Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages

Abstract

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts.

Conflict of interest statement

A.C.Y.C., J.J.Y.O., B.H.T., S.N.A., A.Y., K.B., S.B., A.L., C.B., B.K.S.Y., T.T.D. and P.B were/are employed by and/or shareholders of Novartis Pharma AG. The remaining authors declare no competing interests.

Figures

Fig. 1

Adaptation of P. cynomolgi Berok K4 line to continuous culture. a Schematic diagram of the successful adaptation of P. cynomolgi (Berok) from in vivo to in vitro culture. b Pilot ex vivo culture of P. cynomolgi Berok, B and M strain. The in vitro-cultured Berok K4 had to be sub-cultured at days 1, 3 and 5 due to robust growth, in contrast to the M and B strains where parasitaemias decreased to undetectable levels after day 3. cP. cynomolgi (Berok) in vitro culturing was further optimised to enable substantial multiplication (up to tenfold increase) that necessitated frequent dilution of the cultures when high parasitaemias were reached (red arrowheads). d Mature schizonts of culture adapted P. cynomolgi Berok K4 were enriched and allowed to re-invade fresh red blood cells that were then monitored every 2 h to document the complete asexual erythrocytic cycle in vitro. Scale bar represents 5 μm

Fig. 2

Transmission study from P. cynomolgi Berok K4 continuous culture. a In vivo blood-stage parasitaemia in two rhesus monkeys infected with 100,000 P. cynomolgi Berok K4 sporozoites (because of the use of a log scale for parasitaemia, negative smears are shown as 0.0001% parasitaemia). Both monkeys became blood-stage patent on day 11 post infection (dpi). Arrows indicate drug treatment (black arrows: 5-day chloroquine treatment, red arrow: 7-day primaquine treatment). Monkey 1 was bled for stocks on day 15, and relapsed (measured as thin smear positivity) on days 31 and 52, after which it was treated with chloroquine and primaquine. Monkey 2 was bled on day 19 dpi, and did not relapse during the follow-up period of 102 dpi, after which it was treated with primaquine. b In vitro infection of primary rhesus hepatocytes with P. cynomolgi Berok K4 sporozoites produced both hypnozoites (left panel) and developing liver-stage schizonts (right panel). Cultures were fixed at day 6 dpi, and stained with anti-PcHsp70 and a secondary antibody labelled with Alexa 568 fluorescent dye. Nuclei were stained with DAPI. An average of 16 PE forms per 10,000 inoculated P. cynomolgi Berok K4 sporozoites were observed. Scale bar represents 50 μm. c The total PE forms of in vitro infection rate of various primary rhesus hepatocytes with P. cynomolgi M strain sporozoites and P. cynomolgi Berok K4 line sporozoites. d The percentage of hypnozoites observed in vitro using primary rhesus hepatocytes from different donors infected with P. cynomolgi M strain or P. cynomolgi Berok K4 line sporozoites. The data (c and d) were analysed using the Welch’s t test with the significance level set at P < 0.05. The histograms represent means (n = 3), and the error bars the standard error of the mean (SEM) of replicates

Fig. 3

Phenotypic and rheological characterisation of the Berok K4 line from in vitro culture. aP. cynomolgi Berok K4-infected RBCs exhibit caveolae structures (yellow arrows) that are similar to those in P. vivax-infected RBCs (scanning electron microscopy, scale bars represent 1 µm and 100 nm for area shown at higher magnification in white box). b An atomic force microscope scan of trophozoite-infected human blood cells revealed caveolae occurred at lower frequency when compared with P. vivax. c, d The median (+ /- IQR) dimensions of these caveolae were similar (P. vivax n = 177, P. cynomolgi n = 91). e, f Amnis flow imaging clearly shows that the mature erythrocytic stages P. cynomolgi Berok K4; readily formed rosettes with uninfected red blood cells, which are also a key feature P. vivax (n = 5). g, h A dual micropipette aspiration method was used to demonstrate the rheological stability of the P. cynomolgi Berok K4 rosettes (n = 5). As observed in P. vivax, P. cynomolgi rosettes are tightly attached and the cells require around 400 pN to disrupt the adhesion. The non-parametric data resented in b, d, f and h were analysed using the Mann–Whitney U test with the significance level set at P < 0.05. The histograms and lines on box plots and scatter plots represent medians, and the error bars the interquartile range (IQR)

Fig. 4

Drug susceptibility testing using P. cynomolgi Berok K4 in vitro culture. a Correlation of P. cynomolgi Berok K4 and P. falciparum IC50 values of common antimalarial reference compounds in a SYBR green I proliferation assay. The potency of the compounds was comparable between the two species, except for artemisinin, atovaquone and artesunate which were more potent in P. cynomolgi as compared with P. falciparum. A X = Y line indicates equal inhibition towards compound. b Heatmap showing IC50 (μM) of a representative set of compounds from the Malaria Box. Majority of the compounds showed activity against both P. falciparum and P. cynomolgi, except for six compounds—MMV008127, MMV006250, MMV008270, MMV000563, MMV007839 and MMV008294 which displayed an IC50 > 10 µM for P. cynomolgi, and < 5 µM for P. falciparum. c Heatmap showing percentage of inhibition of the 125 antimalarial compounds from the Pathogen Box in P. cynomolgi Berok K4 and P. falciparum in different serums and concentrations. Nine compounds (MMV676380, MMV023388, MMV026550, MMV007625, MMV023949, MMV007638, MMV676442, MMV006833 and MMV020289) showed more than fourfold difference in inhibition between P. cynomolgi Berok K4 and P. falciparum in their equivalent serums

Fig. 5

Correlation of chloroquine IC50 of P. cynomolgi Berok K4 continuous culture with P. vivax clinical isolates. a Flow cytometry dot plots (Ethidium/Hoechst) of chloroquine-treated P. cynomolgi Berok K4 continuous culture gated for schizonts population. b IC50 determination of chloroquine in P. cynomolgi Berok K4 continuous culture using the schizont maturation assay (n = 3) with error bars representing standard deviation (SD). c IC50 determination of chloroquine in P. cynomolgi Berok K4 continuous culture using SYBR green I proliferation assay (n = 3) with error bars representing standard deviation (SD) d IC50 of chloroquine in P. cynomolgi Berok K4 continuous culture and P. vivax clinical isolates using the schizont maturation assay. The median ( + /− IQR) values for the IC50 of P. vivax clinical isolates and of P. cynomolgi Berok K4 continuous culture were similar. The non-parametric data in d was analysed using the Mann–Whitney U-Test with the significance level set at P < 0.05. The histograms and lines on box plots and scatter plots represent medians (P. vivax n = 10, P. cynomolgi n = 4) and the error bars the interquartile range (IQR)