period.However, some extracellular merozoites  ).As pseudo-rings were not restrained within a parasitophorous vacuole, their pseudopodia became greatly elongated.

Antibody mediated inactivation of extracellular merozoites
In the presence of anti-PfRH5 and -PfCyRPA mAbs, the percentage of merozoites beginning to differentiate into intracellular rings by developing pseudopodia after invasion was very high and comparable to the control (Fig 4A and S4A Table).The percentages of merozoites that differentiated into intracellular pre-rings and into complete-rings in the presence of these mAbs was likewise similar to the EBL 040 control (Fig 4B and 4C, and S4B and S4C Table ).A major difference was observed, however, for extracellular merozoites in R5.004-( 22 ).
To determine if the pseudo-rings were live cells and not lysed merozoites we exposed latestage parasites expressing cytoplasmic GFP to R5.004 at 22.3 μg/mL.After release of the merozoites the pseudo-rings retained their fluorescence even after the extension of pseudopodia (S3A Fig) .If pseudo-ring formation represented a form of cell lysis and dying, we would have expected a loss of the cytoplasm and a strong reduction of GFP fluorescence.Instead, the fluorescence was strongly retained leading us to conclude the pseudo-rings were intact.As a control, we imaged merozoites untreated with IgGs and they retained the normal rounded shapes (S3B Fig). In

Antibody effects on regression
As mentioned previously, in the presence of R5.008- (40) ).
We also noted whether the extracellular merozoites treated with R5.004, R5.008, Cy.009 and the mAb combination required contact with RBCs prior to pseudo-ring development and observed that RBC contact was not required.For EBL 040, Cy.003 and Cy.007-antibody treated merozoites, pseudo-ring formation was infrequent whether RBCs were contacted or not.The only merozoites that reliably became pseudo-rings in Cy.003 and Cy.007 IgGs, were the merozoites that had invaded and then regressed (Fig 4).

Antibody effects on merozoite spinning
As indicated in Fig 1A, the first notable activity performed by merozoites after invasion is their spinning, oscillating, or twisting actions that have been implicated in helping sever the nascent PVM from the host cell plasma membrane [22,32,34].To compare extracellular and intracellular ring development, we measured time from egress (when merozoite exposure to mAbs began) to the start of spinning (the first observable indication of ring conversion).On average, in the presence of EBL 040, intracellular merozoites began spinning 173.50 s (SD 165.70 s] after egress, more quickly than extracellular merozoites at 284.  ).As a general observation, the mean times from egress to spinning for extracellular merozoites were shorter in the presence of the anti-PfRH5 mAbs than the extracellular control but was only significant for the R5.   ).
The duration of spinning in all extracellular merozoites developing into pre-rings was comparable to that observed in intracellular EBL 040 (mean 116.).  ).For most of the antibody-treated extracellular merozoites, the first pseudopod was often visible before the end of spinning compared to intracellular merozoites.This was particularly so for extracellular merozoites treated with Cy.003 or Cy.007 suggesting merozoite development may be more dysregulated for extracellular compared to intracellular merozoites (S4C Fig).

Antibody effects on pre-ring formation
Pre-ring formation was defined as the stage at which the merozoite's ovoid shape became progressively more irregular either by the growth and subsequent branching of the initial pseudopod or by the development of secondary pseudopods (Fig 1A

Antibody effects on complete ring formation
For PfRH5 and PfCyRPA antibody-treated intracellular parasites, the time from pre-ring formation to complete-ring formation was similar to the intracellular EBA 040 control (S4E Fig and S6E Table ).Extracellular merozoites in the presence of R5.004-( 22 The anti-PfRH5-exposed extracellular merozoites, which are efficiently neutralized and converted to pseudo-rings, do so in similar stages and timings as merozoites which have successfully invaded.In contrast, the Cy.003 and Cy.007 treated merozoites both intracellular and extracellular tend to be more dysregulated often taking longer to complete the differentiation steps (S6A-S6E Table ).

