This application is in response to the NIAID RFA-AI-21-075 entitled “Identification and Characterization of
Persistence Mechanisms of Select Protozoan Pathogens”, which explicitly states the study of P. vivax
hypnozoites as a research objective. P. vivax malaria burdens people within a wide global range and is more
pathogenic than previously appreciated. The parasite’s epidemiology and clinical impact is governed by latent
liver stages called hypnozoites, which are the source of relapsing blood stage infection. The molecular regulation
of hypnozoite stage formation, persistence and activation in hepatocytes has until recently remained unstudied,
mainly due to the lack of laboratory tools for this parasite. This has changed of the past ten years with the
development of our robust human liver-chimeric mouse model (FRG huHep mouse) that enables the detailed
analysis of hypnozoite formation and persistence as well the occurrence of relapses and robust in vitro primary
hepatocyte models of infection. In addition, with partners we have recently developed the genetically defined P.
vivax Chesson strain for use in hypnozoite studies, replacing complex field-isolate derived sporozoites for
infection. With these tools, the goals of this application are the identification of the parasite molecular drivers of
hypnozoite formation and persistence and host hepatocyte factors that impact hypnozoite formation and
persistence. We will achieve these goals through three independent but complementary aims. In Aim 1 we will
generate and interrogate hypnozoite gene expression data with emphasis on factors that are known to regulate
quasi-persistence in salivary gland sporozoites. Candidate factors such as AP2 transcription factors, the
eIF2a/IK2/UIS2 translational repression system and the, SAP1 and PUF stress granule mRNA storage system
will undergo comprehensive examination in P. vivax hypnozoites. Implicated factors that might drive and maintain
hypnozoite persistence will then be functionally interrogated using transgene expression and knockout as well
as overexpression in a rodent malaria model. In aim 2, we will establish a P. vivax transgenesis system using
sporozoite transfection and the Saimiri monkey infection model to establish P. vivax reporter gene-expressing
parasite lines, including a line that expresses a hypnozoite-specific marker GRP94, recently identified by us. We
will also functionally analyze factors in P. vivax that show strong persistence phenotypes in aim 1 to directly
interrogate their role in hypnozoite formation and persistence. In aim 3, we will identify and interrogate host
factors with emphasis on host defenses and metabolism in hypnozoite-infected hepatocytes. Host factors that
are associated with hypnozoite formation and persistence will be functionally analyzed using siRNA-mediated
knockdown and overexpression techniques in an in vitro hypnozoite infection model. Together, this application
will gain unprecedented insights into the parasite-intrinsic molecular regulation of liver stage latency as well as
the host factors that can further shape the latency phenotype. The findings might lead to the identification of new
parasite-targeted and host-targeted therapeutics that prevent relapsing malaria infection.
