Investigating the invasion biology of Plasmodium vivax using its sister species, Plasmodium cynomolgi

Abstract

The erythrocytic stages of the relapsing human malaria parasite, Plasmodium vivax (P. vivax), preferentially invades immature CD71+ reticulocytes. This strict tropism for nascent reticulocytes is the primary reason for the absence of P. vivax long term in vitro culture. Plasmodium cynomolgi (P. cynomolgi), a closely related simian malaria parasite, also preferentially invades human CD71+ reticulocytes, however it can be cultured continuously using mature monkey erythrocytes. In this thesis, I investigated the invasion biology of P. vivax using a recently re-established P. cynomolgi strain, P. cynomolgi Berok, which is sustained in Macaca fascicularis erythrocytes for long term in vitro culture.

The first aim of this research was to re-establish and optimise the long term in vitro culture of erythrocytic P. cynomolgi at the University of Otago. Here we not only succeeded in establishing a robust continuous culture of P. cynomolgi in New Zealand, but also developed methods to replace expensive monkey serum with cost effective human serum. We also optimised the enrichment and cryopreservation of cultured P. cynomolgi.

Secondly, the research compared the functional invasion characteristics between P. vivax and P. cynomolgi Berok erythrocytic merozoites. We observed both P. vivax and P. cynomolgi Berok to preferentially invade immature CD71+ human reticulocytes. Furthermore, as P. vivax is duffy-dependent, we incorporated the use of Fy6 2c3 and FyB antibodies to block the extracellular domain of the Duffy receptor (DARC, Fy or CD234) and observed a decrease in percentage of invasion in both P. vivax and P. cynomolgi Berok respectively. Our data shows that P. cynomolgi Berok is a representative model species navigating further exploration of the interaction between the P. vivax Duffy Binding Protein (PvDBP) and the host red cell DARC.

The next section of this study aimed to further characterize P. cynomolgi Berok ligand-receptor interactions with the human reticulocyte surface receptors based on solved P. vivax protein binding complexes to their cognate human host surface proteins. In addition to further investigating the PvDBP-DARC relationship using an in-situ protein binding assay we also endeavoured to study the less known relationship between P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) and the Transferrin receptor 1 (TfR1 or CD71) by co-immunoprecipitation assay. The study explored the binding of these recombinant proteins and their respective homologs in P. cynomolgi Berok to human and macaque recombinant proteins. Interestingly, no interaction was observed between paralog PcyDBP2 and DARC, and ortholog PcyRBP2b and TfR1.

We briefly explored the genotype underlying the expression of Vel blood group using cord blood donors collected at Shoklo Malaria Research Unit, Mae Sod, and Thailand. This newly identified blood group is highly expressed on the surface of immature reticulocytes. We have observed that in the majority of the donors, the intron 2 region of the underlying gene, Small integral membrane protein 1 (SMIM1), harbours the rs143702418C and rs1175550A genotypes.

Following the functional and recombinant protein studies, we compiled a series of transmission electron micrographs (TEMs) to show merozoite invasion with and without Cytochalasin D, trophozoite maturation, asexual multiplication, and cytoadherence to neighboring erythrocytes using the in vitro P. cynomolgi Berok culture.

In summary, our findings emphasized the role for in vitro P. cynomolgi Berok as a laboratory surrogate for erythrocytic P. vivax. The re-established in vitro P. cynomolgi Berok culture has widened our perspective to current P. vivax vaccine candidates (PvDBP and PvRBP2b), and strengthens our ability to develop effective treatments and vaccines to control vivax malaria in the future.