title: Plant expression and characterization of the transmission-blocking vaccine candidate PfGAP50.
abstract: BACKGROUND
  Despite the limited success after decades of intensive research and development efforts, vaccination still represents the most promising strategy to significantly reduce the disease burden in malaria endemic regions. Besides the ultimate goal of inducing sterile protection in vaccinated individuals, the prevention of transmission by so-called transmission blocking vaccines (TBVs) is being regarded as an important feature of an efficient malaria eradication strategy. Recently, Plasmodium falciparum GAP50 (PfGAP50), a 44.6 kDa transmembrane protein that forms an essential part of the invasion machinery (glideosome) multi-protein complex, has been proposed as novel potential transmission-blocking candidate. Plant-based expression systems combine the advantages of eukaryotic expression with a up-scaling potential and a good product safety profile suitable for vaccine production. In this study we investigated the feasibility to use the transient plant expression to produce PfGAP50 suitable for the induction of parasite specific inhibitory antibodies.
  
  
  RESULTS
  We performed the transient expression of recombinant PfGAP50 in Nicotiana benthamiana leaves using endoplasmatic reticulum (ER) and plastid targeting. After IMAC-purification the protein yield and integrity was investigated by SDS-PAGE and Western Blot. Rabbit immune IgG derived by the immunization with the plastid-targeted variant of PfGAP50 was analyzed by immune fluorescence assay (IFA) and zygote inhibition assay (ZIA). PfGAP50 could be produced in both subcellular compartments at different yields IMAC (Immobilized Metal Affinity Chromatography) purification from extract yielded up to 4.1 μg/g recombinant protein per fresh leaf material for ER-retarded and16.2 μg/g recombinant protein per fresh leave material for plasmid targeted PfGAP50, respectively. IgG from rabbit sera generated by immunization with the recombinant protein specifically recognized different parasite stages in immunofluorescence assay. Furthermore up to 55 % inhibition in an in vitro zygote inhibition assay could be achieved using PfGAP50-specific rabbit immune IgG.
  
  
  CONCLUSIONS
  The results of this study demonstrate that the plant-produced PfGAP50 is functional regarding the presentation of inhibitory epitopes and could be considered as component of a transmission-blocking malaria vaccine formulation.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany. veronique.beiss@ime.fraunhofer.de.
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. holger.spiegel@ime.fraunhofer.de.
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: Research Center for Infectious Diseases, University of Wuerzburg, Josef Schneider Str. 2/Bau D15, 97080, Wuerzburg, Germany. matthias.scheuermayer@googlemail.com.
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. andreas.reimann@ime.faunhofer.de.
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. stefan.schillberg@ime.fraunhofer.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074, Aachen, Germany. rainer.fischer@ime.fraunhofer.de.|RWTH Aachen University, Institute for Molecular Biotechnology, Worringer Weg 1, 52074, Aachen, Germany. rainer.fischer@ime.fraunhofer.de.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D019085:Fluorescent Antibody Technique, Direct; D007074:Immunoglobulin G / Q000276:immunology; D008288:Malaria / Q000517:prevention & control*; D017780:Malaria Vaccines / Q000235:genetics* / Q000276:immunology; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D010963:Plasmodium falciparum / Q000235:genetics*; D011817:Rabbits; D014026:Nicotiana / Q000378:metabolism*
chemical_list: D007074:Immunoglobulin G; D017780:Malaria Vaccines; D008565:Membrane Proteins; C509919:glideosome-associated protein 50, Plasmodium falciparum