Evaluating the potential of cross-species neutralization of anti-PfCyRPA and anti-PfRIPR Monoclonal Antibodies

Abstract

PfCyRPA and PfRIPR are promising next-generation malaria blood-stage vaccine candidate antigens that play an essential role in erythrocyte invasion of Plasmodium falciparum. 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 zoonotic disease in South-East 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.

Figure 1.. P. falciparum and P. knowlesi CyRPA and RIPR Gene Alignments

A. Aligned P. falciparum and P. knowlesi CyRPA genes. Alignment is annotated with contact residues between PfCyRPA mAbs and PfCyRPA (Cy.002 - dark red, Cy.003 - red, Cy.004 - blue, Cy.007 - green, and 8A7 - purple) and with PfCyRPA mAbs to specific PfCyRPA binding regions when contact residues are unknown (i.e blade specific – light blue and light purple) (Cy.009 - yellow). CyRPA blades are annotated as Blade number.beta-sheet (e.g. - Blade 1, beta-sheet 2 = B1.2). B. Aligned P. falciparum and P. knowlesi RIPR genes. Alignment is annotated with PfRIPR mAbs to specific PfRIPR binding regions when contact residues are unknown (i.e EGF domain specific - gray) (1C4 - pink, 5G6 - red, 1G12 - blue).

Figure 2.. Assessment of anti-PfCyRPA and anti-PfRIPR monoclonal antibodies against 3D7 P. falciparum and H1 P. knowlesi

A. Growth inhibitory activity of seven anti-PfCyRPA mAbs, alone or in combination, tested against 3D7 P. falciparum. B. Growth inhibitory activity of seven anti-PfCyRPA mAbs, alone or in combination, tested against YH1 P. knowlesi. C. Growth inhibitory activity of three anti-PfRIPR mAbs tested at different concentration against 3D7 P. falciparum. D. Growth inhibitory activity of three anti-PfRIPR mAbs tested at different concentration against YH1 P. knowlesi. Controls include Naïve total IgG, anti-DARC mAb, and anti-BSG mAb. Anti-DARC and anti-BSG mAbs serve as positive and negative controls dependent on Plasmodium species. All experiments were performed with technical duplicates. N = 3 biological replicates were performed for anti-PfCyRPA mAbs and N = 2 biological replicates were performed for anti-PfRIPR mAbs. Error bars show SD.