In vitro Cultivation and Visualization of Malaria Liver Stages in Primary Simian Hepatocytes

[Abstract] Human liver is the primary and obligatory site for malaria infection where sporozoites invade host hepatocytes. Malaria hepatic stages are asymptomatic and represent an attractive target for development of anti-malarial interventions and vaccines. However, owing to lack of robust and reproducible in vitro culture system, it is difficult to target and study this imperative malaria liver stage. Here, we describe a procedure that allow cultivation and visualization of malaria hepatic stages including dormant hypnozoites using primary simian hepatocytes. This method enables sensitive and quantitative assessment of different hepatic stages in vitro .


Immunofluorescence assay
[Background] Malaria is transmitted to humans after bite of female Anopheles mosquito injecting sporozoites into the bloodstream, which migrate to the liver and invade host hepatocytes. Inside hepatocytes, sporozoites undergo first round of asexual multiplication and transformed into multinucleated hepatic schizonts. Fully matured hepatic schizonts burst and release merozoites that enter bloodstream and infect red blood cells (RBCs). Inside the RBCs, the parasites undergo second round of asexual multiplication and completion of blood stage cycle eventually elicit the clinical symptoms associated with malaria. Exceptionally, of all the Plasmodium species, sporozoites of P. vivax, P. cynomolgi and P. ovale can produce dormant hepatic forms known as hypnozoites (Prudêncio et al., 2011). P. vivax is the second major malaria parasite with a broader geographical distribution of all the malaria species including tropics, sub-tropics and temperate climate. The biggest challenge to elimination of P.
vivax malaria is the periodic malaria relapse caused by activation of dormant hypnozoites that initiate onset of liver stage proliferation (Wells et al., 2010). Due to lack of robust in vitro culture system, we have little understanding of the mechanism regulating dormancy and hypnozoite activation. As a result, there are no rationale target-based drug discovery approaches to develop therapeutics to treat and prevent relapsing P. vivax malaria. The P. cynomolgi simian malaria parasite, genetically related to P. vivax, is an excellent model that has been previously employed to study hypnozoite biology (Voorbergvan der Wel et al., 2017 and 2020) as well as used for discovery of liver stage active compounds (Zeeman et al., 2014) and novel anti-relapse compounds (Campo et al., 2015). Currently, primaquine and tafenoquine are the only two commercially approved drugs that can eliminate hepatic hypnozoites and consequently prevent relapses of P. vivax malaria. However, both drugs have major limitations, as they are contraindicated in pregnancy, lactating mothers, and patients with glucose-6-phosphate 2 www.bio-protocol.org/e3722 dehydrogenase (G6PD) deficiency (Baird et al., 2019). In addition, primaquine requires CYP2D6 bioactivation in the liver, and thus cannot be administered to poor CYP2D6 metabolizers (Baird et al., 2018).
Hence, novel radical cure anti-malarial drugs with improved safety profile are critically needed to enable malaria elimination.
Due to the inherent difficulties associated with studying relapsing parasites there is a dearth of stage specific protein markers. Transcriptomics (Voorberg-van der Wel et al., 2017) studies with relapsing malaria parasites yielded some differential markers of schizont development and hypnozoite maintenance. Ferredoxin, an apicoplast electron transport chain protein and GAP45 (Glideosome Associated Protein) an inner membrane complex (IMC) protein expressed differentially in liver stages.
Antibodies generated against these proteins revealed a contrasting staining pattern in P. cynomolgi, Recently, we demonstrated that LISP2 (Liver specific protein 2) expression coincides with liver stage development and serves as a molecular marker to differentiate hypnozoites from earliest developing hepatic stages or schizonts in relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax) .
We further demonstrated that LISP2 could be used as an early marker suitable for the development of the drug discovery assays predictive of anti-relapse activity (Gupta et al., 2019). Here, we describe the detailed protocol to cultivate and visualize P. cynomolgi malaria liver stages in simian primary hepatocytes including hypnozoites.    8. Invert the tube once to gently mix the cells with the media. 9. Spin down at 100 x g (677 rpm), 28 °C for 4 min (Acceleration = 9, Brake = 9). 10. Carefully aspirate the supernatant with a serological pipette. 11. With a serological pipette, gently re-suspend the cell pellet with sufficient warm plating medium.

Materials and Reagents
12. Count the cells with Trypan blue and a Neubauer improved hemocytometer chip.
13. Adjust hepatocyte suspension to 1 x 10 6 cells/ml with additional pre-warmed plating medium, if needed.

Note: Spin down at low brake to settle the sporozoites onto the cells.
4. Cautiously remove the plates from the centrifuge and incubate at 37 °C, 5% CO2 for three hours before the first medium replacement. Limit access to the incubator. 5. After three hours of incubation, cautiously remove medium from the wells with a multichannel pipette and add 150 µl of fresh medium (maintenance medium + Amp B) into every well.