Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi

[Abstract] Malaria remains a major cause of morbidity and mortality globally. Clinical symptoms of the disease arise from the growth and multiplication of Plasmodium parasites within the blood of the host. Thus in vitro assays to determine how drug, antibody and genetic perturbations affect the growth rate of Plasmodium parasites are essential for the development of new therapeutics and improving our understanding of parasite biology. As both P. falciparum and P. knowlesi can be maintained in culture with human red blood cells, the effect of antimalarial drugs and inhibitory antibodies that target the invasion or growth capacity of Plasmodium parasites are routinely investigated by using multiplication assays or growth inhibition activity (GIA) assays against these two species. This protocol gives detailed step-by-step procedures to carry out flow cytometry-based multiplication assays and growth inhibition activity assays to test neutralizing antibodies based on the activity of the parasite enzyme lactate dehydrogenase of Plasmodium knowlesi adapted to human red blood cell culture. Whilst similar assays are well established for P. falciparum, P. knowlesi is more closely related to all other human infective species (Pacheco et al., 2018) and so can be used as a surrogate for testing drugs and vaccines for other malaria species such as P. vivax, which is the most widespread cause of malaria outside of Africa, but cannot yet be cultured under laboratory conditions.


cytometry-based invasion/multiplication rate assays have previously been described for various
Plasmodium species (Basco et al., 1995;Bhakdi et al., 2007;Xie et al., 2007;Izumiyama et al., 2009;Bei et al., 2010;Moon et al., 2013). The advantage of flow cytometry-based assays is that they provide absolute measurements of parasite numbers, so are particularly good for examining changes in parasitemia between different parasite lines. As parasitemia increase only occurs after the release of merozoites from schizonts resulting in invasion and formation of new ring stage parasites-the flow cytometry-based assays are particularly useful for examining this transition. They can be expressed either as % of control, or as an absolute value like fold-growth or parasite multiplication rate.
The activity of the enzyme lactate dehydrogenase (LDH) was first measured as a means to detect the presence of Plasmodium falciparum as a fast alternative to microscopy screening (Makler and Hinrichs, 1993). The LDH assay is based on the fact that Plasmodium LDH can rapidly convert lactate to pyruvate by employing the NAD analog 3-acetylpyridine NAD (APAD) as a coenzyme, whereas human erythrocyte LDH uses APAD instead of NAD at a much smaller rate (200-fold slower). Measuring the malarial LDH activity in the presence of APAD is a specific and sensitive method for the detection of Plasmodium parasites (Basco et al., 1995).
The standardized P. falciparum growth inhibition activity (GIA) assay based on the LDH activity is used as standard to investigate vaccine candidate antigen activity (Kennedy et al., 2002) and routinely used for analysis of clinical trials with the international GIA Reference Laboratory at the NIH, USA (Malkin et al., 2005). LDH activity assays are often simpler than flow cytometry-based assays, but measure the presence of parasite derived LDH rather than directly measuring the number of infected cells. As such they can only provide relative growth rate values, so are best suited for comparison of multiple treatments of specific parasite lines e.g. for drug or antibody inhibition assays. Relative growth rates are normally expressed as a % of an untreated control.
This protocol describes the methodology for both multiplication assays based on flow cytometry and GIA assays based on LDH activity of Plasmodium blood stage parasites ( Figure 1). Here, the focus is on the simian malaria parasite Plasmodium knowlesi, but they can easily be adapted to other Plasmodium species by adjusting incubation times based on the life cycle length and specific culture conditions. P. knowlesi has recently been adapted to grow in human Duffy positive blood  and is therefore the second human malaria parasite with a long-term in vitro culture system, next to P. falciparum. Due to its close ancestry and biology, it is a suitable model to study invasion genes of P. vivax, which lacks a long-term in vitro culture system. 4 www.bio-protocol.org/e3743

