title: Assessment of Pfs25 expressed from multiple soluble expression platforms for use as transmission-blocking vaccine candidates.
abstract: 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. 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.
  
  
  
  While 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.
  
  
  
  Through numerous attempts, soluble, monomeric Pfs25 derived from Escherichia coli was not achieved, while Pichia pastoris 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.
  
  
  
  In 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.
authors: PATH Malaria Vaccine Initiative (MVI), 455 Massachusetts Avenue NW, Suite 1000, Washington, DC, 20001-2621, USA. smlee@path.org.
authors: Shwu-Maan
authors: 0000-0003-0819-0569
authors: SM
authors: Lee
authors: PATH Malaria Vaccine Initiative (MVI), 455 Massachusetts Avenue NW, Suite 1000, Washington, DC, 20001-2621, USA.
authors: Chia-Kuei
authors: CK
authors: Wu
authors: PATH Malaria Vaccine Initiative (MVI), 455 Massachusetts Avenue NW, Suite 1000, Washington, DC, 20001-2621, USA.
authors: Jordan
authors: J
authors: Plieskatt
authors: PATH, 2201 Westlake Avenue, Suite 200, Seattle, WA, 98121, USA.
authors: David H
authors: DH
authors: McAdams
authors: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 20852, USA.
authors: Kazutoyo
authors: K
authors: Miura
authors: PATH Malaria Vaccine Initiative (MVI), 455 Massachusetts Avenue NW, Suite 1000, Washington, DC, 20001-2621, USA.
authors: Chris
authors: C
authors: Ockenhouse
authors: PATH Malaria Vaccine Initiative (MVI), 455 Massachusetts Avenue NW, Suite 1000, Washington, DC, 20001-2621, USA.
authors: C Richter
authors: CR
authors: King
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D018562:Disease Transmission, Infectious / Q000517:prevention & control*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008288:Malaria / Q000517:prevention & control*; D017780:Malaria Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000913:Antibodies, Protozoan; D017780:Malaria Vaccines; C071284:Pfs25 protein, Plasmodium falciparum; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic
keywords: Baculovirus; Glycosylation; Malaria; Pfs25; Pichia; Plasmodium falciparum; Recombinant protein; Transmission blocking vaccine