title: Expression, purification, and evaluation of recombinant LecA as a candidate for an amebic colitis vaccine.
abstract: Entamoeba histolytica, which causes amebic colitis and liver abscess, is considered a major enteric pathogen in residents and travelers to developing countries where the disease is endemic. Interaction of this protozoan parasite with the intestine is mediated through the binding of the trophozoite stage to intestinal mucin and epithelium via a galactose and N-acetyl-d-galactosamine (Gal/GalNAc) lectin comprised of a disulfide linked heavy (ca. 180 kDa) and light chain (ca. 35 kDa) and a noncovalently bound intermediate subunit (ca. 150 kDa). Our efforts to develop a vaccine against this pathogen have focused on an internal 578 amino acid fragment, designated LecA, located within the cysteine-rich region of the heavy chain subunit because: (i) it is a major target of adherence-blocking antibodies of seropositive individuals and (ii) vaccination with his-tagged LecA provides protection in animal models. We developed a purification process for preparing highly purified non-tagged LecA using a codon-optimized gene expressed in Escherichia coli. The process consisted of: (i) cell lysis, collection and washing of inclusion bodies; (ii) solubilization and refolding of denatured LecA; and (iii) a polishing gel filtration step. The purified fragment existed primarily as a random coil with β-sheet structure, contained low endotoxin and nucleic acid, was highly immunoreactive, and elicited antibodies that recognized native lectin and that inhibited in vitro adherence of trophozoites to CHO cells. Immunization of CBA mice with LecA resulted in significant protection against cecal colitis. Our procedure yields sufficient amounts of highly purified LecA for future studies on stability, immunogenicity, and protection with protein-adjuvant formulations.
authors.affiliation: TECHLAB, Inc., 2001 Kraft Drive, Blacksburg, VA 24060-6158, USA.
authors.forename: L
authors.initials: L
authors.lastname: Barroso
authors.affiliation: Division of Infectious Diseases and International Health University of Virginia Health System, Carter Harrison Building, Room 1709A, 345 Crispell Drive, P.O. Box 801340, Charlottesville, VA 22908-1340, USA.
authors.forename: M
authors.initials: M
authors.lastname: Abhyankar
authors.affiliation: Division of Infectious Diseases and International Health University of Virginia Health System, Carter Harrison Building, Room 1709A, 345 Crispell Drive, P.O. Box 801340, Charlottesville, VA 22908-1340, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Noor
authors.affiliation: TECHLAB, Inc., 2001 Kraft Drive, Blacksburg, VA 24060-6158, USA.
authors.forename: K
authors.initials: K
authors.lastname: Read
authors.affiliation: TECHLAB, Inc., 2001 Kraft Drive, Blacksburg, VA 24060-6158, USA.
authors.forename: K
authors.initials: K
authors.lastname: Pedersen
authors.affiliation: TECHLAB, Inc., 2001 Kraft Drive, Blacksburg, VA 24060-6158, USA.
authors.forename: R
authors.initials: R
authors.lastname: White
authors.affiliation: Infectious Disease Research Institute, Seattle, WA, USA.
authors.forename: C
authors.initials: C
authors.lastname: Fox
authors.affiliation: Division of Infectious Diseases and International Health University of Virginia Health System, Carter Harrison Building, Room 1709A, 345 Crispell Drive, P.O. Box 801340, Charlottesville, VA 22908-1340, USA.
authors.forename: W A
authors.initials: WA
authors.lastname: Petri
authors.affiliation: TECHLAB, Inc., 2001 Kraft Drive, Blacksburg, VA 24060-6158, USA. Electronic address: dlyerly@techlab.com.
authors.forename: D
authors.initials: D
authors.lastname: Lyerly
mesh_terms: D000818:Animals; D004404:Dysentery, Amebic / Q000517:prevention & control*; D004748:Entamoeba histolytica; D037102:Lectins / Q000276:immunology / Q000302:isolation & purification*; D008297:Male; D051379:Mice; D008808:Mice, Inbred CBA; D015800:Protozoan Proteins / Q000276:immunology / Q000302:isolation & purification*; D016052:Protozoan Vaccines / Q000276:immunology*
chemical_list: D037102:Lectins; D015800:Protozoan Proteins; D016052:Protozoan Vaccines
keywords: Amebiasis; Diarrhea; Entamoeba histolytica; LecA vaccine; Lectin