Sporozoite motility as a quantitative readout for anti-CSP antibody inhibition

Abstract

Antibodies can prevent malaria by neutralizing the infectious Plasmodium falciparum sporozoites (SPZ) before they establish an infection in the liver. Circumsporozoite protein (CSP), the most abundant surface protein of SPZ is the leading candidate for passive (and subunit) immunization approaches against malaria. Comprehensive assessment of the parasite-inhibitory capacity of anti-CSP monoclonal antibodies (mAbs) is an important step in advancing CSP-based immunization strategies. In this study, we employed a quantitative imaging-based motility assay to quantify the effect of anti-CSP mAbs on SPZ motility, both in vitro and in human skin.Our assay provided a quantitative measure of mAb parasite-inhibitory capacity through measurement of the half-maximal motility inhibitory concentration (IC_50M) value for anti-CSP mAbs (IC_50M 2A10: 24 nM, IC_50M 3SP2: 71 nM). We found a sevenfold discrepancy between the IC_50M and the binding saturation concentration measured by ELISA, possibly related to the observed shedding of CSP-mAb complexes during SPZ movement. In a subset of SPZ (5%), in vitro motility was unaffected by the presence of 2A10 while 3SP2 was able to completely block movement. In our ex vivo skin explant model, SPZ proved less susceptible to anti-CSP mAbs compared to SPZ in an in vitro environment. By quantitatively assessing motility, we created a valuable tool that can be used for comprehensive assessment of anti-CSP mAb potency. Insight that will help deepen our understanding of anti-CSP mAb potency and guide selection of the most promising anti-CSP mAbs for downstream clinical development.

Figure 1

Anti-CSP mAb reactivity to whole SPZ lysate. (a) ELISA curve of anti-CSP mAbs binding at different concentrations to whole SPZ lysate to determine mAb affinity (expressed as K_D). (b) ELISA curve of the effect of a serial dilution of ammonium thiocyanate on anti-CSP mAbs binding to whole SPZ lysate to determine mAb avidity (expressed as IC_50A).

Figure 2

Effect of anti-CSP mAb on SPZ movement and attachment. (b) The movement pattern distribution for control SPZ and SPZ exposed to a high concentration (> 100 nM) of the anti-CSP mAbs 2A10 and 3SP2. (b) The normalized percentage of moving SPZ plotted against the concentration of mAb to determine the IC_50M value at which movement of SPZ was 50% reduced. (c) Tracks of SPZ moving in vitro on a glass surface, color-coded for velocity using color range: purple (low velocity) to yellow (high velocity). (d) The probability distribution plotted of the velocity of control SPZ (median: 2.0 (IQR: 1.5–2.6) μm/s), SPZ exposed to low concentrations of 2A10 and 3SP2 (7 nM) (median velocity 2A10: 2.1 (IQR: 1.4–2.6) μm/s, median velocity 3SP2: 2.1 (IQR: 1.4–2.8) μm/s ) and SPZ exposed to approximately their IC_50M concentration (median velocity while exposed to 33 nM 2A10: 1.4 (IQR: 1.0–2.2) μm/s, median velocity while exposed to 66 nM 3SP2: 1.1 (IQR: 0.9–1.6) μm/s).

Figure 3

Localization of anti-CSP mAb (a) Schematic overview of the localization of bound anti-CSP mAbs using a fluorescent secondary Ab. (b) Visualization of the presence of the anti-CSP mAbs 2A10 and 3SP2; the mAbs staining is shown in green, the location of the SPZ is depicted with an arrow and the zigzag line indicates at which concentration the mAbs started to induce protrusions. Scalebar: 10 μm. (c) Visualization of the presence and the effect of 2A10 and 3PS2 on SPZ; to the left an overlay of the brightfield image with the SPZ counterstained with Cy5-Methyl-Methyl (shown in red) and Hoechst (shown in blue) and to the right an overlay of the mAbs staining (shown in green) and the counterstained SPZ. Scalebar: 20 µm.

Figure 4

SPZ velocity in human skin explant (a) Tracks of SPZ moving in human skin explant, color-coded for velocity using colour range: purple (low velocity) to yellow (high velocity). Scalebar: 100 µm. (b) The probability distribution plotted of the velocity of control SPZ (median: 1.8 (IQR: 1.1–2.3) μm/s, median annotated with grey dashed line) and SPZ exposed to increasing concentrations (67, 167, 333 nM) of 2A10 (median velocity SPZ exposed to 333 nM: 1.0 (IQR: 0.7–1.3) μm/s, median annotated with red dashed line). (c) The probability distribution plotted of the velocity of control SPZ and SPZ exposed to increasing concentrations (67, 167, 333 nM) of 3SP2 (median velocity SPZ exposed to 333 nM: 1.4 (IQR: 0.8–2.0) μm/s, median annotated with blue dashed line).

Figure 5

Effect of anti-CSP mAb on HC-04.J7 infection rate (a) Immunofluorescent staining of schizonts in HC-04.J7 cells by anti-Hsp70 staining (orange) and counterstaining with Hoechst (blue). Scale bar: 30 µm. (b) Images of the liver stage development assay representative for exposure to 333 nM 2A10 and 3SP2. Fluorescent anti-Hsp70 staining (orange) and counterstaining with Hoechst (blue). Scale bar: 30 µm. (c) The Ct (cycle threshold) value of 6 wells (open symbols) and the median (closed symbols) obtained by RT-PCR plotted against the concentration of mAb. The Ct values that represent a decrease of 50% and 90% in liver stage development are annotated.