Cryopreservation of Plasmodium Sporozoites

Abstract

Malaria is a deadly disease caused by the parasite, Plasmodium, and impacts the lives of millions of people around the world. Following inoculation into mammalian hosts by infected mosquitoes, the sporozoite stage of Plasmodium undergoes obligate development in the liver before infecting erythrocytes and causing clinical malaria. The most promising vaccine candidates for malaria rely on the use of attenuated live sporozoites to induce protective immune responses. The scope of widespread testing or clinical use of such vaccines is limited by the absence of efficient, reliable, or transparent strategies for the long-term preservation of live sporozoites. Here we outline a method to cryopreserve the sporozoites of various human and murine Plasmodium species. We found that the structural integrity, viability, and in vivo or in vitro infectiousness were conserved in the recovered cryopreserved sporozoites. Cryopreservation using our approach also retained the transgenic properties of sporozoites and immunization with cryopreserved radiation attenuated sporozoites (RAS) elicited strong immune responses. Our work offers a reliable protocol for the long-term storage and recovery of human and murine Plasmodium sporozoites and lays the groundwork for the widespread use of live sporozoites for research and clinical applications.

Keywords: Plasmodium; RAS vaccination; cryopreservation; freezing; malaria; sporozoite.

Figure 1

Cryopreservation protocol for Plasmodium sporozoites. Mice are infected with the murine Plasmodium spp. of interest (1). Once parasitemia reaches 2–4%, female mosquitos (Anopheles stephensi) are allowed to feed on the infected mice to transfer the infection (2). In the case of human malaria parasites, mosquitos are fed on parasitized red blood cells instead. The infected mosquitos are dissected and salivary glands removed (3), the glands disrupted using a 30-gauge syringe and resuspended in 100 µL media containing 1% mouse serum (4). An equal volume (100 μL) of Fraction A (Table 1) of the freezing medium is added to the isolated sporozoites in media (5) and equilibrated at 4 °C for 2 h (6). An equal volume of cold (4 °C) Fraction B (Table 1, 200 μL) is added to the tube containing the sporozoites with Fraction A (7). The sporozoites in the media (400 μL) are subsequently frozen down in the vapor phase of LN2 (8). After at least 30 min, the tubes containing sporozoites are transferred to the liquid phase of LN2 for long-term storage (9).

Figure 2

Cryopreservation retains the vitality of P. berghei and P. yoelii sporozoites. Bar graphs depicting the integrity, viability, and infectiousness of freshly isolated or cryopreserved (9 months) wild type P. berghei and P. yoelii sporozoites. All data collected from in vitro (integrity, viability) or in vivo (infectiousness) assays with at least three technical or biological replicates. Infectiousness indicates the frequency of mice exhibiting blood-stage infection following inoculation with 1 × 10³ freshly isolated or cryopreserved sporozoites at 7–10 d.p.i. Data presented as mean+ s.e.m and analyzed using 2-tailed t-tests, n.s = p > 0.05.

Figure 3

Cryopreservation retains infectiousness and function in sporozoites. (A,B) Microscopy images showing different developmental stages- sporozoites (A) and the extra-erythrocytic form in development (B) in in vitro cultured hepatocytes in recovered Pb-GFP-Luc after cryopreservation for 14 days. DIC: Differential Interference Contrast image. (C) Representative image depicting luminescence signal detected in B6 mice inoculated with cryopreserved Pb-GFP-Luc sporozoites (5 × 10⁴/mouse), 44 h post-inoculation. (D) Representative image depicting luminescence signal detected in B6 mice inoculated with cryopreserved Pb-GFP-Luc sporozoites (5 × 10⁴/mouse), 7 days post-inoculation. (E) Representative flow plot indicating the frequency of infected RBCs in a B6 mouse inoculated with cryopreserved Pb-GFP-Luc sporozoites (5 × 10⁴/mouse), 10 days post-inoculation. Data presented as mean + s.e.m from two separate replicate experiments, with 3 mice per group.

Figure 4

Cryopreserved radiation-attenuated sporozoites (RAS) induce strong immune responses. (A) Schematic depicting the generation of cryopreserved RAS vaccine. Plasmodium sporozoites isolated from freshly dissected mosquitos are attenuated through irradiation and preserved using the protocol outlined in Figure 1. Recovered sporozoites are inoculated into mice intravenously. Seven days after RAS vaccination, blood is collected and screened by flow cytometry. (B) Representative flow plots showing the frequency of activated (CD11a^hiCD8^lo) CD8 T cells (upper panel) or GAP50 epitope-specific CD8 T cells (lower panel) in B6 mice following vaccination with 1 × 10⁴ cryopreserved P. yoelii RAS, at 7-day post inoculation, presented as percentages. Naïve mice served as controls. Data represent two biological replicate experiments with at least 3 mice per group and presented as mean + s.e.m. The mean frequencies of activated and GAP50-specific CD8 T cell frequencies in B6 mice immunized with freshly isolated RAS were respectively 14.0 ± 4.5% and 2.26 ± 0.8%.