Abstract
Despite decades of intensive research efforts there is currently no vaccine that provides sustained sterile immunity against malaria. In this context, a large number of targets from the different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates. None of these candidates has fulfilled expectations, and as long as we lack a single target that induces strain-transcending protective immune responses, combining key antigens from different life cycle stages seems to be the most promising route toward the development of efficacious malaria vaccines. After the identification of potential targets using approaches such as omics-based technology and reverse immunology, the rapid expression, purification, and characterization of these proteins, as well as the generation and analysis of fusion constructs combining different promising antigens or antigen domains before committing to expensive and time consuming clinical development, represents one of the bottlenecks in the vaccine development pipeline. The production of recombinant proteins by transient gene expression in plants is a robust and versatile alternative to cell-based microbial and eukaryotic production platforms. The transfection of plant tissues and/or whole plants using Agrobacterium tumefaciens offers a low technical entry barrier, low costs, and a high degree of flexibility embedded within a rapid and scalable workflow. Recombinant proteins can easily be targeted to different subcellular compartments according to their physicochemical requirements, including post-translational modifications, to ensure optimal yields of high quality product, and to support simple and economical downstream processing. Here, we demonstrate the use of a plant transient expression platform based on transfection with A. tumefaciens as essential component of a malaria vaccine development workflow involving screens for expression, solubility, and stability using fluorescent fusion proteins. Our results have been implemented for the evidence-based iterative design and expression of vaccine candidates combining suitable P. falciparum antigen domains. The antigens were also produced, purified, and characterized in further studies by taking advantage of the scalability of this platform.
Highlights
Even though the first malaria vaccine, GSK′s Plasmodium falciparum circumsporozoite protein (CSP)-based Mosquirix (RTS,S), (Wilby et al, 2012) is expected to enter the market within the 6–12 months, there is still an urgent demand for a malaria vaccine that reliably delivers long lasting protection against infection, clinical manifestation and transmission of the disease
According to Bally et al N. benthamiana isolates vary in the RNA-dependent RNA polymerase 1 gene (Rdr1) and a 72 bp insertion leading to a truncated version of the enzyme has been reported
The heat treatment of clarified or crude plant extracts is a valuable tool to simplify the downstream purification of recombinant protein from plant material because this step removes the majority of host cell proteins (HCPs) including RuBisCo (Buyel et al, 2014) but this approach is only suitable for recombinant proteins that resist temperature-induced denaturation
Summary
Even though the first malaria vaccine, GSK′s Plasmodium falciparum circumsporozoite protein (CSP)-based Mosquirix (RTS,S), (Wilby et al, 2012) is expected to enter the market within the 6–12 months, there is still an urgent demand for a malaria vaccine that reliably delivers long lasting protection against infection, clinical manifestation and transmission of the disease. RTS′S (Chattopadhyay et al, 2003; Richards et al, 2013; Penny et al, 2015; Reddy et al, 2015; RTS,S Clinical Trials Partnership, 2015) presents pre-erythrocytic stage epitopes and targets the parasite in an early phase of its life cycle within the human host. Various strategies must be considered to develop a better malaria vaccine, including the optimization of immune responses and immune memory against well-known and well-characterized targets like Pf CSP, by testing different presentation strategies (e.g., viral vectored vaccines, virus-like particles), formulations (virsosomes), delivery, and immunization schemes, as well as the identification of alternative and/or additional antigens to target the blood and sexual stages of the parasite life cycle to overcome allelic diversity and to prevent immune evasion. The available data indicate that the development of better malaria vaccines face two major challenges, i.e., the induction of a strong and long-lasting (durable) response, and the identification and combination of appropriate epitopes as targets to induce a neutralizing response
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