Abstract
BackgroundTransmission-blocking vaccines (TBVs) have become a focus of strategies to control and eventually eliminate malaria as they target the entry of sexual stage into the Anopheles stephensi mosquito thereby preventing transmission, an essential component of the parasite life cycle. Such vaccines are envisioned as complements to vaccines that target human infection, such as RTS,S as well as drug treatment, and vector control strategies. A number of conserved proteins, including Pfs25, have been identified as promising TBV targets in research or early stage development. Pfs25 is a 25 kDa protein of Plasmodium falciparum expressed on the surface of zygotes and ookinetes. Its complex tertiary structure, including numerous cysteines, has led to difficulties in the expression of a recombinant protein that is homogeneous, with proper conformation, and free of glycosylation, a phenomenon not found in native parasite machinery.MethodsWhile the expression and purification of Pfs25 in various systems, has been previously independently reported, here a parallel analysis of Pfs25 is presented to inform on the biochemical features of Pfs25 and their impact on functionality. Three scalable expression systems were used to express, purify, and evaluate Pfs25 both in vitro and in vivo, including the ability of each protein to produce functional antibodies through the standard membrane feeding assay.ResultsThrough numerous attempts, soluble, monomeric Pfs25 derived from Escherichia coli was not achieved, while Pichiapastoris presented Pfs25 as an inhomogeneous product with glycosylation. In comparison, baculovirus produced a pure, monomeric protein free of glycosylation. The glycosylation present for Pichia produced Pfs25, showed no notable decrease in the ability to elicit transmission reducing antibodies in functional evaluation, while a reduced and alkylated Pfs25 (derived from plant and used as a control) was found to have significantly decreased transmission reducing activity, emphasizing the importance of ensuring correct disulfide stabilized conformation during vaccine design and production.ConclusionsIn this study, the biochemical features of Pfs25, produced from different expression systems, are described along with their impact on the ability of the protein to elicit functional antibodies. Pfs25 expressed using baculovirus and Pichia showed promise as candidates for vaccine development.
Highlights
Transmission-blocking vaccines (TBVs) have become a focus of strategies to control and eventually eliminate malaria as they target the entry of sexual stage into the Anopheles stephensi mosquito thereby preventing transmission, an essential component of the parasite life cycle
Pfs25 expressed as soluble protein in Pichia and baculovirus Recombinant His-tagged Pfs25 was produced as an intracellular protein in E. coli, and as secreted proteins in Pichia and baculovirus at small scale and purified by Ni–NTA chromatography and gel filtration for subsequent evaluation
The purified Pfs25 protein presented as a single band of ~20 kDa and greater than 90 % purity (Fig. 1c), with identity confirmed with the 4B7 mAb (Fig. 1d)
Summary
Transmission-blocking vaccines (TBVs) have become a focus of strategies to control and eventually eliminate malaria as they target the entry of sexual stage into the Anopheles stephensi mosquito thereby preventing transmission, an essential component of the parasite life cycle. Valuable would be a vaccine that blocks parasite function at multiple stages of the life cycle including transmission from humans to mosquitoes [3] Such transmission-blocking vaccines (TBVs) would not block disease in the vaccine recipients directly but rather would reduce the prevalence of malaria in a population thereby complementing current vector control strategies and increasing the efficacy of the RTS,S vaccine which blocks infection from mosquito to human [4]. To advance such TBVs, the identification of appropriate target antigens, their expression, characterization, and preparation for experimental clinical testing is underway
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