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.

Keywords: Nicotiana benthamiana plants; Plasmodium falciparum; expression screening; heat stability; multi domain-fusion antigens; red fluorescent protein.

Figure 1

Plant expression cassette. Schematic presentation of the expression cassettes in the plant binary expression vector pTRAkc-ERH. CaMV 35Spromoter and terminator: promoter with duplicated enhancer and terminator of the Cauliflower mosaic virus (CaMV) 35S RNA gene; 5′ untranslated region: 5′-UTR of the chalcone synthase gene from Petroselinum crispum; signal peptide sequence: transit peptide sequence of the murine antibody heavy chain; RFP, red fluorescent protein from Discosoma spp. (Bevis and Glick, 2002); GOI, Gene of interest. The restriction sites used to insert the GOI into the plant expression vector are indicated; His₆ tag: sequence encoding the six histidine affinity purification tag; ER-retention signal: sequence encoding the SEKDEL ER-retention signal.

Figure 2

Visualization of RFP accumulation in infiltrated N. benthamiana leaves. Accumulation of RFP-antigen fusion proteins can be visualized under green light using a red filter (Rademacher et al., 2008). Non-infiltrated (wt) and infiltrated N. benthamiana leaves infiltrated with different RFP-fusion constructs under white light (A) and under green light (B).

Figure 3

Quantification of RFP-fusion proteins in plant extracts before and after heat treatment by fluorescence detection. The accumulation of RFP-fusion proteins was determined by fluorescence quantification compared to affinity-purified RFP-derived calibration curve. Values are expressed as mean of three biological replicates including standard deviations. Gray columns, crude extract; red columns, extract after heat treatment; lanes 3–16 contain the samples identified in Table 1. FLW, fresh leaf weight.

Figure 4

Visualization of insoluble aggregate formation. Analysis of selected RFP-fusion proteins after purification from plant crude extracts using Ni-NTA magnetic agarose beads, showing the difference between soluble and insoluble proteins. Wt, non-infiltrated leaf extract; RFP, RFP construct; 19_1,RFP-Pf Msp1-19_EGF1 construct; ST, RFP-Pf SPATR_TSR; RFP-ZenH; 10_1, RFP-Pf Msp10_EGF1. Left panel, Transmission image; right panel, fluorescence image.

Figure 5

SDS-PAGE and western blot analysis of RFP-fusion proteins. SDS-PAGE (A) and western blot analysis (B) of heat-treated plant extracts obtained from infiltrated N. benthamiana leaves under reducing conditions. Proteins were detected using rabbit anti-His₆ and alkaline phosphatase-labeled goat anti-rabbit antiserum. M, PageRuler™ pre-stained protein ladder (Fermentas); wt, non-infiltrated leaf extract; lane 1, RFP; lane 2, RFP-ZenH, lanes 3–16 contain the samples identified in Table 1. ^*Putative monomeric RFP/RFP-fusions; ^**putative cross-linked dimeric forms of RFP-fusions. D1-4, putative degradation products.

Figure 6

Construction and expression of the pre-erythrocytic stage candidate (variants P1–P3). (A) Construct architecture. For antigen abbreviations refer to Table 1. (B) SDS-PAGE and western blot analysis of pre-erythrocytic stage candidate variants. Heat-treated crude extracts were separated under reducing conditions and analyzed by staining with Coomassie (left panel) or by western blot using rabbit anti-His₆ and alkaline phosphatase-labeled goat anti-rabbit antiserum. M, PageRuler™ pre-stained protein ladder (Fermentas); wt, non-infiltrated leaf extract.

Figure 7

Construction and expression of the multi-EGF dual-stage vaccine candidate (variants E1–E5). (A) Construct architecture. For antigen abbreviations refer to Table 1. (B) SDS-PAGE and western blot analysis of dual-stage candidate variants. Heat-treated crude extracts were separated under reducing conditions and analyzed by staining with Coomassie (left panel) or by western blot using rabbit anti-His₆ and alkaline phosphatase-labeled goat anti-rabbit antiserum. M, PageRuler™ pre-stained protein ladder (Fermentas); wt, non-infiltrated leaf extract. (C) Dot-blot analysis of heat-treated crude extracts detected under non-reducing conditions using a Pf Msp1-19_EGF1-specific monoclonal antibody to confirm minimal expression of the single EGF1 domain of PfMsp1-19 and the proper folding of this domain. Binding was detected using goat anti-mouse alkaline phosphatase-labeled antiserum.

Figure 8

Initial characterization of E5-specific immune responses. (A) SDS-PAGE analysis of purified E5. M, PageRuler™ pre-stained protein ladder (Fermentas); E5, 15 μg of purified E5 under reducing conditions. (B) Domain-specific titer analysis of E5-specific murine immune sera. Titers were derived by direct-coating ELISA against purified single-domain RFP-fusions (data not shown) and are defined as the dilution that gives more than twice the value of pre-immune serum. Titers for the individual animals are given (M1–M3) as well as the geometric mean (horizontal solid black line). For domain identification refer to Table 1. (C,D) Immunofluorescence assay of P. falciparum NF54 parasites at two different stages. (C) Schizonts (blood stage) and (D) macrogametes (sexual stage) were fixed with methanol on the surface of a slide. Detection with IgGs from mice immunized with E5 is shown as a representative example. Rabbit antisera, Pf Msp1-19 (schizonts) and Pfs25 (magrogametes) were used as positive controls. Rabbit controls were visualized with an anti-rabbit secondary antibody labeled with Alexa Fluor 594 (red) whereas murine immune IgG was visualized with a secondary Alexa Fluor 488 labeled anti-murine antibody (green). (I) murine immune IgG; (II) counterstaining with stage-specific rabbit antiserum; (III) neutral mouse serum; (IV) counterstaining with stage-specific rabbit antiserum.

Figure 9

Construction and expression of the multi-domain, multi-stage vaccine candidate (variants M1–M8). (A) Construct architecture. For antigen abbreviations refer to Table 1. (B) SDS-PAGE and western blot analysis of multi-domain, multi-stage candidate variants. Heat-treated crude extracts were separated under reducing conditions and analyzed by staining with Coomassie (left panel) or by western blot using rabbit anti-His₆ and alkaline phosphatase-labeled goat anti-rabbit antiserum. M, PageRuler™ pre-stained protein ladder (Fermentas); wt, non-infiltrated leaf extract.