Ageing of Plasmodium falciparum malaria sporozoites alters their motility, infectivity and reduces immune activation in vitro

Abstract

Background: Sporozoites (SPZ), the infective form of Plasmodium falciparum malaria, can be inoculated into the human host skin by Anopheline mosquitoes. These SPZ migrate at approximately 1 µm/s to find a blood vessel and travel to the liver where they infect hepatocytes and multiply. In the skin they are still low in number (50-100 SPZ) and vulnerable to immune attack by antibodies and skin macrophages. This is why whole SPZ and SPZ proteins are used as the basis for most malaria vaccines currently deployed and undergoing late clinical testing. Mosquitoes typically inoculate SPZ into a human host between 14 and 25 days after their previous infective blood meal. However, it is unknown whether residing time within the mosquito affects SPZ condition, infectivity or immunogenicity. This study aimed to unravel how the age of P. falciparum SPZ in salivary glands (14, 17, or 20 days post blood meal) affects their infectivity and the ensuing immune responses.

Methods: SPZ numbers, viability by live/dead staining, motility using dedicated sporozoite motility orienting and organizing tool software (SMOOT), and infectivity of HC-04.j7 liver cells at 14, 17 and 20 days after mosquito feeding have been investigated. In vitro co-culture assays with SPZ stimulated monocyte-derived macrophages (MoMɸ) and CD8⁺ T-cells, analysed by flow cytometry, were used to investigate immune responses.

Results: SPZ age did not result in different SPZ numbers or viability. However, a markedly different motility pattern, whereby motility decreased from 89% at day 14 to 80% at day 17 and 71% at day 20 was observed (p ≤ 0.0001). Similarly, infectivity of day 20 SPZ dropped to ~ 50% compared with day 14 SPZ (p = 0.004). MoMɸ were better able to take up day 14 SPZ than day 20 SPZ (from 7.6% to 4.1%, p = 0.03) and displayed an increased expression of pro-inflammatory CD80, IL-6 (p = 0.005), regulatory markers PDL1 (p = 0.02), IL-10 (p = 0.009) and cytokines upon phagocytosis of younger SPZ. Interestingly, co-culture of these cells with CD8⁺ T-cells revealed a decreased expression of activation marker CD137 and cytokine IFNγ compared to their day 20 counterparts. These findings suggest that older (day 17-20) P. falciparum SPZ are less infectious and have decreased immune regulatory potential.

Conclusion: Overall, this data is a first step in enhancing the understanding of how mosquito residing time affects P. falciparum SPZ and could impact the understanding of the P. falciparum infectious reservoir and the potency of whole SPZ vaccines.

Keywords: Plasmodium falciparum; Immunogenicity; Malaria; Motility; Sporozoites.

Fig. 1

Experimental setup. Plasmodium falciparum infected mosquitoes were dissected at day 14, 17 or 20 post blood meal. To determine the number of SPZ, they were counted by light microscopy. Viability was determined by counting after Propidium Iodide staining using a confocal microscope. Motility of SPZ was analysed by traditional gliding assay and automated sporozoite motility orienting and organizing tool (SMOOT) analyses of confocal videos. Metabolism of SPZ was measured by real-time cell metabolic analyses. Infectivity was measured by in vitro infection of HC-04.j7 cells and analysed by immunofluorescence stains and real-time PCR of P. falciparum targets. Immune responses were assessed by SPZ stimulation of monocyte-derived macrophages (MoMϕs) for assessment of uptake (1 h) by confocal microscopy, phenotype (24 h) by flowcytometry and function (24 h) by ELISA. To investigate the activation of CD8⁺ T-cells, SPZ-stimulated MoMϕs and monocyte-derived dendritic cells (MoDCs) were co-cultured with circumsporozoite protein (CSP) specific CD8⁺ T-cells and analysed by flowcytometry

Fig. 2

Number and viability of SPZ. A Average number of SPZ per mosquito after 14 days (light blue), 17 days (blue) or 20 days (dark blue), post blood meal (A). B Percentage of live and dead SPZ 14 days (light blue), 17 days (blue) or 20 days (dark blue), post blood meal. Analysis using Mann Whitney U test, Fishers exact. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005 and ****p ≤ 0.0001

