A rapid and scalable density gradient purification method for Plasmodium sporozoites

Abstract

Background: Malaria remains a major human health problem, with no licensed vaccine currently available. Malaria infections initiate when infectious Plasmodium sporozoites are transmitted by Anopheline mosquitoes during their blood meal. Investigations of the malaria sporozoite are, therefore, of clear medical importance. However, sporozoites can only be produced in and isolated from mosquitoes, and their isolation results in large amounts of accompanying mosquito debris and contaminating microbes.

Methods: Here is described a discontinuous density gradient purification method for Plasmodium sporozoites that maintains parasite infectivity in vitro and in vivo and greatly reduces mosquito and microbial contaminants.

Results: This method provides clear advantages over previous approaches: it is rapid, requires no serum components, and can be scaled to purify >107 sporozoites with minimal operator involvement. Moreover, it can be effectively applied to both human (Plasmodium falciparum, Plasmodium vivax) and rodent (Plasmodium yoelii) infective species with excellent recovery rates.

Conclusions: This novel method effectively purifies viable malaria sporozoites by greatly reducing contaminating mosquito debris and microbial burdens associated with parasite isolation. Large-scale preparations of purified sporozoites will allow for enhanced in vitro infections, proteomics, and biochemical characterizations. In conjunction with aseptic mosquito rearing techniques, this purification technique will also support production of live attenuated sporozoites for vaccination.

Figure 1

Accudenz density gradient purification of Plasmodium sporozoites. A. Photographs of a bilayer discontinuous Accudenz density gradient overlayed with P. yoelii sporozoites in RPMI before and after centrifugation. Mosquito debris pellets to the bottom of the gradient, mosquito lipids float to the top of the gradient, and sporozoites accumulate at the bilayer interface. B. Representative DIC images of unpurified and purified sporozoites before and after centrifugation show a significant reduction in mosquito material. Scale bar is 5 microns.

Figure 2

Purified P. falciparum and P. vivax sporozoites remain infectious in vitro. A. HC-04 cells were infected with unpurified or purified P. falciparum sporozoites for 24 hours and were then subjected to an IFA (green: anti-PfCSP (2A10), blue: DAPI). Scale bar is 10 microns. B. Representative plots of sporozoite infectivity are provided from flow cytometric measurements of anti-Pf CSP antibody staining of HC-04 cells C. A graphical representation of two biological replicates of infections from (B) demonstrates that there is no infectivity defect of purified sporozoites. D. Purified P. vivax sporozoites were observed by IFA (green: anti-PvCSP 210 or anti-PvCSP247, blue: DAPI). No gross differences in parasite morphology were detected. Scale bar is 5 microns. E. HC-04 cells were infected with unpurified and purified P. vivax sporozoites for four days and developing liver stage parasites were observed by IFA (green: anti-PbHsp70, red: Phalloidin, blue: DAPI). No gross differences in parasite morphology were detected. Scale bar is 5 microns.

Figure 3

Purified P. falciparum sporozoites remain infectious to a humanized mouse. Two representative IFA panels of late liver stage P. falciparum parasites are shown using antibodies to human FAH (green), PfMSP1 (red) or with DAPI (blue). These images indicate that purified sporozoites are able to fully progress through liver stage development. Scale bar is 10 microns.