Discussion
Live cell invasion imaging has previously played an important role in establishing how the invasion slowing anti-PfRH5 human mAb R5.011 could boost the potency of the invasionneutralizing human mAb R5.016 [4].Indeed, these data identified a mechanism of synergy between non-competing vaccine-induced antibody clones that bound different epitopes on

PLOS PATHOGENS
The dual action of human antibodies specific to Plasmodium falciparum PfRH5 and PfCyRPA PfRH5 [4].We thus decided to apply this approach to study potential synergies between anti-PfRH5 and anti-PfCyRPA antibodies.We discovered that anti-PfRH5 mAbs generally appear to slow aspects of the pre-invasion complex phase immediately prior to tight junction formation, while anti-Cy.003and -Cy.007 mAbs slow the downstream internalization stage where the merozoite penetrates its erythrocyte.An exception was Cy.009 which often behaved more like an anti-PfRH5 mAb rather than the other anti-PfCyRPA mAbs.Titration of R5.008 alone and in combination with Cy.009 indicated this anti-PfCyRPA mAb was able to synergistically boost the inhibitory capacity of R5.008 both in GIA assays, and this synergy was confirmed by live cell imaging.Hopefully this synergistic effect can be clinically achieved considering recent experimental vaccinations of rats with PfRH5 and PfCyRPA antigen combinations did not produce IgGs that inhibited in vitro parasite growth much better than PfRH5 antigen antibody alone [35].
We also observed for the first time that anti-PfRH5 and PfCyRPA antibodies have effects on the post-invasion period (where newly invaded intraerythrocytic merozoites differentiate into ring-stage parasites).Unexpectedly, we found that the anti-PfRH5 mAbs and Cy.009 caused most of the merozoites that had not invaded to rapidly differentiate into extracellular amoeboid forms, which we have termed pseudo-rings.It is anticipated that the rapid induction of extracellular merozoites into pseudo-rings would block the capacity of the parasites to attempt re-invasion and could increase the potency of antibodies that target PfRH5 and PfCyRPA.Re-examination of previous invasion videos of parasites treated with PfRH5-binding R5.011 and R5.016 mAbs [4], indicated that pseudo-ring formation for extracellular merozoites was particularly strong for the highly growth inhibitory mAb combination (500 μg/mL R5.011 and 10 μg/mL R5.016) and intermediate for less growth inhibitory individual mAbs at these concentrations.
One of the most inhibitory anti-PfRH5 human mAbs reported is R5.004 [4], which binds directly to the basigin binding site of PfRH5.R5.008 binds near the basigin binding site of PfRH5, probably sterically hindering PfRH5's access to basigin.Using several measures, both PfRH5 mAbs reduced the number of invasions (both per egress and invasions per merozoiteerythrocyte contact).This also applied to productive invasions where the merozoites did not regress during the observation period.The degree of invasion inhibition observed by video microscopy at the antibody concentrations used here is on average double that of the growth inhibitory assay data for the PfCyRPA antibodies shown here and for PfRH5 antibodies in Alanine, 2019 [4].This is likely due to the merozoites taking longer to find suitable target RBCs in the monolayer of cells observed by microscopy than in the packed cells used for the growth inhibition assays.
For the limited number of invasions that were successful, our live cell imaging indicated both mAbs caused significant delay between the end of deformation and the start of internalization when PfRH5 binds basigin as part of the PCRCR complex [6,8,9,20].It therefore seems likely that during erythrocyte contact, there is competition between basigin and the anti-PfRH5 mAbs to gain access to PfRH5 with a threshold level of interaction between PfRH5 and basigin required for successful invasion.Competition between basigin and the anti-PfRH5 mAbs for PfRH5 likely delays time to reach the invasion threshold.
