Late killing of Plasmodium berghei sporozoites in the liver by an anti-circumsporozoite protein antibody

Abstract

Plasmodium sporozoites are inoculated into the skin during the bite of an infected mosquito. This motile stage invades cutaneous blood vessels to reach the liver and infect hepatocytes. The circumsporozoite protein (CSP) on the sporozoite surface is an important antigen targeted by protective antibodies (Abs) in immunoprophylaxis or elicited by vaccination. Antibody-mediated protection mainly unfolds during parasite skin migration, but rare and potent protective Abs additionally neutralize sporozoite in the liver. Here, using a rodent malaria model, microscopy and bioluminescence imaging, we show a late-neutralizing effect of 3D11 anti-CSP monoclonal antibody (mAb) in the liver. The need for several hours to eliminate parasites in the liver was associated with an accumulation of 3D11 effects, starting with the inhibition of sporozoite motility, sinusoidal extravasation, cell invasion, and terminating with the parasite killing inside the invaded cell. This late-neutralizing activity could be helpful to identify more potent therapeutic mAbs with stronger activity in the liver.

Keywords: Plasmodium; infectious disease; malaria; microbiology; monoclonal antibodies.

Figure 1.. 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 monoclonal antibody (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 hr 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.

Figure 2.. 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 third 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/s/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 two independent experiments. Data are presented as means ± SD. The cartoon was created with BioRender.com.

Figure 2—figure supplement 1.. Comparison of the ratio of total flux between 3D11-treated and control mice 24, 44, and 72 hr post-infection (n = 3 animals).

The analyzed region of interest (ROI) at 24 and 44 hr was located in the upper abdominal region. In comparison, at 72 hr, 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.

Figure 3.. 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. Ten minutes later, the animals were placed on the microscope stage and susceptible (Pb mCherry) and control sporozoites (PbPf GFP) were co-inoculated i.v. into the tail vein. For ex vivo experiments, 3D11 was transferred 10 min before sporozoite injection, and 1 hr later the liver was dissected and imaged. (B) Quantification of motility inhibition over time. Data from four 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. Scale bar: 10µm. (D) Left panel: Quantification of the localization of Pb mCherry (red) and PbPf GFP (black) sporozoites determined in vivo between 30 min to 3 hr post-inoculation. Data from two independent experiments including a total of three animals. Statistical significance was determined by the Chi-square test. Right panel: Quantification of sporozoite localization ex vivo determined 1 hr 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.

Figure 3—figure supplement 1.. Experimental settings and additional results related to Figure 3.

(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.

Figure 3—figure supplement 2.. Representative images of one focal plane showing PbPf GFP sporozoites inside the vessel or the hepatic parenchyma.

Figure 4.. 3D11 inhibits sporozoite invasion and development in vitro.

(A) Experimental setup to measure the impact of 3D11 on sporozoite invasion and exoerythrocytic form (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 hr and development at 15 and 44 hr post-infection. Cells, as well as the supernatant, were analyzed by flow cytometry (B). Inhibition of HepG2 invasion by sporozoites was measured 2 hr 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 2 hr relative to the control (yellow triangle). (C) In addition to the invasion assay, development inhibition was quantified at 15 (pink inverted triangle) and 44 hr (blue rhombus) after sporozoite addition to the cells. (D) Comparison between the percentage of infected HepG2 cells at 44 and 2 hr. (E) Mean fluorescent intensity (MFI) of EEFs 44 hr post-infection. (B–E) Data were combined from three to seven independent experiments. Bars represent 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 44 hr 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 circumsporozoite protein (CSP) expression (green). Scale bar: 10µm. (G) The effect of 3D11 after sporozoite invasion was measured by adding 3D11 2 hr after sporozoites were added to the culture (purple triangle, 2 hr). 3D11 was added simultaneously with the sporozoites for 2 hr in the positive control group (green circle, 0 hr). Symbols represent two independent experiments, each with two technical replicates. Schematic created using BioRender.com.

Figure 4—figure supplement 1.. Gating strategy to analyze parasite mortality, invasion, and development in the absence or presence of 3D11.

After 2 hr, 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 4A). (A) Gating strategy used to detect sporozoites that did not attach to or invaded cells at 2 hr 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) Forty-four hours later, only the exoerythrocytic forms (EEFs) inside the cells (GFP+ cells) were quantified.

Figure 5.. In vitro effect of 3D11 on intracellular parasites.

HepG2 cells were incubated for 2 hr in the absence (Control) or presence (3D11) of 1.25 µg/ml 3D11 antibody. Cells were PFA fixed at 2 and 44 hr. Extracellular sporozoites were stained using 1 µg/ml of 3D11. UIS4 was labeled using polyclonal antibodies to assess the formation of the parasitophorous vacuole membrane (PVM). (A) Representative maximum projections of the double staining with 3D11 to differentiate between intra- and extracellular parasites and UIS4 at 2 and 44 hr 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 hr after sporozoites were added into the culture. (C) Representative images of intracellular parasites at 44 hr in the presence or absence of 3D11 for (D) comparison of their area, the mean fluorescence intensity of the UIS4, 3D11 staining, and GFP.