Antibody-Mediated Protection against Plasmodium Sporozoites Begins at the Dermal Inoculation Site

Abstract

Plasmodium sporozoites are injected into the skin as mosquitoes probe for blood. From here, they migrate through the dermis to find blood vessels which they enter in order to be rapidly carried to the liver, where they invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic stage. Once sporozoites enter the blood circulation, they are found in hepatocytes within minutes. In contrast, sporozoite exit from the inoculation site resembles a slow trickle and occurs over several hours. Thus, sporozoites spend the majority of their extracellular time at the inoculation site, raising the hypothesis that this is when the malarial parasite is most vulnerable to antibody-mediated destruction. Here, we investigate this hypothesis and demonstrate that the neutralizing capacity of circulating antibodies is greater at the inoculation site than in the blood circulation. Furthermore, these antibodies are working, at least in part, by impacting sporozoite motility at the inoculation site. Using actively and passively immunized mice, we found that most parasites are either immobilized at the site of injection or display reduced motility, particularly in their net displacement. We also found that antibodies severely impair the entry of sporozoites into the bloodstream. Overall, our data suggest that antibodies targeting the migratory sporozoite exert a large proportion of their protective effect at the inoculation site.IMPORTANCE Studies in experimental animal models and humans have shown that antibodies against Plasmodium sporozoites abolish parasite infectivity and provide sterile immunity. While it is well documented that these antibodies can be induced after immunization with attenuated parasites or subunit vaccines, the mechanisms by and location in which they neutralize parasites have not been fully elucidated. Here, we report studies indicating that these antibodies display a significant portion of their protective effect in the skin after injection of sporozoites and that one mechanism by which they work is by impairing sporozoite motility, thus diminishing their ability to reach blood vessels. These results suggest that immune protection against malaria begins at the earliest stages of parasite infection and emphasize the need of performing parasite challenge in the skin for the evaluation of protective immunity.

Keywords: antibodies; malaria; preerythrocytic; skin; sporozoites; vaccine.

FIG 1

Sporozoites inoculated into the skin of irradiated-sporozoite immunized wild-type mice exhibit impaired motility. Wild-type mice were immunized with irradiated P. berghei sporozoites. Three weeks after the last boost, P. berghei mCherry sporozoites were inoculated into immunized or naive mice and imaged 10 min after inoculation by confocal microscopy. (A) Motile and nonmotile sporozoites were manually counted, and shown is the percent motile and nonmotile sporozoites in immunized and naive control mice. Data from 3 independent experiments were pooled and are shown are means ± standard deviations. The proportions of motile to nonmotile sporozoites in naive and immunized mice are significantly different by Fisher’s exact test (P < 0.001). (B and C) Sporozoite movement in the skin of immunized and naive mice was recorded at 10 min after sporozoite inoculation, and trajectories were analyzed. Shown are net displacement (B) and average speed (C) of motile sporozoites over the entire length of the movie. Movies from 3 independent experiments were analyzed, and shown is a representative experiment. Statistical comparisons of net displacement and speed in naive and immunized mice were performed using a linear mixed-effects model on the pooled data. Data from all 3 experiments are shown in Fig. S2. ***, P < 0.001.

FIG 2

Mice were immunized with irradiated P. berghei (Pb) or P. berghei expressing P. falciparum CSP (PbPfCSP) sporozoites, and 3 weeks after the last boost, PbPfCSP sporozoites were inoculated into naive and immunized mice and imaged at 10 min post-inoculation. Videos were recorded and sporozoite motility was analyzed. Shown are net displacement and average speed of motile sporozoites over the entire length of the movie. Movies from 2 independent experiments were pooled. Statistical comparisons were performed using a linear mixed-effects model. ***, P < 0.001; ns, not significant.

FIG 3

Sporozoites inoculated into the skin of mice passively immunized with MAb 3D11 exhibit impaired motility. Mice were passively immunized by i.v. inoculation of 150 µg of MAb 3D11. Sixteen hours later, P. berghei mCherry sporozoites were injected intradermally, and their movement in the skin was recorded in 5-min videos and analyzed. (A) Motile and nonmotile sporozoites were manually counted at the indicated time points after sporozoite inoculation, and shown are the percentages of motile and nonmotile sporozoites in MAb 3D11-immunized and naive control mice at each time point. At least 100 sporozoites were imaged per movie per time point. Results from 2 time courses were pooled, and shown is the mean ± standard deviation. The proportions of motile to nonmotile sporozoites between naive and MAb 3D11 inoculated mice were statistically significantly different at all time points (Fisher’s exact test, P < 0.001). (B) The nature of sporozoite trajectories at 10 min post-sporozoite inoculation into control and passively immunized mice was determined. Shown is the percentage of motile sporozoites that were exhibiting more linear meandering trajectories (left) or circular trajectories (right). Shown is the mean ± standard deviation of 3 biological replicates. *, P < 0.05 by the Mann-Whitney test. (C) Net displacement and speed of motile sporozoites in passively immunized and naive control mice at 10 min post-sporozoite inoculation measured over the entire length of the 5-min movie. Three independent experiments were performed, and shown is a representative experiment. Statistical comparisons of net displacement and speed in naive and immunized mice were performed using a linear mixed-effects model on the pooled data. Data from all 3 experiments are shown in Fig. S4. ***, P < 0.001.

