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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

A rapid and scalable density gradient purification method for Plasmodium sporozoites

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Mark Kennedy, Matthew E Fishbaugher, Ashley M Vaughan, Rapatbhorn Patrapuvich, Rachasak Boonhok, Narathatai Yimamnuaychok, Nastaran Rezakhani, Peter Metzger, Marisa Ponpuak, Jetsumon Sattabongkot, Stefan H Kappe, Jen CC Hume, Scott E Lindner
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-421) contains supplementary material, which is available to authorized users.

Competing interests

A provisional patent has been filed on this purification method on August 1st, 2012 (US Patent Application Number 61678541).

Authors’ contributions

Developed purification method: MK, SEL; Assessed P. yoelii purification and infectivity: MK, MF; Assessed P. falciparum purification and infectivity: AMV, NR, PM, SEL; Assessed P. vivax purification and infectivity: RP, RB NY and SEL; Assessed reductions of microbial burden: MF; Provided project supervision: MP, JS, SHK, JCCH, SEL; Wrote the Manuscript and Analysed the Data: MK, AMV, JCCH, SEL. All authors read and approved the final manuscript.

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.
Zusatzmaterial
Additional file 1: Representative pre- and post-purification sporozoite numbers tested in optimal conditions.(PDF 179 KB)
12936_2012_2582_MOESM1_ESM.pdf
Additional file 2: The composition of the solvent used to produce the Accudenz gradient greatly affects sporozoite recovery.(PDF 248 KB)
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Additional file 3: Representative pre- and post-purification sporozoite numbers tested with different accudenz concentrations.(PDF 169 KB)
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Additional file 4: The relative centrifugal force applied to the Accudenz gradient greatly affects sporozoite recovery.(PDF 247 KB)
12936_2012_2582_MOESM4_ESM.pdf
Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
12936_2012_2582_MOESM7_ESM.tiff
Literatur
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