Tissue-dependent neutralization of Pb sporozoites.

(A) Comparison of the log of percentual parasitemia at day 5 post-challenge and (B) survival curves of animals that received 100 µg of the isotype control (W24) or 3D11 mAb, followed by inoculation of sporozoites, in the tail vein (i.v.), micro-injected in the skin or by mosquito bite (bite). For i.v. and skin n=7 mice. For mosquito bite n=6 mice. N.I: non-infected. (C) Titration of the protection elicited by 3D11 after the i.v. challenge, 24 h after intraperitoneal transfer of the antibody (n =3). (A, C) Statistical significance was determined by one-way ANOVA with Holm-Šídák correction for multiple comparisons.

Kinetics of Pb sporozoite elimination in the liver.

(A-E) To assess the kinetics of parasite killing by 3D11, either PBS (control) or 100 µg of 3D11 mAb (3D11) were i.v. injected in mice 10-15 min before the i.v. inoculation of sporozoites expressing GFP-luciferase. Parasite load in the liver was assessed by bioluminescence. (A) Top: schematic representation of sporozoite homing, invasion, and development in the liver after i.v. injection. Bottom: Time schedule of the experiment. spz: sporozoites. (B) Exemplary picture of the recorded bioluminescence images and selected regions of interest (in red, ROIs). The signal in the 3rd quadrant was used to measure liver infection. Measurement was performed 7 min after spz inoculation in control and mice passively transferred with 3D11. Scale in radiance (photons/sec/cm2/sr). (C) Quantification of the percentage of the signal collected from the liver compared to the whole body. Statistical significance was analyzed using the unpaired t-test. (D) Total flux (photons/s) in the liver of mice from control and 3D11 groups. Statistical significance was determined by Kruskal-Wallis test. BG: background signal. (E) The ratio of the total flux in the liver between control and 3D11mAb-treated mice. (C-E) n=5 mice from 3 independent experiments. Data are presented as means ± standard deviation (SD). The cartoon was created with biorender.com.

3D11 inhibits sporozoite motility and partially blocks entry into the liver parenchyma.

(A) Graphical representation of the experimental setup to analyze sporozoite localization in the liver. For intravital experiments (in vivo), flk1-GFP female mice were transferred intravenously with 100 µg of 3D11. 10 min later, the animals were placed on the microscope stage and susceptible (Pb mCherry) and control sporozoites (PbPf GFP) were coinoculated i.v. into the tail vein. For ex vivo experiments, 3D11 was transferred 10 min before sporozoite injection, and 1 h later the liver was dissected and imaged. (B) Quantification of motility inhibition over time. Data from 4 independent experiments. (C) Representative images of one focal plane showing sporozoites inside the sinusoid lumen (left panel) or the hepatic parenchyma (right panel). Pb mCherry is shown in red and the GFP expressed by liver sinusoidal endothelial cells in white. (D) Left panel: Quantification of the localization of Pb mCherry (red) and PbPf GFP (black) sporozoites determined in vivo between 30 min to 3 h post-inoculation. Data from 2 independent experiments including a total of 3 animals. Statistical significance was determined by the Chi-square test. Right panel: quantification of sporozoite localization ex vivo determined 1 h post-inoculation. Data from two independent experiments. On top of the graphs is represented the number of quantified sporozoites. Data are presented as means ± SD. Cartoon was done with biorender.com.

3D11 inhibits sporozoite invasion and development in vitro.

