Generation of a transgenic P. cynomolgi parasite expressing P. vivax circumsporozoite protein for testing pre-erythrocytic malaria vaccines in non-human primates

Malaria, caused by infection with Plasmodium parasites, exacts a heavy toll worldwide. There are two licensed vaccines for malaria as well as two monoclonal antibodies that have shown promising efficacy in field trials. Both vaccines and monoclonals target the major surface protein (circumsporozoite protein, CSP) of Plasmodium falciparum. Yet Pf is only one of the four major species of Plasmodium that infects humans. Plasmodium vivax is the second leading cause of malaria but Pv vaccine and monoclonal development lags far behind P. falciparum owing to the lack of basic preclinical tools such as in vitro culture or mouse models that replicate the key biological features of P. vivax. Notably among these features is the ability to form dormant liver stages (hypnozoites) that reactivate and drive the majority of P. vivax malaria burden. Plasmodium cynomolgi is a simian parasite which is genotypically and phenotypically very close to P. vivax, can infect common research non-human primates and replicates many features of Pv including relapsing hypnozoites. Recently, a strain of Pc has been adapted to in vitro culture allowing parasite transgenesis. Here, we created a transgenic P. cynomolgi parasite in which the endogenous Pc CSP has been replaced with Pv CSP with the goal of enabling preclinical study of anti-Pv CSP interventions to protect against primary and relapse infections. We show that the in vitro-generated transgenic Pc[PvCSP] parasite expresses both serotypes of Pv CSP and retains full functionality in vivo including the ability to transmit to laboratory-reared Anopheles mosquitos and cause relapsing infection in rhesus macaques. To our knowledge, this is the first gene replacement in a relapsing Plasmodium species. This work can directly enable in vivo development of anti-Pv CSP interventions and provide a blueprint for the study of relapsing malaria through reverse genetics.