Abstract
Malaria, is a life-threatening disease caused by the intracellular protozoan Plasmodium spp. While the majority of the ~200 million malaria cases in 2021, were caused by the deadliest form of malaria Plasmodium falciparum, Plasmodium vivax is the most widespread. Over 40% of the world is at risk of P. vivax infection and as a relapsing form of malaria (due to dormant liver stages), its diagnosis and treatment are challenging. Unlike P. falciparum which has been in vitro cultured since the 1970s, we are still unable to continuously culture in vitro P. vivax, hindering our understanding of its biology and the development of new drug and vaccine targets.
A tractable model for P. vivax is its closely related sister taxon Plasmodium cynomolgi, whose usual host is the old-world monkey Macca fascicularis. Importantly P. cynomolgi shares many phenotypic and genetic characteristics of P. vivax. Unlike P. vivax, the Berok strain of P. cynomolgi can be cultured in vitro. However, the in vitro culture of P. cynomolgi is expensive (requiring the importation of monkey red cells) and its culture conditions, are fastidious and time consuming.
One of the major goals of this study was to optimise P. cynomolgi culturing methods with a focus on erythrocytic stage synchronisation to facilitate future transfection technologies. Therefore, the initial stages of this study involved mastering the fundamental skills needed to culture P. cynomolgi before examining new culture synchronisation techniques needed to conduct red cell invasion assays and transfections. Using a combination of Sorbitol, Nycodenz and magnetic sorting, we successfully achieved highly synchronised, high yield schizont stages which were used to quantify the invasion efficiency of P. cynomolgi Berok K4A7 laboratory clone. We found the K4A7 clone mean invasion rate was 31.27%, almost 10-fold higher than invasion rates of P. vivax and P. cynomolgi Bastianelli strain.
A secondly goal was to investigate the Duffy antigen receptor chemokine (DARC) and PcyRBP1, PcyDBP1 and PcyDBP2 ligands and their involvement in the invasion of P. cynomolgi into the M. fascicularis red blood cell (RBC). This was achieved through the use of enzyme treatments and monoclonal and polyclonal antibodies targeting the DARC receptor and PcyRBP1, PcyDBP1 and PcyDBP2 ligands. One of the key novel findings from this work was that the high efficacy (89.77%) of antibodies targeting PcyDBP2 in abrogating RBC invasion.
Finally, we sought to investigate rbp1a, rbp2d, dbp1 and dbp2 genes and their roles in P. cynomolgi RBC invasion, by tagging the proteins with a fluorescent reporter to be able to visualise them under an electron microscope. Despite numerous challenges, we were able to successfully construct the RBP2d plasmid tagged with an mCherry reporter.
The cost-effective in vitro culture and manipulation of P. cynomolgi provides a much-needed model to investigate the biology of P. vivax. Here we show that although many challenges face the genetic manipulation of this species, its high RBC invasion efficacy provides for an important platform to develop vaccines targeting the RBC stages of P. vivax.