title: Heat-precipitation allows the efficient purification of a functional plant-derived malaria transmission-blocking vaccine candidate fusion protein.
abstract: Malaria is a vector-borne disease affecting more than two million people and accounting for more than 600,000 deaths each year, especially in developing countries. The most serious form of malaria is caused by Plasmodium falciparum. The complex life cycle of this parasite, involving pre-erythrocytic, asexual and sexual stages, makes vaccine development cumbersome but also offers a broad spectrum of vaccine candidates targeting exactly those stages. Vaccines targeting the sexual stage of P. falciparum are called transmission-blocking vaccines (TBVs). They do not confer protection for the vaccinated individual but aim to reduce or prevent the transmission of the parasite within a population and are therefore regarded as an essential tool in the fight against the disease. Malaria predominantly affects large populations in developing countries, so TBVs need to be produced in large quantities at low cost. Combining the advantages of eukaryotic expression with a virtually unlimited upscaling potential and a good product safety profile, plant-based expression systems represent a suitable alternative for the production of TBVs. We report here the high level (300 μg/g fresh leaf weight (FLW)) transient expression in Nicotiana benthamiana leaves of an effective TBV candidate based on a fusion protein F0 comprising Pfs25 and the C0-domain of Pfs230, and the implementation of a simple and cost-effective heat treatment step for purification that yields intact recombinant protein at >90% purity with a recovery rate of >70%. The immunization of mice clearly showed that antibodies raised against plant-derived F0 completely blocked the formation of oocysts in a malaria transmission-blocking assay (TBA) making F0 an interesting TBV candidate or a component of a multi-stage malaria vaccine cocktail.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany.
authors.forename: Veronique
authors.initials: V
authors.lastname: Beiss
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Spiegel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany. alexander.boes@ime.fraunhofer.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Boes
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kapelski
authors.affiliation: Research Center for Infectious Diseases, University of Wuerzburg, Josef Schneider Str. 2/Bau D15, 97080, Wuerzburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Scheuermayer
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany.
authors.forename: Gueven
authors.initials: G
authors.lastname: Edgue
authors.affiliation: Institute for Molecular Biotechnology, RWTH Aachen University, Worringer Weg 1, 52074, Aachen, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Sack
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Fendel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Reimann
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
authors.affiliation: Research Center for Infectious Diseases, University of Wuerzburg, Josef Schneider Str. 2/Bau D15, 97080, Wuerzburg, Germany.|Institute for Molecular Biotechnology, RWTH Aachen University, Worringer Weg 1, 52074, Aachen, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Pradel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany.|Institute for Molecular Biotechnology, RWTH Aachen University, Worringer Weg 1, 52074, Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D005590:Fractional Precipitation; D006358:Hot Temperature; D017780:Malaria Vaccines / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D030821:Plants, Genetically Modified / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014611:Vaccination / Q000379:methods
chemical_list: D000953:Antigens, Protozoan; D017780:Malaria Vaccines; C430263:Pfs230 antigen, Plasmodium falciparum; C071284:Pfs25 protein, Plasmodium falciparum; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins
keywords: Plasmodium falciparum; agroinfiltration; downstream processing; host cell protein removal; plant-made vaccine