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Plasmodium vivax is the most widespread of the different Plasmodium species able to infect humans and is responsible for most malaria cases outside Africa. An effective, strain-transcending vaccine that alleviates or suppresses erythrocyte invasion would be a game-changer in eliminating vivax malaria. Recently, the binding of P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) to human Transferrin receptor (TfR1) has been described as essential for reticulocyte invasion, making this parasite protein an appealing vaccine candidate. Here, using P. vivax Cambodian clinical isolates in robust ex vivo invasion assays, we show that anti-PvRBP2b polyclonal and monoclonal antibodies that inhibit binding of PvRBP2b to TfR1 do not block P. vivax invasion into reticulocytes even at high concentrations. Anti-TfR1 antibodies do not inhibit P. vivax invasion either. Combinations at high concentrations of human monoclonal antibodies targeting different PvRBP2b epitopes do not inhibit invasion. Combinations of anti-PvRBP2b with anti-PvDBP do not enhance invasion inhibition caused by anti-PvDBP alone. We also show that the invasion of Cambodian P. vivax is trypsin-resistant while TfR1 is trypsin-sensitive, and we demonstrate that TfR1 is not recycled following trypsin treatment. We determined the PvRBP2b sequence of all isolates used in the invasion assays and analyzed polymorphism within epitopes recognized by anti-PvRBP2b antibodies. We show that polymorphism does not explain the absence of neutralization. Anti-PvRBP2b polyclonal antibodies recognized all four isolates tested in immunofluorescence assays while not inhibiting P. vivax invasion. Overall, our results demonstrate that PvRBP2b binding to TfR1 is not essential for invasion into reticulocytes of P. vivax Cambodian strains questioning the relevance of PvRBP2b as vaccine candidate., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade., (© 2021. The Author(s).)