PfCyRPA and PfRIPR are promising next-generation blood-stage vaccine candidates that play a crucial role in erythrocyte invasion of Plasmodium species. CyRPA and RIPR orthologs are present in all human-infecting Plasmodium species, suggesting the potential for a cross-species vaccine. Using Growth Inhibition Assays (GIA), this study investigates seven anti-PfCyRPA and three anti-PfRIPR monoclonal antibodies targeting P. falciparum for their inhibitory activity against P. knowlesi, a non-falciparum species that contributes to a significant burden of disease in South Asia, shares some biological features with Plasmodium vivax,  and has a robust in vitro culture system. Despite their efficacy against P. falciparum and partially conserved epitopes, these antibodies exhibited minimal inhibition of P. knowlesi. Understanding the antigenic diversity and immune mechanisms across Plasmodium species is critical for advancing pan-species vaccine strategies.

Monoclonal antibody production

Seven previously characterized anti-PfCyRPA mAbs (Cy.002, Cy.003, Cy.004, Cy.007, Cy.009, 8A7, and 5B12) and three previously characterized anti-PfRIPR mAbs (1C4, 5G6, 1G12) were produced as previously described). Briefly, Cy.003, Cy.004, Cy.007, and Cy.009 mAbs were produced by Icosagen using HybriFree Technology. Spleen cells from immunized chickens were panned against antigen-coated immune modules. RNA that was extracted from bound cells were used to synthesize cDNA and amplify variable light and heavy chains. Amplified variable light and heavy chains were purified and cloned into human immunoglobulin G1 expression vectors. Cy.002, 8A7, and 5B12 were produced from immunized mice, while 1C4, 5G6, and 1G12 were produced from immunized rabbits. Spleen cells from immunized mice or rabbits were fused with myeloma cells and positive antibody-producing hybridoma cell lines were identified using ELISA. For 8A7, 5B12, 1C4, 5G6, and 1G12, positive hybridomas were sub-cloned to obtain monoclonal cell lines. For Cy.002 and 8A7, variable domains of the heavy and light chains were sequenced, synthesized, and cloned into AbVec-hIgG1/AbVec-hIgG1-kappa vectors.

Parasite Culture and growth inhibition assays (GIA)

3D7 P. falciparum cultures were maintained at 0.5-1.5% parasitemia and 4% hematocrit in supplemented parasite medium (RPMI 1640 containing 25 mM HEPES, 0.1 mg/mL Hypoxanthine, and 50 μg/mL Gentamicin and supplemented with Albumax II (0.5% v/v), 2 mg/mL sodium bicarbonate, and heat-inactivated human AB serum (5% v/v). H1 P. knowlesi cultures (human-RBC adapted clone YH1) were maintained at 0.5-1.5% parasitemia and 4% hematocrit in supplemented parasite medium (RPMI 1640 containing 25 mM HEPES, 0.1mg/ml Hypoxanthine, and 50 μg/mL Gentamicin and supplemented with Albumax II (0.5% v/v), 0.292 mg/mL of L- glutamine, 2 mg/mL sodium bicarbonate, and horse serum (10% v/v)). Human erythrocytes of blood group O+ donors were used for all cultures and cultures were incubated in an atmosphere of 1% O₂, 5% CO₂, and 94% N₂ at 37 °C.

Growth inhibition assays (GIAs) to quantify the anti-merozoite inhibitory functionality of antibodies

Prior to setting up GIAs, YH1 P. knowlesi cultures were synchronized with a 280 mM D-sorbitol, 20 mM Na-HEPES, and 0.1 mg/ml BSA solution (“P. knowlesi sorbitol”), while 3D7 P. falciparum cultures were synchronized with 5% D-sorbitol to kill non-ring blood stages. For GIAs, mAbs were added to 1% parasitemia at 4% hematocrit cultures in 96 well half-well area plates (for a final concentration of 1% parasitemia and 2% hematocrit) with a total volume of 40µL and incubated for one full erythrocytic cycle (~24 for P. knowlesi YH1 and ~48 hours for P. falciparum 3D7). Anti-PfCyRPA mAbs were tested in triplicates at final antibody concentrations of 200 ug/ml, 100 ug/ml, 50 ug/ml, and 25 ug/ml. To evaluate synergistic antibody effect, determining if combing mAbs resulted in a greater inhibition effect than if the mAbs were evaluated alone, combinations of anti-PfCyRPA mAb were performed. Anti-PfCyRPA mAbs used in combination (Cy.003 : Cy.009, Cy.004 : Cy.007, Cy.007 : Cy.009) were combined at equal concentrations (1 : 1) to achieve the same final antibody concentrations previously mentioned (i.e. Cy.003 100 ug/ml + Cy.009 100 ug/ml = Cy.003 : Cy.009 200 ug/ml). Anti-PfRIPR mAbs were tested in duplicate at concentrations of 400 ug/ml, 200 ug/ml, 100 ug/ml, and 50 ug/ml). For controls, a-BSG was tested at 10ug/ml, 1ug/ml, and 0.1ug/ml and anti-DARC (2C3, Novus Biologicals) was tested at 10ug/ml, 1ug/ml, and 0.1ug/ml as well as 200 ug/ml, 100 ug/ml, 50 ug/ml, and 25 ug/ml for two of the H1 P. knowlesi and 3D7 P. falciparum biological triplicate experiments. Naive IgG was tested at all the listed concentrations. After at least 95% of the parasites reinvaded in control wells with no antibody (RPMI only), the assays were harvested. Parasites cultures were transferred to 96 well U-bottom plates, washed once with 1X PBS-1% BSA, stained with a dilution of 1/2000 SYBR Green I (10,000X stock, Invitrogen) in PBS for 20 minutes, washed twice with 1X PBS-1% BSA, and resuspended in 1X PBS. Parasitemia for each well was acquired using a Cytoflex cytometer (Beckman Coulter), with 100,000 events (erythrocytes) per sample. Flow cytometry data was analyzed using Flow Jo_v10.8.1 software. Percent inhibition was calculated as 100-[(Parasitemia of test Antibody)/(Parasitemia of Naïve IgG control)x100].

Methods for processing the data

Flow data were processed using Flow Jo_v10.8.1 to calculate parasitemia. Parasitemia data, invasion percentages, and inhibition percentages were analyzed using Microsoft Excel and GraphPad Prism software.

Standards and calibration information

Growth inhibition activity assays controls included an Anti-Duffy mAb for P. knowlesi, as P. knowlesi relies on the duffy antigen receptor for chemokines (DARC) as an invasion receptor, and an Anti-Basigin mAb for P. falciparum, as P. falciparum relies on basigin (BSG,) an invasion receptor. Additionally, Naïve IgG was included.

Quality-assurance procedures performed on the data

For an assay to meet the quality-assurance threshold required for a successful assay, the parasite multiplication rate (PMR) must exceed 1 between the final parasitemia in RPMI control wells and initial parasitemia in the RPMI control wells, meaning the parasites have re-invaded at a higher parasitemia than the assay seeding parasitemia. Positive and negative controls were included in every experiment. For growth inhibition activity assays, biological replicates were performed to confirm results inhibition results.