Clustering and Erratic Movement Patterns of Syringe-Injected versus Mosquito-Inoculated Malaria Sporozoites Underlie Decreased Infectivity

Abstract

Malaria vaccine candidates based on live, attenuated sporozoites have led to high levels of protection. However, their efficacy critically depends on the sporozoites' ability to reach and infect the host liver. Administration via mosquito inoculation is by far the most potent method for inducing immunity but highly impractical. Here, we observed that intradermal syringe-injected Plasmodium berghei sporozoites (^syrSPZ) were 3-fold less efficient in migrating to and infecting mouse liver than mosquito-inoculated sporozoites (^msqSPZ). This was related to a clustered dermal distribution (2-fold-decreased median distance between ^syrSPZ and ^msqSPZ) and, more importantly, a 1.4-fold (significantly)-slower and more erratic movement pattern. These erratic movement patterns were likely caused by alteration of dermal tissue morphology (>15-μm intercellular gaps) due to injection of fluid and may critically decrease sporozoite infectivity. These results suggest that novel microvolume-based administration technologies hold promise for replicating the success of mosquito-inoculated live, attenuated sporozoite vaccines.IMPORTANCE Malaria still causes a major burden on global health and the economy. The efficacy of live, attenuated malaria sporozoites as vaccine candidates critically depends on their ability to migrate to and infect the host liver. This work sheds light on the effect of different administration routes on sporozoite migration. We show that the delivery of sporozoites via mosquito inoculation is more efficient than syringe injection; however, this route of administration is highly impractical for vaccine purposes. Using confocal microscopy and automated imaging software, we demonstrate that syringe-injected sporozoites do cluster, move more slowly, and display more erratic movement due to alterations in tissue morphology. These findings indicate that microneedle-based engineering solutions hold promise for replicating the success of mosquito-inoculated live, attenuated sporozoite vaccines.

Keywords: Plasmodium berghei; fluorescent microscopy; malaria; motility; sporozoite.

FIG 1

Study design. Sporozoites were administered via mosquito inoculation or intradermal syringe injection. Liver load was subsequently assessed by bioluminescence and blood smear patency. Detailed analysis of the appearance of the dermal site, the sporozoite distribution, and sporozoite migration behavior was performed to reveal underlying mechanisms of decreased infectivity.

FIG 2

Outcomes for different administration routes. (A) In vivo images which show the liver load 44 h postinfection via 33 (IQR, 30 to 33) mosquito bites or intradermal syringe injection of 3,000 sporozoites. (B) Average of the luciferase activities in the liver 44 h after challenge by mosquito inoculation (median, 2.7 × 10⁵ RLU; IQR, 1.8 × 10⁵ to 3.7 × 10⁵ RLU) or intradermal syringe injection (median, 7.9 × 10⁴ RLU; IQR, 5.9 × 10⁴ to 8.5 × 10⁴ RLU) (*, P = 0.011; Mann-Whitney U test). (C) A calibration curve was generated based on a syringe-injected concentration range of sporozoites in skin (n = 3 in duplicate) to estimate the number of sporozoites delivered by 30 mosquito bites (median, 6,060 [2,203 to 13,481] sporozoites). Ct, threshold cycle.

FIG 3

Overview of dermal site appearance. (A and B) Overview of the inoculation site after sporozoite delivery by mosquito (A) and of the injection site after sporozoite delivery by intradermal syringe injection (B), shown as an overlay of a bright-field images and the sporozoite distribution (pseudocolored, blurred fluorescent image), accompanied by zoom-in images showing individual sporozoites (i to iv). (C) Plot of the nearest-neighbor distance for ^msqSPZ (yellow; median, 55 μm; IQR, 18 to 132 μm) or ^syrSPZ (purple; median, 23 μm; IQR, 13 to 43 μm). The overlap of the two distributions is plotted in orange.

FIG 4

Magnifications of dermal tissue morphology after sporozoite delivery. (A) Magnification of the tissue morphology of the inoculation site after sporozoite delivery by mosquito and of the injection site after sporozoite delivery by intradermal syringe injection. Based on the bright-field images, the cells (depicted in white) and the interstitial space (depicted in blue) were segmented. (B) Quantification of the cell shapes found after mosquito inoculation (n = 164) and syringe injection (n = 203), using roundness (panel i) and Feret’s diameter (the longest distance between any two points along the cell membrane) (ii) as measures. ***, P < 0.001; independent sample t test. (C) Overview of the dermal site shown as an overlay of a bright-field image and a map of mosquito-inoculated and syringe-injected sporozoite tracks (depicted in green).

FIG 5

Movement pattern of sporozoites. (A) Overview of the inoculation site after sporozoite delivery by mosquito and the injection site after sporozoite delivery by intradermal syringe injection, shown as a map of sporozoite tracks, color-coded for movement pattern (sharp turns in yellow, slight turns in blue, linear movement in red). (B) Quantification of the different aspects of sporozoite motility after administration by mosquito or syringe: (i) the movement pattern distribution based at frames, (ii) the percentage of clockwise and counter clockwise segments, (iii) the straightness index of the tracks (low: <0.5, high: >0.5), and (iv) the angular dispersion of the tracks (low, <0.5; high, >0.5). P values obtained by chi-square test.

FIG 6

Velocity of sporozoites. (A) Overview of the inoculation site after sporozoite delivery by mosquito inoculation and the injection site after sporozoite delivery by intradermal syringe injection, shown as a map of sporozoite tracks, color coded for velocity (yellow sections correspond to high velocity, purple sections correspond to a lower velocity). (B) Distributions of the average track velocities, including a probability density function, with its mean determined using expectation-maximization-based fitting of a mixture of 2 normal distributions, one describing the slow-moving sporozoite fraction (depicted in red, accounting for 38% of the ^msqSPZ and 23% of the ^syrSPZ) and one describing the fast-moving sporozoite fraction (depicted in green, accounting for 62% of the ^msqSPZ and 77% of the ^syrSPZ).

FIG 7

Interplay between motility parameters. (A) Density plot of all sporozoite tracks, including both ^msqSPZ (n = 778) and ^syrSPZ (n = 778), in a matrix of angular dispersion and the straightness index. (B) Representative example movement patterns associated with every quarter of the density plot. Group Q1, low angular dispersion, high straightness index (short, erratic tracks); Q2, high angular dispersion, high straightness index (short, straight tracks); Q3, high angular dispersion, low straightness index (consistently turning tracks); Q4, low angular dispersion, low straightness index (erratically turning tracks). (C) Density plot of all low (i)-, intermediate (ii)-, and high (iii)-velocity tracks on the same matrix of angular dispersion and the straightness index, resulting in 12 subsets (S1 to S12). Velocity categories are defined as slow (<1 μm/s, depicted in purple), intermediate (1 to 2 μm/s, depicted in orange), and fast (>2 μm/s, depicted in yellow). Percentages of the overall number of sporozoite tracks in every quarter of the density plots are given. (D) Comparison of the distributions (as a percentage of total tracks) for ^msqSPZ and ^syrSPZ tracks across subsets S1 to S12, with significant differences between S8 and S11 (P < 0.001, chi-square test).