Fig. 3

SPZ motility by immunofluorescent staining of tracks. A Representative image of SPZ tracks obtained from SPZ harvested at day 14, day 17 and day 20 post blood meal. B Percentage (%) of moving SPZ and stationary SPZ at day 14 (light blue), day 17 (blue) and day 20 (dark blue). C Distance travelled by motile SPZ determined by counting circles, n = 3 with day 14 (light blue) 794 SPZ tracks, day 17 (blue) 469 SPZ tracks and day 20 (dark blue) 775 SPZ tracks. Analysis using Mann Whitney U test. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005 and ****p ≤ 0.0001

Fig. 4

Motility by SMOOT. A PLS-DA of day 14 SPZ (light blue), day 17 SPZ (blue) and day 20 SPZ (dark blue). B Loading plot for PLS-DA. C tSNE of day 14 SPZ (light blue), day 17 SPZ (blue) and day 20 SPZ (dark blue). D Percentage of SPZ from each timepoint per cluster plotted in stacked bars. E Features measured and analysed by SMOOT: frames, velocity, angular dispersion and straightness index. PLS-DA performed with R studio, tSNE with Matlab

Fig. 5

Infectivity of SPZ to HC-04.j7 hepatocytes. A Representative image of overlay 3 × 3 fields by ImageXpress of liver schizonts in HC-04.j7 cells, 72 h post infection with day 14, 17 and 20 SPZ. HC-04.j7 nucleus in blue, liver schizont double positive, HSP70 in green, GAPDH in red and overlay in yellow. B Number of HSP70 and GAPDH expressing exoerythrocytic forms (EEFs) 72 h after infection with day 14 SPZ (light blue), day 17 SPZ (blue) and day 20 SPZ (dark blue). C 18S PCR of infection assay of HCO4.j7 cells infected with SPZ at day 14 (light blue), day 17 (blue) and day 20 (dark blue). D Exp1 PCR of infection assay with HCO4.j7 cells. Analysis using Mann Whitney U test. Individual experiments indicated with different shapes (circle, square or triangle). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005 and ****p ≤ 0.0001

Fig. 6

Immune response. A Confocal microscopy image of SPZ uptake by MoMɸs. MoMɸs membrane in green, nucleus in blue and SPZ in red. B Percentage of P. falciparum SPZ uptake by MoMɸs of day 14 (light blue), day 17 (blue) and day 20 (dark blue) SPZ. C Expression of PD-L1 after stimulation with lipopolysaccharide (LPS) as a positive control (grey), and SPZ used at 14 days (light blue), 17 days (blue) and 20 days (dark blue) post blood meal. Data shown as median fluorescence intensity (MFI) fold change relative to medium stimulated control. D Expression of CD80 after stimulation with lipopolysaccharide (LPS) as a positive control (black), and SPZ at 14 days (light blue), 17 days (blue) and 20 days (dark blue) post blood meal. Data shown as median fluorescence intensity (MFI) fold change relative to medium stimulated control. E Production of IL-6 by MoMɸs after 24 h in pg/mL, fold change relative to medium stimulated control. F production of IL-10 by MoMɸs after 24 h. Day 14 N = 13, Day 17 N = 6, Day 20 N = 15. Analysis using Mann Whitney U test. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005 and ****p ≤ 0.0001

Fig. 7

CD8⁺ T-cell response and immunogenicity. A Percentage of CD137⁺ CD8⁺ T-cells. CSP (black), day 14 SPZ (light blue), day 20 SPZ (dark blue), day 14 and 20 dead SPZ (grey striped bar). Data shown as fold change relative to medium stimulated control. B Percentage of IFNγ⁺ CD8⁺ T-cells. C Ratio of day 14 divided by day 20. In purple ratio score above 1, day 14 SPZ show an increased pattern compared with day 20 SPZ. In yellow ratio score below 1, day 14 SPZ show a decreased pattern compared with day 20 SPZ. Calculated for MoMɸs experiments: uptake, expression of PDL1 and CD80 and cytokine production of IL10 and IL6, and for CD8⁺ T-cell co-culture activation markers CD137 and IFNγ. Analysis using Mann Whitney U test. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005 and ****p ≤ 0.0001