The anti-PfCyRPA mAbs were also very potent at inhibiting invasion using the invasions per egress and invasions per contact measurements with the Cy.007 Fabs being especially effective.This indicates that the Cy.007 Fab may be able to access its epitope far more effectively in the confined space between the merozoite apex and the RBC bound basigin complex [29] than the whole 150 kDa IgG even though the Fab would have less avidity than the parental mAb.Of the invasions which did occur, Cy.009 behaved (in a dose-dependent manner) similarly to the anti-PfRH5 mAbs by delaying the start of invasion after the end of deformation, suggesting Cy.009 may inhibit PCRCR complex formation or the binding of PCRCR to basigin [4,6].Although Cy.009 does not occupy basigin's binding site on PfRH5 it could sterically inhibit PfRH5 from binding to basigin, particularly in the crowded environment at the erythrocyte membrane where basigin binds RBC partner proteins PCMA or MCT1 [8,29].Cy.003 and Cy.007 on the other hand did not delay PCRCR complex formation or the binding of PCRCR to basigin but rather slowed down merozoite internalization.This could be due to the mAbs slowing movement of the merozoite through the tight junction into the erythrocyte.The combination of the R5.008 and Cy.009 mAbs so greatly inhibited invasion that only one event was recorded.Here, Cy.009 appeared to function similarly to the potentiating mAb R5.011, in that it slowed the pre-invasion phase allowing more time for the neutralizing PfRH5 mAb to function [4].Although Cy.009 did not appear to increase pre-invasion times as much as R5.011 [4], Cy.009 was much more inhibitory by itself and proved particularly effective in combination with R5.008.How mAb-induced interference of the PCRCR complex mechanistically blocks invasion will be explored later during the discussion of extracellular differentiation into rings.
Once merozoite internalization is complete, the erythrocyte starts to become an echinocyte about half a minute later.This is thought to be due to the deposition of lipids and other materials from the parasite rhoptries into the erythrocyte membrane causing asymmetry in the lipid bilayer, producing outward bending protrusions of the erythrocyte surface [20,32].Recently, high resolution lattice light sheet microscopy has revealed that echinocytosis probably begins much sooner after invasion than previously thought, evident as undulations of the erythrocyte membrane [32].The time required for echinocytosis to reach its maximum, duration of maximum echinocytosis, and time required for the erythrocyte to return to its normal biconcave shape vary broadly [20,32].The only consistently observed effect of the antibodies upon echinocytosis was that it appeared to initiate more rapidly during treatment with the anti-PfRH5 and Cy.009 mAbs after invasion, possibly because invasion had been delayed.
The transformation of newly invaded merozoites into ring-stage parasites was described several decades ago for P. knowlesi with the characteristic steps of merozoite spinning, pseudopod appearance and transformation of the ovoid-shaped merozoite into an amoeboid ring [22,33].Here we examined the timing of these events in P. falciparum and found the differentiation steps were conserved and that most invaded merozoites converted into rings.Spinning or oscillation of newly invaded P. falciparum merozoites have been previously noted and are thought to be a mechanical mechanism to promote severance of the parasitophorous vacuole membrane from the host cell membrane [32,34].Although we know little about merozoite spinning and the various downstream steps that result in conversion into rings, it is interesting that anti-PfRH5 and -PfCyRPA antibodies, particularly Cy.003 and Cy.007, could still influence ring differentiation (e.g., increasing spinning duration) despite the merozoites having already invaded.Whether the antibodies ultimately reduce the successful transformation and growth of intraerythrocytic parasites is not yet known.
