PfAMA1-expressing chimeric rodent malaria parasites provide an in vivo platform for evaluating multistage interventions against malaria
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Apical membrane antigen 1 (AMA1) is expressed in the merozoite and sporozoite infectious stages of the malaria parasite, and upon secretion plays essential roles during host cell invasion. AMA1 is a leading candidate for vaccine development, although specific antibodies frequently fail to inhibit the growth of field-isolated Plasmodium falciparum malaria parasites, likely due to the high diversity of polymorphisms in AMA1 surface antigens. A key step for efficient invasion of target cells is tight junction formation through interaction of merozoite-surface AMA1 and rhoptry neck protein 2 (RON2), which is secreted and embedded within the erythrocyte membrane. Antibodies or reagents that disrupt the AMA1-RON2 interaction represent interventions to reduce parasite transmission to humans, as well as to repress clinical symptoms. To create a mouse model system for the evaluation of reagents against P. falciparum AMA1 (PfAMA1), we generated CRISPR/Cas9-engineered rodent malaria parasites in which the endogenous Plasmodium berghei AMA1 (PbAMA1) was replaced with PfAMA1, resulting in a chimeric line Pb_PfAMA1. Pb_PfAMA1 parasites infect mouse liver and erythrocytes as efficiently as the parental line, demonstrating that PfAMA1 functionally complements the essential roles of PbAMA1. AlphaFold-based structure modeling suggested structural compatibility of the heterologous PfAMA1-PbRON2 interaction, and co-immunoprecipitation analyses supported the functional association of the PfAMA1 and PbRON complex required for merozoite invasion of erythrocytes. Utilizing the interaction-inhibitor R1 peptide with Pb_PfAMA1 sporozoites, we demonstrated that the AMA1-RON2 interaction is crucial for sporozoite invasion of hepatocytes. Repeated infection with Pb_PfAMA1 elicited PfAMA1-reactive antibodies, and immune sera inhibited the growth of the P. falciparum lines Pf3D7 and PfHB3B; suggesting that naturally processed parasite-derived PfAMA1 induces antibodies which recognize conserved conformational epitopes. To expand this platform, we replaced circumsporozoite protein PbCSP with PfCSP, to generate dual-chimeric Pb_PfCSP+PfAMA1 parasites. Together, these chimeric parasites establish an in vivo platform for evaluating multistage and multi-antigen interventions against malaria.
Author summary
Malaria remains a major global health problem despite the recent introduction of the first WHO-recommended malaria vaccine. The vaccine targets sporozoites before they establish liver infection, and interventions are needed which act at additional stages of the parasite life cycle. Apical membrane antigen 1 (AMA1) is a promising candidate because it is expressed in both merozoites and sporozoites, and participates in host-cell infection at both stages. To enable in vivo evaluation of PfAMA1-targeted interventions, we generated genetically engineered rodent malaria parasites expressing Plasmodium falciparum AMA1 in place of the endogenous protein. The chimeric parasites infected mouse livers and erythrocytes comparable to the parental line, demonstrating functional complementation by PfAMA1 in the rodent malaria parasite. Blocking the AMA1–RON2 interaction inhibited sporozoite infection of the liver, highlighting the importance of this interaction at multiple stages of infection. Repeated infection with the chimeric parasite induced antibodies that reduced the growth of two genetically distinct P. falciparum strains in vitro, suggesting recognition of native conformational epitopes. This chimeric parasite model provides a practical in vivo platform for developing and evaluating multistage malaria interventions targeting AMA1 and its interaction with RON2.
