Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P . falciparum , the P . vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P . vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P . vivax , however, lags considerably behind P . falciparum , which has many identified targets with several having transitioned to Phase II testing. By contrast only one P . vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P . vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P . vivax and P . knowlesi , for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P . vivax blood-stage targets. A panel of P . vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P . knowlesi . These antibodies were then tested for inhibition of growth and invasion of both wild type P . knowlesi and chimeric P . knowlesi lines modified using CRISPR/Cas9 to exchange P . knowlesi genes with their P . vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P . knowlesi as a model for P . vivax vaccine development and prioritizing antigens for further follow up.