title: Production of biologically active scFv and VHH antibody fragments in Bifidobacterium longum.
abstract: Bifidobacteria constitute a significant part of healthy intestinal microbiota in adults and infants and present a promising platform for construction of genetically modified probiotic agents for treatment of gastrointestinal disorders. In this study, three strains of Bifidobacterium longum were constructed that express and secrete biologically active single-chain antibodies against human TNF-α and Clostridium difficile exotoxin A. Anti-TNF-α scFv antibody D2E7 was produced at the level of 25 μg L(-1) in broth culture and was mostly retained in the cytoplasm, while VHH-type antibodies A20.1 and A26.8 against C. difficile exotoxin A were produced at the levels of 0.3-1 mg L(-1) and secreted very efficiently. The biological activity of both antibody types was demonstrated in the mammalian cell-based assays. Expression of A20.1 and A26.8 was also observed in vivo after intragastric administration of transformed B. longum strains to (C57/BL6 × DBA/2)F1 mice. The obtained B. longum strains may serve as prototypes for construction of novel probiotic medications against inflammatory bowel disease and C. difficile-associated disease.
authors.affiliation: Department of Microbiology and Virology, Pirogov Russian National Research Medical University, Ostrovitjanova str. 1, Moscow 117997, Russia Pharmbacter LLC, Skladochnaya ul., 1 - 1, Moscow 127018, Russia a.shkoporov@gmail.com.
authors.forename: A N
authors.initials: AN
authors.lastname: Shkoporov
authors.affiliation: Department of Microbiology and Virology, Pirogov Russian National Research Medical University, Ostrovitjanova str. 1, Moscow 117997, Russia.
authors.forename: E V
authors.initials: EV
authors.lastname: Khokhlova
authors.affiliation: Pharmbacter LLC, Skladochnaya ul., 1 - 1, Moscow 127018, Russia.
authors.forename: K A
authors.initials: KA
authors.lastname: Savochkin
authors.affiliation: Department of Microbiology and Virology, Pirogov Russian National Research Medical University, Ostrovitjanova str. 1, Moscow 117997, Russia.
authors.forename: L I
authors.initials: LI
authors.lastname: Kafarskaia
authors.affiliation: Department of Microbiology and Virology, Pirogov Russian National Research Medical University, Ostrovitjanova str. 1, Moscow 117997, Russia.
authors.forename: B A
authors.initials: BA
authors.lastname: Efimov
mesh_terms: D000068879:Adalimumab / Q000096:biosynthesis*; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis*; D001644:Bifidobacterium / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D004731:Endotoxins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D030781:Organisms, Genetically Modified
chemical_list: D000907:Antibodies, Bacterial; D004731:Endotoxins; D000068879:Adalimumab
keywords: Bifidobacterium; VHH; antibody; scFv; transformation; vector