The most interesting phenomenon observed in the presence of the antibodies was the rapid transformation of extracellular merozoites into pseudo-rings which our data indicate are equivalent to normal intraerythrocytic rings since GFP expressing pseudo-rings remained intact.This was most evident in the presence of anti-PfRH5 mAbs and Cy.009, where 40-100% of extracellular merozoites began to differentiate into pseudo-rings compared to around 8-18% in the presence of the EBL 040 IgG control and the other anti-PfCyRPA mAbs.In the presence of anti-PfRH5 mAbs, Cy.009, and the R5.008 + Cy.009 combination, the time from merozoite egress and hence antibody exposure, to the start transformation into pseudo-rings occurred with similar timing as successfully invaded merozoites.Pseudo-ring development is particularly interesting because it would presumably inactivate and neutralize the extracellular merozoites, preventing them from attempting another round of invasion.As egressed merozoites have been estimated to have a half-life of five minutes, this could mean a substantial proportion of the extracellular merozoites could be inactivated while they are still invasion competent [36].
The mechanism by which the anti-PfRH5 and Cy.009 antibodies trigger ring development in extracellular merozoites is not yet understood but it is possible that induction is potentially mimicking some naturally occurring step that takes place during normal invasion.It has been shown that the PCRCR complex and basigin are involved in triggering a calcium ion flux event at the apical end of merozoites that are about to invade erythrocytes [9,20,32].This event is thought to be part of tight junction formation whereby the rhoptries and micronemes release proteins that form the ring-like junction with the RBC surface through which the merozoite propels itself into the erythrocyte [9,20,27,37,38].It is possible that binding of IgGs to PfRH5 and subsequent crosslinking mediated by the bivalent IgG molecules may mimic binding of PfRH5 with basigin at the RBC surface and trigger a merozoite apical calcium ion flux that initiates a signaling cascade in the merozoite leading to ring development.Contact with RBCs is not required for pseudo-ring formation and experiments with merozoites treated with fluorescent calcium dyes could help resolve if the PfRH5 mAbs are triggering apical calcium fluxes.We note that merozoites treated with RON2L peptide which blocks tight junction formation, remain attached to the RBC surface unable to invade [20].In these merozoites in which the upstream PfRh5 -basigin interaction still presumably occurs, pseudo-ring formation takes place as rapidly as normal ring formation in parasites not treated with RON2L peptide.The putative role of basigin binding to PfRH5 in triggering pseudo-ring development could be investigated using recombinant basigin to mimic that present on the RBC surface.It will be particularly interesting to discover if basigin must be bound to the RBC membrane to trigger synapse formation with merozoite bound PfRH5 and its PCRCR partners to stimulate downstream invasion events and merozoite differentiation.
It is curious that the Cy.009 antibody also promotes pseudo-ring development given the Cy.003 and Cy.007 are not any more effective than EBL 040 control.Cy.009 binds to the B1 and B2 propeller regions of PfCyRPA like the other PfCyRPA mAbs, so perhaps subtle differences in its angle of binding are responsible for Cy.009's pseudo-ring promoting activity [7].It is therefore important to understand the fine specificity of the anti-PfCyRPA mAbs for their epitopes because even though they bind to same PfCyRPA blades, the epitopes do not overlap, and this could have important biological implications as observed here.

