Application of a Scalable Plant Transient Gene Expression Platform for Malaria Vaccine Development

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.

The Last Ten Years of Advancements in Plant-Derived Recombinant Vaccines against Hepatitis B

“…Transient production using an Agrobacterium tumefaciens- mediated transfer-DNA delivery system (Agro-infiltration) and/or virus-based replicating systems, the two dominant approaches, guarantees both the quality of the resulting purified products and the speed of development [57,58]. Spiegel et al [59] demonstrate the application of the classical Nicotiana benthamiana/A. tumefaciens transient expression system to accelerate the development of a malaria vaccine candidate, with screens for expression, solubility, and stability using fluorescent fusion proteins.…”

Media development for large scale Agrobacterium tumefaciens culture

“…Agrobacterium C58C1 pTFS40 was grown in media with phosphate concentration (Na 2 HPO 4 /KH 2 PO 4 ) varying from 0.01 to 0.1 mol PO 4 ‐/L with other media components held constant (4 g/L sucrose, 0.6 g/L MgSO 4 *7H 2 O, 0.025 g/L CaCl 2 *2H 2 O, 0.01 g/L FeSO 4 *7H 2 O, 0.0025 MnSO 4 *H 2 O) (Supporting information, Figure 4); results indicated that peak final biomass concentration was achieved when phosphate concentration was in the range of 0.04 to 0.085 mol PO 4 ‐/L.…”

“…To determine whether growing Agrobacterium in media lacking zinc or calcium affected transient expression level, strain C58C1 pTFS40 containing the GUS marker gene was grown in defined media lacking these components, and GUS expression was measured in detached N. benthamiana leaves. The base media used in this experiment contained 4 g/L sucrose, 2.5 g/L (NH 4 ) 2 SO 4 , 0.6 g/L MgSO 4 *7H 2 O, 0.01 g/L FeSO 4 *7H 2 O, 0.0025 g/L MnSO 4 *H 2 O, and phosphate buffer at pH 7 at 0.05 mol PO 4 ‐/L; GUS expression induced by Agrobacterium grown in this media was compared with Agrobacterium grown in media supplemented with either 0.06 g/L CaCl 2 *2H 2 O or 0.001 g/L ZnSO 4 *7H 2 O. Lack of Ca 2+ or Zn 2+ in media did not have a detectable effect on transient expression level when compared with bacteria grown in media containing these elements (Figure ).…”

“…Transient expression of recombinant proteins in plants is a rapid and easily‐scalable method of production that in many cases can offer an alternative to conventional mammalian cell culture systems. While proteins produced in plants can exhibit some structural differences to those produced in mammalian systems, transient expression in plants has been shown to be a viable method of producing active forms of a variety of commercially significant proteins, from vaccines to enzymes for cellulosic biofuel production . Large scale production facilities employing this production method have been described .…”

Platforms for Plant-Based Protein Production

“…Plant-produced influenza vaccines are regarded as quicker to develop and potentially cheaper than egg-produced vaccines. Many other therapeutic proteins reportedly produced by the transient expression platform include IgG and IgA antibodies [209,210], vaccine antigens against malaria, influenza and HIV [211][212][213][214][215], and therapeutic enzymes treating lysosomal storage diseases [216]; see recent reviews [140,195,217,218].…”