Article
Evaluating PfGAP50 as a component of novel recombinant subunit vaccines derived from plants
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Published: | January 29, 2014 |
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One key objective in the Malaria Project of the Fraunhofer Future Foundation is the development and production of novel malaria vaccine candidates using plant- based systems. Plants are ideal manufacturing platforms for these purposes because their quality control and subcellular targeting capabilities allow the efficient production of novel, multi-stage, multi-component subunit vaccines in the optimal biochemical environment.
The 44.6-kDa transmembrane protein PfGAP50 of Plasmodium falciparum has previously been described as part of the inner membrane complex, anchoring the actin-myosin motor of the invasive stages. Recently, we showed that during gametogenesis in the gut of the mosquito vector, PfGAP50 relocates from the inner membrane complex to the plasma membrane. Here it functions as a receptor for the complement regulatory protein Factor H, which the newly formed gametes bind from the mosquito blood meal to evade lysis by human complement. Because antibodies against PfGAP50 can synergistically activate the classical and the alternative complement pathway, the transmembrane protein represents a promising novel candidate for transmission blocking vaccines.
We have now successfully produced His6-tagged variants of PfGAP50 variants by transient expression in Nicotiana benthamiana leaves, which are targeted to the ER, the apoplast and the plastids of the plant cells. The corresponding yields were 60 mg/kg (ER variant), 80 mg/kg (apoplast variant) and up to 120 mg/kg (plastid variant). All three proteins were harvested 5 days after infiltration and were efficiently extracted by blending with two volumes of PBS containing 2 µM CoSO4 and 500 mM NaCl. After centrifugation to remove debris, the proteins were recovered from the supernatant by Ni2+ IMAC, reaching 85% purity. The ER and plastid variants were tested for their ability to bind Factor H and used for the immunization of rabbits. The resulting sera will then be tested for transmission- blocking activity using a standard membrane feeding assay.
Our results demonstrate that functional PfGAP50 can be produced efficiently in plants, further adding to our portfolio of Plasmodium spp. antigens that can be used to develop the next generation of combination vaccines against malaria.