Production, characterisation and immunogenicity of a plant-made Plasmodium antigen—the 19 kDa C-terminal fragment of Plasmodium yoelii merozoite surface protein 1

Abstract

Development of a safe, effective and affordable malaria vaccine is central to global disease control efforts. One of the most highly regarded proteins for inclusion in an asexual blood stage subunit vaccine is the 19-kDa C-terminal fragment of merozoite surface protein 1 (MSP1(19)). As production of vaccine antigens in plants can potentially overcome cost and delivery hurdles, we set out to produce MSP1(19) in plants, characterise the protein and test its immunogenicity using a mouse model. Plasmodium yoelii MSP1(19) (PyMSP1(19)) was produced in Nicotiana benthamiana using the MagnICON® deconstructed TMV-based viral vector. PyMSP1(19) yield of at least 23% total soluble protein (TSP;3-4 mg/g Fwt) were achieved using a codon-optimised construct that was targeted to the apoplast. Freeze-dried leaf powder contained at least 20 mg PyMSP1(19) per gram dry weight and the protein retained immunogenicity in this form for more than 2 years. Characterisation studies, including SDS-PAGE, mass spectrometry and circular dichroism, indicated that the plant-expressed PyMSP1(19) was similar to its Escherichia coli- and Saccharomyces cerevisiae-expressed counterparts. Purified plant-made PyMSP1(19) induced strong immune responses following intraperitoneal immunisation, although titres were lower than those induced by an equivalent dose of purified E. coli-expressed PyMSP1(19). The reason for this is uncertain but may be due to differences in the oligomerisation profile of the vaccines. The plant-made PyMSP1(19) vaccine was also found to be orally immunogenic when delivered alone or following immunisation with a PyMSP1(19) DNA vaccine. This study adds to an increasing body of research supporting the feasibility of plants as both a factory for the production of malaria antigens, and as a safe and affordable platform for oral delivery of a temperature-stable malaria vaccine.

Overview of Plant-Made Vaccine Antigens against Malaria

“…Similar results were obtained by Ma et al [65]: although the plant-made PyMSP1 19 was immunogenic when delivered i.p. with Freund's adjuvant or orally without any adjuvant, the specific antibody titers were lower than those induced with the same dose as that of E. coli -made antigens [65].…”

“…Apical membrane antigen-1 (AMA1), merozoite surface protein-1 (MSP1), and erythrocyte surface antigens are under clinical trials [11, 60–64]. Some of them have been expressed in plants, and their immunogenicity assayed (Table 1) [25, 33, 35, 36, 39, 65]. …”

“…The use of magnICON has also been found to increase the expression level of the plant codon-optimized P. yoelii MSP1 19 (PyMSP1 19 ) to at least 23% TSP (~3-4 mg/g of fresh weight) [65]. All together, these results show that not only codon optimization but also the viral provector system improves the expression level of plant-made malaria antigens [33, 65].…”

“…The use of magnICON has also been found to increase the expression level of the plant codon-optimized P. yoelii MSP1 19 (PyMSP1 19 ) to at least 23% TSP (~3-4 mg/g of fresh weight) [65]. All together, these results show that not only codon optimization but also the viral provector system improves the expression level of plant-made malaria antigens [33, 65]. Indeed, plant codon optimization combined with the magnICON expression system has been shown to allow an increase of up to 100 fold with respect to the transgenic tobacco system [25, 33].…”

“…or oral vaccination in a murine model [25, 33, 36, 39, 65]. These findings provide a rationale for the development of a plant-made oral vaccine against malaria.…”