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

Abstract

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.

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. 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.

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 merozoite internalization period. (F) Cy.007 antibodies decrease the numbers of echinocytosis events per egress and (G-H) R5.008 (pink) and Cy.009 (green) decrease the time from the end of invasion/deformation to echinocytosis. Full antibody names and concentrations (μg/mL) are indicated below bottom graphs. Antibody 8 indicates Cy.007 Fab-(133). Symbols represent each event, and the median is shown by black bars. 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 control with *p<0.05, **p<0.01 and ***p<0.001.

Fig 4. The effects of anti-PfRH5 and PfCyRPA antibodies on the differentiation of intracellular and extracellular merozoites into ring-stage parasites.

(A-C) The ability of the antibodies to stimulate or inhibit the differentiation of intracellular, extracellular and regressed merozoites into (A) early, (B) pre-ring and (C) complete ring-stage parasites was assessed from observing live cell imaging videos of Plasmodium falciparum parasites. Full antibody names and concentrations (μg/mL) are indicated below bottom graph. Each event is represented with a symbol and bars indicate 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.