Conclusions
Here we have demonstrated that antibodies to PfRH5 and PfCyRPA block merozoite invasion and that mAbs R5.008 and Cy.009, that are specific for each of these respective proteins, function synergistically.Live cell imaging in the presence of antibodies at concentrations that partly reduce invasion indicate that the anti-PfRH5 and Cy.009 mAbs increase the period of PfRH5-basigin complex formation suggesting they might reduce the efficiency with which PfRH5 can functionally access basigin.The other anti-PfCyRPA mAbs increase the time taken for merozoites to internalize, suggesting they could sterically inhibit the speed with which the merozoite passes through the tight junction which could indicate the PCRCR complex still persists at the tight junction during invasion.The mAbs also influence speed and efficiency with which invaded merozoites can differentiate into intraerythrocytic ring-stage parasites which is surprising as the PCRCR complex functions well before this.The most unexpected finding was that anti-PfRH5 and Cy.009 mAbs trigger rapid differentiation of extracellular uninvaded merozoites into pseudo-rings.We next aim to determine if the differentiation of invasion competent merozoites into invasion-incapable pseudo-rings is rapid enough and occurs at antibody concentrations low enough to majorly boost the protective immunity of PfRH5 and PfCyRPA antigen-based human vaccines and how this can be improved.

Growth Inhibition Activity assays
Growth Inhibition activity (GIA) assays were performed according to the protocol of the international GIA reference center at NIAID, NIH, USA [39].Parasite cultures were synchronized using treatment with 5% sorbitol on the day before GIA assay set up.One-cycle GIA assays were performed at the indicated concentrations of mAbs.Biochemical measurement using a P. falciparum lactate dehydrogenase assay was used to quantify endpoint parasitemia which has been described previously [40].Percent GIA was calculated using the following equation where RBC are red blood cells: % GIA ¼ 100 À 100 * ðA650 sample À A650 uninfected RBC Þ=ðA650 infected control À A650 uninfected RBC Þ Assays were quality-controlled by inclusion of the anti-PfRH5 mAb 2AC7 as an in-plate control, and the anti-PfRH5 mAbs 2AC7, QA5, 9AD4 as external plate controls [41].5 mM EDTA was included as a positive control and a Zaire Ebolavirus glycoprotein-67 reactive IgG1 mAb (EBL 040) [21] was used as a negative isotype control for mAb samples.
To assess synergy/antagonism/additivity, two antibodies were used in combination.In all cases, one mAb was held at a constant concentration that on its own would be predicted to yield approximately 30% inhibition.A second mAb was then titrated alone to generate an inhibition curve.These data for the two mAbs (one titrated and one fixed concentration) were then used to calculate the predicted GIA of the combination by Bliss additivity [42].This prediction was then compared to the real GIA result of the mAb mixture tested in parallel using the combination concentration of the two mAbs (i.e. the fixed + titrated amounts).

Imaging of GFP-expressing pseudo-rings
Parasites expressing cytoplasmic GFP [44] were treated with 25 nM ML10 (Lifearc) to synchronize them at late schizonts.The 4% hematocrit culture was then diluted 1/50 in complete RPMI containing 10 mM ascorbic acid, 2 μg/mL Hoechst and +/-22 μg/mL R5.004 IgG and after being placed into an eight well chambered coverslip (Ibdi) they were imaged with a Zeiss AxioObserver Z1 fluorescence microscope equipped a heated environmental chamber.Parasite GFP fluorescence was captured for 1 s for consistency across the treatments.

Statistical analysis
Data were analyzed using GraphPad Prism (GraphPad Software, version 9).For all unpaired t tests, two-tailed p values were considered significant if �0.05.

Fig 1 .
Fig 1.The major stages of invasion of Plasmodium falciparum blood stage parasites and growth inhibitory activity (GIA) of anti-PfRH5 and -PfCyRPA IgGs and Fab fragments.(A) Several videos of P. falciparum in the presence of 400 μg/mL EBL 040 control IgG were analyzed to derive the average times for each of the major steps of pre-invasion, internalization, merozoite to ring transition and echinocytosis (EC) of the infected erythrocyte.After egress, merozoites took 42.0 s to contact the erythrocyte they invaded (all times are means).Following an initial contact of 0.9 s, the merozoites deformed their erythrocytes for 52.6 s followed by the quiescent complex stage of 1.8 s, when it is believed the PCRCR complex and tight junction form ("Rh5-basigin stage").Merozoite internalization or invasion takes 10.3 s after which the merozoite starts to spin 16.4 s later.Spinning lasts 116.3 s and once finished, a pseudopod emerges from the merozoite 159.2 s later.57.2 s after this, the merozoite membrane starts to become irregular to form a pre-ring.After 638.0 s, a fully formed complete ring is evident.40.9 s after invasion is complete, the host erythrocyte develops membranous protrusions in a process called echinocytosis.These protrusions increase in their extent reaching a maximum after 44.2 s.This state continues for 395.9 s until the erythrocyte returns to its usual biconcave shape over 305.9 s.Of note, the timing and duration of echinocytosis is highly variable.SD, range and number (N) of events are indicated in S1 Table.(B)In vitro single cycle growth inhibition activity (GIA) assay dilution series of R5.008 mAb alone (pink), or in combination with Cy.009 mAb held at 0.13 mg/mL (blue), against 3D7 parasites.Predicted Bliss additivity is indicated (grey).The solid green line indicates the GIA of mAb Cy.009 held alone at a fixed concentration of 0.13 mg/ mL.Dotted line indicates 50% GIA.(C) Comparison of the GIA of the Cy.007 mAb (purple) with its Fab fragment (light pink) indicating the latter is much more potent.Assay performed as in B.

