A Plant-Based Transient Expression System for the Rapid Production of Malaria Vaccine Candidates
There are currently no vaccines that provide sterile immunity against malaria. Various proteins from different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates, but none of them have fulfilled expectations. Therefor
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Introduction The limited success of past and current malaria vaccine candidates [1] indicates the need for intensive and accelerated research to identify and characterize new antigens that confer protection against infection, clinical manifestation, and even the transmission of malaria [2]. Furthermore, multi-stage-specific cocktails combining key antigens from the different stages of the Plasmodium falciparum life cycle may be essential for the development of efficacious malaria vaccines [3]. To determine the suitability of novel vaccine candidates as components of vaccine cocktails, the antigens must be rapidly produced, purified, and characterized in terms of their protective efficacy in animal experiments and/or in vitro assays. Agrobacterium tumefaciens-based transient expression in plants, using either classical T-DNA vectors [4] or amplification systems based on viral replicons [5], is one of the quickest and most versatile strategies for the production of recombinant proteins [6–8]. Although used predominantly for research and development, these systems have also been implemented for the manufacturing of clinical-grade materials, e.g., the experimental antibody cocktail ZMapp, comprising three chimeric monoclonal antibodies against the Ebola virus surface glycoprotein (EBOV-GP) [9], virus-like particles based on human influenza virus hemagglutinin (HA) [10], and the malaria transmissionblocking vaccine candidate Pfs25 [11]. These emerging applications of transient expression are driven by a desire for rapid and inexpensive vaccine development against poverty-related diseases such as malaria [12, 13].
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_39, © Springer Science+Business Media New York 2016
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Alexander Boes et al.
Here we present a well-established and versatile workflow for A. tumefaciens-based transient expression in plants in the context of vaccine development, using the expression of single, multidomain, and fluorescence-labeled malaria vaccine candidates as case studies. The combination of transient expression with a modular and flexible cloning strategy allows the rapid cloning and expression of multi-domain antigens or DsRed reporter gene fusions by gene stacking, and small to medium scale production without expensive and specialized equipment. We have used this workflow successfully to produce several single and multi-domain malaria vaccine candidates [14–16]. However, the technology is generic and can be applied in any vaccine development scenario where progress is dependent on the rapid production of different candidate antigens for analysis and characterization. Because the downstream purification strategies and functionality assays are highly dependent on the specific antigen, these procedures are not covered in this chapter, but examples of such methods can be found in several reports describing the characterization of plantderived vaccine candidat
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