FIG 4

Lower doses of mAb 3D11 impact sporozoite motility at the inoculation site. Mice were passively immunized by i.v. inoculation of either 25 µg or 50 µg of MAb 3D11, and 16 h later, P. berghei mCherry sporozoites were injected intradermally and imaged by confocal microscopy at 10 min post-sporozoite inoculation. (A) Motile and nonmotile sporozoites were manually counted, and shown are the percentages of motile and nonmotile sporozoites in immunized and naive control mice. Data from 2 independent experiments were pooled, and shown is the mean ± standard deviation. The proportion of motile to nonmotile sporozoites between naive and 25 µg or 50 µg MAb 3D11 was statistically significant, as was the difference between 25 µg and 50 µg MAb 3D11 (Fisher’s exact test, ***, P < 0.01). (B and C) Sporozoite trajectories were analyzed; shown is the net displacement (B) and speed (C) of motile sporozoites over the entire length of the 5-min movies. Data from two biological replicates were pooled, and statistical analysis was performed using a linear mixed-effects model. ***, P < 0.001; ns, not significant.

FIG 5

Sporozoites inoculated into passively immunized mice exhibit decreased blood vessel invasion. Mice were passively immunized by i.v. inoculation of 150 µg of MAb 3D11, and 16 h later, P. berghei sporozoites expressing mCherry were injected intradermally and imaged by confocal microscopy. Five-minute movies were recorded at the indicated time points, and the percentage of motile sporozoites entering into the blood circulation was scored. Data were pooled from 4 videos per condition per time point, and shown is the mean ± standard deviation of the percentage of motile sporozoites that entered blood vessels. Statistical analysis was performed using Fisher’s exact test on the pooled data. *, P < 0.05; **, P < 0.01.

FIG 6

Antibody has greater inhibitory activity in mice infected by mosquito bites. Mice were passively immunized by i.v. inoculation of 50, 25, or 12.5 µg MAb 3D11 and challenged 24 h later with 250 to 500 P. berghei sporozoites inoculated i.v. or by 8 infected mosquito bites. Control groups received equivalent doses of mouse IgG and were challenged in the same manner. Forty hours post-sporozoite inoculation, livers were harvested, and the parasite burden was quantified by RT-qPCR. Data were pooled from 3 to 4 independent experiments, with 4 to 6 mice/group per experiment. As shown, the difference between the passively immunized groups challenged with i.v. versus mosquito bites is statistically significant for each concentration (***, P < 0.001). The difference between the control groups (mIgG, i.v. versus mosquito bites [MB]) is not statistically significant for the 50-µg or 12.5-µg groups but is significant for the 25-µg group (*, P < 0.05). There were no statistically significant differences in the inhibition observed between the 50-, 25-, and 12.5-µg MAb 3D11 groups, challenged either by i.v. or MB inoculation of sporozoites. Statistical comparisons were performed using a linear mixed-effects model on the pooled data.

FIG 7

Comparative efficacy of MAb 3D11 after intravenous or mosquito-delivered sporozoite challenge. Data from experiments shown in Fig. 6 were analyzed to calculate the average fold reduction in the 3D11 groups compared to the mIgG controls, for i.v.-inoculated sporozoites (left) and mosquito-inoculated sporozoites (right). Numbers above each bar indicate the average fold reduction for each group. Average fold reduction was calculated by taking the negative inverse of the ratio [−1/(3D11:mIgG)] of the average P. berghei 18S rRNA copies in the 3D11 and mIgG groups for each concentration of MAb 3D11. Data from four independent experiments for 50 µg MAb 3D11 (n = 20 to 21 mice per group) and three independent experiments each for 25 µg (n = 15 mice per group) and 12.5 µg MAb 3D11 (n = 13 to 15 mice per group) were used to calculate the average liver parasite burden for each group.