(A) Experimental setup to measure the impact of 3D11 on sporozoite invasion and EEF development in HepG2 cells. Each experiment was performed in triplicates in the presence or absence of different concentrations of 3D11. Two identical plates were prepared to assess invasion at 2 h and development at 15 and 44h post-infection. Cells, as well as the supernatant, were analyzed by flow cytometry (B). Inhibition of HepG2 invasion by sporozoites was measured 2 h after parasites were added to the culture in the presence of 3D11 (blue square). In the same assay, sporozoite viability was quantified as the percentage of viable sporozoites after 2h relative to the control (yellow triangle). (C) In addition to the invasion assay, development inhibition was quantified at 15 (pink inverted triangle) and 44h (blue rhombus) after sporozoite addition to the cells. (D) Comparison between the percentage of infected HepG2 cells at 44 and 2 h. (E) Mean fluorescent intensity (MFI) of EEFs 44h post-infection. (B-E). Data were combined from 3-4 independent experiments. Bars represent the mean ± standard error of the mean (SEM). (F) Left: Representative images of intracellular parasites after being treated with 1.25 µg/ml 3D11. For treated parasites: 2, 15 and 44h after sporozoite and 3D11 addition, the cultures were washed, fixed and permeabilized, allowing the internalized 3D11 to be revealed using a goat anti-mouse 647 antibody (cyan). Right: Control parasites were labeled post-permeabilization with 3D11 to visualize CSP expression (green). Scale bar 10 µm. (G) The effect of 3D11 after sporozoite invasion was measured by adding 3D11 2 h after sporozoites were added to the culture (purple triangle, 2h). 3D11 was added simultaneously with the sporozoites for 2 h in the positive control group (green circle, 0h). Symbols represent two independent experiments, each with two technical replicates. Schematic created using Biorender.com.

In vitro effect of 3D11 on intracellular parasites.

HepG2 cells were incubated for 2 h in the absence (Control) or presence (3D11) of 1.25 µg/ml 3D11 antibody. Cells were PFA fixed at 2 and 44h. Extracellular sporozoites were stained using 1 µg/mL of 3D11. UIS4 was labeled using polyclonal antibodies to assess the formation of the PVM. (A) Representative maximum projections of the double staining with 3D11 to differentiate between intra-and extracellular parasites and UIS4 at 2 and 44 h post sporozoite addition. Scale bar 10 µm. (B) Quantification of the number of intracellular parasites with or without a positive UIS4 staining 2, 4, and 44 h after sporozoites were added into the culture. (C) Representative images of intracellular parasites at 44 h in the presence or absence of 3D11 for (D) comparison of their area, the Mean Fluorescence Intensity of the UIS4, 3D11 staining, and GFP.

Comparison of the ratio of total flux between 3D11-treated and control mice 24, 44, and 72 h post-infection (n= 3 animals). The analyzed ROI at 24 and 44 h was located in the upper abdominal region. In comparison, at 72 h, we analyzed the signal of the whole body since, at this time point, the parasites are outside the liver and have invaded the erythrocytes. The Kruskal-Wallis test determined statistical significance. Data are presented as means ± SD.

(A) The total amount of sporozoites as well as the percentage of Pb mCherry or PbPf GFP sporozoites that were injected in each experiment in Figure 3. 8.2,8.3, 8.4, 8.6: motility analysis. 8.2, 8.3: intravital imagining experiments. 8.7 and 8.10: ex vivo imaging. (B) log percentage parasitemia of mice used for intravital imaging experiments 3 days after sporozoites injection. Statistical significance was determined using the paired t-test.

Representative images of one focal plane showing PbPf GFP sporozoites inside the vessel or the hepatic parenchyma.

Gating strategy to analyze parasite mortality, invasion and development in the absence or presence of 3D11.

After 2 h, sporozoites can be found in the supernatant in the culture, attached to the cells, or inside the cells. We used the following gating strategy to analyze these three populations using the supernatant and the trypsinized cells (Figure 3A). (A) Gating strategy used to detect sporozoites that did not attach to or invaded cells at 2 h in samples supernatant. This parasite population was quantified using the green gate (SPZ outside HepG2). (B) After trypsinization, we used the same gating strategy to quantify the sporozoites inside (invaded, orange gate) or outside (attached, green gate) the HepG2 cells. The total number of sporozoites was calculated using these three populations: parasites found outside the cells in the supernatant and inside and outside of HepG2 cells after trypsinization. (C) 44 h later, only the EEFs inside the cells (GFP+ cells) were quantified.