Procedure
The protocol of thawing Plasmodium knowlesi A1-H.1 parasite depends on the source of parasites and needs to be checked with the person that froze the sample. Parasites are maintained in a flask gassed with a mixture of 90% N2, 5% O2 and 5% CO2 at 37 °C, monitored by microscopy using Giemsa-stained thin films, and parasitaemia maintained at between 0.5% and 10%. All procedures need to be carried out with sterile equipment, materials and reagents with aseptic techniques. Local safety policies must be followed for all work involving human infectious agents.
A. Parasite multiplication assay 1. Synchronize P. knowlesi parasites via purification with Nycodenz a. Transfer 5 ml of 55% Nycodenz working solution to a 15 ml conical tube and warm up to room temperature (check Note 1).
b. Centrifuge down a high parasitemia (4-10%) P. knowlesi 50 ml culture with 2% hematocrit at 900 x g for 4 min at high brake/acceleration at room temperature.
c. Resuspend parasite pellet at 50% hematocrit in RPMI. d. Carefully lay 2 ml of this culture onto 5 ml Nycodenz in a 15 ml tube.
e. Centrifuge at 900 x g for 12 min with low brake/acceleration (check Note 2).
f. Transfer the brownish colored top layer schizonts to a new conical tube and wash with RPMI to remove Nycodenz (see Figure 2). g. Incubate schizonts in culture media with 1 μM Compound 2 for 2-3 h (check Note 3).
h. Wash off Compound 2 with RPMI (centrifuge at 900 x g for 4 min at high brake/acceleration at room temperature) and transfer schizonts back to culture (with 2% hematocrit red blood cells). The number of harvested schizonts depends on the starting parasitemia and age of the parasites (check Note 4). 2. In the following cycle, when parasites are again reaching the schizont stage, set up the multiplication assay in a 96-well plate (see Figure 3 for the plate layout).
a. Fill out all outer wells in the 96-well plate with 100 μl of RPMI or sterile water. This ensures inner wells are not affected by evaporation.
b. Plate out 75 μl complete growth media alone (or with vehicle control) or with 2x concentration of drug or antibody to be tested. Plate out 75 µl of the parasite culture per well as indicated in the plate layout. The plate layout can be customized depending on the individual experiment (see Figure 3 for plate layout).
c. Prepare a culture with young schizonts at around 1% parasitemia and 4% hematocrit.
i. Purify synchronous schizonts with Nycodenz as described in Step A1.

d. Open a Histogram for R1 and generate a gate for parasites (H-2). X-axis is SYBR-Green signal 530/30 488F-A and y-axis is number of cells. Compare RBC only control with a
parasite containing well to find the right areas on the x-axis where RBCs and parasites need to be separated (Figure 4). Use the same gates for all wells. e. Save data as xls file and open it with excel.
f. Calculate ratios (H-2/R1*100) and subtract the ratio of RBC only control. Calculate the ratio of End (24 h incubation, plate 2) and Start (0 h incubation, plate 1) to determine the multiplication rate (Example in Figure 5).

Notes
1. 5 ml Nycodenz tube is required to purify parasites from 1 ml packed red blood cells at 4-10% parasitemia (e.g., 50 ml culture maintained at 2% hematocrit and 4-10% parasitemia will yield 1 ml of packed red blood cells after centrifugation). Add 1 ml of medium to 1 ml of packed red blood cells to make a 2 ml culture with 50% hematocrit.
2. Uninfected red cells and ring-stage parasites will sink to the bottom and schizonts form a layer on top of the Nycodenz.
3. Compound 2 is a PKG inhibitor that reversibly blocks merozoite egress. This step is optional but will help to maximize yield of late schizonts and also provides the user with some flexibility in timing for subsequent steps. Viability of parasites will dramatically decline for incubations longer than 3 h. As an alternative to Compound 2 a highly specific and potent derivative of Compound 2, referred to as ML10, can be used as well, which is available from LifeArc (Ressurreição et al., b. Whilst the initial starting parasitemia should be fixed for all samples, it is still critical to obtain a timepoint 0 for parasitemia (i.e., plate 1 for multiplication assay). This is because small variations in starting parasitemia can have a significant effect on the calculated growth rateusing different RBCs between samples can also alter the precise starting parasitemia.
Whilst here we use a second time point of 24 h, various other timepoints could be used depending on experimental aims. A timepoint of 24 h works well because all parasites would have undergone reinvasion and progressed to schizonts again, which are very easily identified using the flow cytometry assay. The downside of this is that both invasion efficiency and viability of developing rings and trophozoites are measured. Shorter timepoints (e.g., 2-6 h) can be used to more specifically look at invasion and early ring formation, but these require very synchronous and late stage schizonts, as any schizonts that have not yet progressed to form new rings at the specified timepoint will affect interpretation of the data. 12 www.bio-protocol.org/e3743 7. Assays can also be set up in 24-well plates with final volume of 1 ml.
8. For polyclonal antibodies; purified IgG from serum is typically used and no background inhibition is usually seen from negative control samples (N.B. mouse IgG can sometimes be problematic in P. falciparum GIA assays; but all other species tested have been OK), purified IgG from plasma cannot typically be used as the anticoagulant (EDTA or heparin) cannot be fully removed even with IgG purification, leading to background GIA. P. falciparum GIA assays using diluted serum are reported, but are less common. Monoclonal antibodies work well when purified.
9. If necessary, use a pipette to resuspend, but this poses the risk of introducing bubbles. Do not vortex).