Fig 2 .
Fig 2. Parasite specific mAbs to PfRH5 and PfCyRPA inhibit Plasmodium falciparum invasion of human RBCs.(A-F) Several live cell videos of P. falciparum merozoites egressing and attempting to invade erythrocytes in the presence of each of the antibodies (concentrations and combinations indicated) were analyzed.The number of successful events is presented for each parameter indicated by the y axis.Full antibody names and concentrations (μg/mL) are indicated below bottom graphs.Each event is represented by a symbol and bars indicate the median number of events (A,B,E) or the percentage of events (C,D,F).Statistical analyses were performed using unpaired t tests in GraphPad Prism V 9.0.The asterisks indicate where parasite mAbs have altered the number or percentage of events significantly from the EBL 040 control with *p<0.05,**p<0.01 and ***p<0.001.https://doi.org/10.1371/journal.ppat.1011182.g002 ) (mean 4.76 s [SD 1.63]) and R5.008-(40) (mean 4.39s [SD 2.34]), as well as Cy.009-(60 and 200) (mean 4.20 s [SD 2.55] and mean 9.88 s [SD 4.96], respectively) caused a delay at this stage, that was dose dependent in the case of Cy.009 (Fig 3D and S3D Table

Fig 3 .
Fig 3. Antibodies to PfRH5 and PfCyRPA modify temporal aspects of the pre-and post-invasion phases of Plasmodium falciparum into human RBCs.(A-E) The invasion steps being monitored from live cell videos of antibody-treated parasites are indicated on the y-axes.The antibody types and concentrations are indicated on the x-axes.Anti-PfRH5 (orange, pink) and Cy.009 (green) antibodies increase the length of the preinvasion phase from first erythrocyte contact to the start of merozoite penetration.The Cy.003 (dark blue) and Cy.007 IgG and Fabs (purples) tend to increase the length of the ) (mean 15.40 s [SD 4.76]), Cy.009-(200) (mean 18.80 s [SD 7.97]), Cy.003-(250) (mean 20.62 s [SD 9.30]) and Cy.007-(400) (mean 22.85 s [SD 10.11]) compared to the control (mean 11.89 s [SD 2.33]) (S2C Fig and S5C Table

Table and
Table and S7-S10 Videos).Cy.007 Fab-(400) was noteworthy as no extracellular merozoites reached the amoeboid ring stage (Fig 4C). the median.Statistical analyses were performed using unpaired t tests in GraphPad Prism V 9.0.The asterisks indicate where parasite mAbs have altered the number of events significantly from the EBL 040 intracellular or extracellular control (arrows) with *p<0.05,**p<0.01 and ***p<0.001.(D) Diagram summarizing the effects of the anti-PfRH5 and -PfCyRPA antibodies on ring differentiation for intracellular and extracellular merozoites.https://doi.org/10.1371/journal.ppat.1011182.g004PLOS PATHOGENS The dual action of human antibodies specific to Plasmodium falciparum PfRH5 and PfCyRPA While most conditions caused either nearly complete or close to no extracellular ring development, Cy.009-(60) caused intermediate levels of extracellular pseudopod (mean 41.38% [SD 19.21]), preliminary ring (mean 36.85%[SD 17.30]), and amoeboid ring (mean 24.20% [SD 19.63]) development, leaving this group differing from the binary situation evident with other conditions (Fig 4A-4D and S4A-S4C Table).