title: Preparation and preclinical evaluation of a freeze-dried formulation of a novel combined multivalent whole-cell/B-subunit oral vaccine against enterotoxigenic Escherichia coli diarrhea.
abstract: A promising liquid killed multivalent whole-cell plus enterotoxin B-subunit oral vaccine against enterotoxigenic Escherichia coli (ETEC), the primary cause of diarrhea among children in low-income countries and travelers to these areas, has recently been developed and tested in preclinical and phase-I and phase-II clinical studies. The vaccine contains killed E. coli bacteria over-expressing the main ETEC colonization factors (CFs) CFA/I, CS3, C5 and C6, and a recombinant enterotoxin B subunit protein (LCTBA) given together with a recently developed enterotoxin-derived adjuvant, dmLT. A dry-powder vaccine formulation should be advantageous especially for use in low-income countries. Here we describe a method to produce a dry-powder formulation by freeze-drying of the vaccine using inulin as stabilizer. Although not completely preventing aggregation of bacteria during freeze-drying, the stabilizer provided both improved overall bacterial morphology and almost complete recovery of the CF and B subunit antigens. Most importantly, oral-intragastric immunization of mice with the freeze-dried vaccine together with dmLT adjuvant elicited strong intestinal mucosal and serum antibody responses against all vaccine antigens, which were comparable to those achieved with the liquid vaccine. Our results indicate the feasibility to use freeze-drying with inulin as stabilizer for preparing a dry-powder formulation of the novel ETEC vaccine with retained oral-mucosal immunogenicity compared to the liquid formulation.
authors.affiliation: University of Gothenburg Vaccine Research Institute (GUVAX), Department of Microbiology and Immunology, The Sahlgrenska Academy of University of Gothenburg, SE-40530 Gothenburg, Sweden. Electronic address: annika.borde@astrazeneca.com.
authors.forename: Annika
authors.initials: A
authors.lastname: Borde
authors.affiliation: University of Gothenburg Vaccine Research Institute (GUVAX), Department of Microbiology and Immunology, The Sahlgrenska Academy of University of Gothenburg, SE-40530 Gothenburg, Sweden. Electronic address: annelie.ekman@microbio.gu.se.
authors.forename: Annelie
authors.initials: A
authors.lastname: Ekman
authors.affiliation: Pharmaceutical Technology, Chemical and Biological Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden. Electronic address: anette.larsson@chalmers.se.
authors.forename: Anette
authors.initials: A
authors.lastname: Larsson
authors.affiliation: Scandinavian Biopharma, Gunnar Asplunds allé 16, 171 63 Solna, Sweden. Electronic address: nils.carlin@etvax.se.
authors.forename: Nils
authors.initials: N
authors.lastname: Carlin
authors.affiliation: University of Gothenburg Vaccine Research Institute (GUVAX), Department of Microbiology and Immunology, The Sahlgrenska Academy of University of Gothenburg, SE-40530 Gothenburg, Sweden. Electronic address: jan.holmgren@microbio.gu.se.
authors.forename: Jan
authors.initials: J
authors.lastname: Holmgren
authors.affiliation: University of Gothenburg Vaccine Research Institute (GUVAX), Department of Microbiology and Immunology, The Sahlgrenska Academy of University of Gothenburg, SE-40530 Gothenburg, Sweden. Electronic address: joshua.tobias@microbio.gu.se.
authors.forename: Joshua
authors.initials: J
authors.lastname: Tobias
mesh_terms: D000284:Administration, Oral; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000917:Antibody Formation; D000941:Antigens / Q000737:chemistry; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000737:chemistry*; D003967:Diarrhea / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*; D004353:Drug Evaluation, Preclinical; D054307:Enterotoxigenic Escherichia coli / Q000276:immunology; D004768:Enterotoxins / Q000276:immunology; D004927:Escherichia coli Infections / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*; D029968:Escherichia coli Proteins / Q000276:immunology; D005260:Female; D036781:Fimbriae Proteins / Q000276:immunology; D005612:Freeze Drying; D007114:Immunization / Q000379:methods; D007444:Inulin / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D011208:Powders; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000906:Antibodies; D000941:Antigens; D001428:Bacterial Vaccines; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011208:Powders; D011994:Recombinant Proteins; D036781:Fimbriae Proteins; D007444:Inulin
keywords: ETEC; Freeze-dried; Immunogenicity; Inulin; Stabilizer; Vaccine