Generation of a Transgenic Plasmodium cynomolgi Parasite Expressing Plasmodium vivax Circumsporozoite Protein for Testing P. vivax CSP-Based Malaria Vaccines in Non-Human Primates

<b>Background/Objectives</b>: Malaria, caused by infection with <i>Plasmodium</i> 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. The vaccines and monoclonal antibodies target the major surface protein (circumsporozoite protein, CSP) of <i>Plasmodium falciparum</i>. Yet <i>P. falciparum</i> is only one of the four major species of <i>Plasmodium</i> that infect humans. <i>Plasmodium vivax</i> is the second leading cause of malaria, but the <i>P. vivax</i> vaccine and monoclonal development lags far behind that for <i>P. falciparum</i> owing to the lack of basic preclinical tools such as in vitro culture or mouse models that replicate the key biological features of <i>P. vivax</i>. Notably among these features is the ability to form dormant liver stages (hypnozoites) that reactivate and drive the majority of the <i>P. vivax</i> malaria burden. <i>Plasmodium cynomolgi</i> is a simian parasite which is genotypically very close and phenotypically similar to <i>P. vivax</i>; it can infect non-human primates commonly used in research and replicates many features of <i>P. vivax</i>, including relapsing hypnozoites. <b>Methods</b>: Recently, a strain of <i>P. cynomolgi</i> has been adapted to in vitro cultures allowing parasite transgenesis. Here, we created a transgenic <i>P. cynomolgi</i> parasite in which the endogenous <i>P. cynomolgi</i> CSP has been replaced with <i>P. vivax</i> CSP, with the goal of enabling the preclinical study of anti-<i>P. vivax</i> CSP interventions to protect against primary and relapse infections. <b>Results</b>: We show that the in vitro-generated transgenic <i>Pcy</i>[<i>Pv</i>CSP] parasite expresses both serotypes of <i>P. vivax</i> CSP and retains full functionality in vivo, including the ability to transmit to laboratory-reared <i>Anopheles</i> mosquitoes and cause relapsing infections in rhesus macaques. To our knowledge, this is the first gene replacement in a relapsing <i>Plasmodium</i> species. <b>Conclusions</b>: This work can directly enable the in vivo development of anti-<i>P. vivax</i> CSP interventions and provide a blueprint for the study of relapsing malaria through reverse genetics.