Fig 1.
Human hepatocyte cell lines can be traversed by P. falciparum sporozoites.
Traversal was assessed by flow cytometry as an ability of cells to uptake and retain fluorescent high molecular weight dextran during sporozoite co-incubation for 6 hours. (A) Representative plots for each cell line are shown, 1.5:1 sporozoite-to-hepatocyte ratio was used. Numbers indicate percentage of dextran-positive cells. (B) Percentage of traversed cells normalized to HC-04 obtained from three sporozoite preparations, each experiment was conducted in triplicate. Mean +/- SD shown.
Fig 2.
Identification of hepatocytes infected with P. falciparum 3D7HT-GFP parasites by flow cytometry.
Sporozoites were added at a 2.3:1 sporozoite-to-hepatocyte ratio. Infected cells were identified by flow cytometry at 96 hours postinfection as GFP-positive events in PI-negative (viable) cell populations. Uninfected cultures propagated in parallel with infected cultures were used to define specificity of GFP-positive events. A similar number of total events were acquired from infected and uninfected cultures of each cell lines. (A) Data from one representative culture is shown for each cell line. Event number corresponding to the GFP-positive gate (middle panels) and the geometric mean of events in the GFP-positive gate is indicated (right panels). (B) Percentage of GFP-positive PI-negative events. (C) Number of GFP events per well (D) Geometric MFI in FL-1 of GFP-positive PI-negative events. Mean +/- SD shown for all bar graphs (n = 6 per cell line).
Fig 3.
Quantification of parasite-encoded 18S rRNA in cultures of human hepatocytes infected with P. falciparum.
Sporozoites were added at a 2.3:1 sporozoite-to-hepatocyte ratio. Real-time PCR analysis of 18S rRNA expression was done at 48 and 96 hours postinfection. Each culture (n = 3) was analyzed in technical triplicate. (A) 18S copy number normalized using CT values from GAPDH (B) 18S copy number per culture. (C) Growth curve of uninfected hepatocyte cell lines (n = 3 per time point). Mean +/- SD shown for all graphs.
Fig 4.
Visualization of parasites in HC-04 cells infected with P. falciparum.
GFP-positive PI-negative events were isolated by flow cytometry-based cell sorting 96 hours postinfection and cytospun. Representative micrographs of EEFs show DNA staining with DAPI, GFP expression and immunofluorescent detection of PfHsp70 by anti-Hsp70 monoclonal antibody (Blue = DAPI, Green = GFP, Red = PfHsp70. Scale bars indicate 10 μm).
Fig 5.
Comparison of detection of P. falciparum infection in primary human hepatocytes and HC-04.
Sporozoites were added at a 1:1 sporozoite-to-hepatocyte ratio. Infected cells were identified by flow cytometry at 96 hours postinfection as GFP-positive events in PI-negative (viable) cell populations (n = 3). Uninfected cultures were used to define the positive gates. (A) Representative gating for detection of GFP events using three human hepatocyte donors and HC-04. Number of GFP-positive PI-negative events indicated (middle panels). Geometric mean of GFP-positive PI-negative events indicated (right panels). (B) Comparisons of the percentage of GFP-positive events and (C) total number of GFP events obtained per well. Mean +/- SD shown. (D) Representative examples of GFP-positive PI-negative events isolated by flow cytometry-based cell sorting 96 hours postinfection and cytospun (Scale bars indicate 10 μm).
Fig 6.
Parasite morphology and size in cryopreserved human hepatocytes from three donors.
Sporozoites were added at a 1:1 sporozoite-to-hepatocyte ratio. Cultures were fixed and stained directly on coverslips 96 hours postinfection. (A) Representative micrographs of EEFs observed from each donor. DAPI (blue) represents host and Plasmodium nuclei and PfHsp70 (red) denotes EEF. Arrows indicate EEF in phase contrast micrographs (Scale bar = 10 μM). EEF size was measured by (B) surface area and (C) maximum diameter. (D) Proportion of EEFs observed to be directly adjacent to host nucleus. (E) Of EEFs distant from host nucleus, the distance to the closest nucleus is indicated. Mean shown (horizontal line) for individual dot plots.
Fig 7.
Parasite detection and persistence over time.
Sporozoites were added at 0.5:1 sporozoite-to-hepatocyte ratio. Cultures were infected and collected for flow cytometry starting at 48 hours postinfection in duplicate. Plots are shown for the number of GFP-positive events acquired at each time point and the geometric MFI of these events for (A) HC-04 and (B) primary donor 4051 (shading indicates time when a portion of the GFP population is obscured). Mean +/- SD shown.
Fig 8.
Expression of surface CD81 by human hepatocyte cell lines and primary hepatocyte donors.
Surface CD81 was stained using specific antibodies or an isotype control followed by flow cytometry for detection. (A) Representative plots for all cells are shown and geometric MFI is indicated for both isotype (black) and anti-CD81 staining (red). (B) Surface staining of a mock and transient transfection of HC-04. Transiently transfected HC-04 were infected and run on flow cytometry 96 hours postinfection; (C) GFP-positive number and (D) geometric MFI shown. Mean +/- SD shown.
Fig 9.
Influence of CD81 blocking by mAb 1D6 on P. falciparum hepatocyte infection.
Sporozoites were added at a 3:1 sporozoite-to-hepatocyte ratio. 1D6 or isotype were added to cultures at 10μg/ml prior to infection (-2 to 0 hours), during invasion (0 to 6 hours) or after invasion (6 to 24 hours). Representative flow plots shown for (A) HC-04 48 hours postinfection and (B) number and percentage of GFP-positive events in duplicate. (C) Flow plots for donor 4051 96 hours postinfection and (D) graphs indicated the number and percentage of GFP-positive events in duplicate. Mean +/- SD shown on all graphs.
Fig 10.
Influence of a humanized anti-CSP mAb 2A10 on P. falciparum traversal and invasion.
Sporozoites were added at a 4.5:1 sporozoite-to-hepatocyte ratio. Cells were infected in the presence of h2A10 or pre-2A10-AAV serum at varying dilutions. (A) Percentage of traversed cells measured by dextran uptake 6 hours postinfection. Representative flow plots for (B) HC-04 48 hours postinfection and (C) donor 4051 96 hours postinfection. Graphs indicated the number, percentage and geometric MFI of GFP-positive events in duplicate. Mean +/- SD shown on all graphs.