FEMS Microbiology Letters Advance Access published May 19, 2015
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Production of biologically active scFv and VHH antibody fragments in Bifidobacterium
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longum
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Shkoporov A.N.1,2*, Khokhlova E.V.1, Savochkin K.A.2, Kafarskaya L.I. 1, and Efimov B.A. 1
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1
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Department of Microbiology and Virology, Pirogov Russian National Research Medical
University, Moscow 117997, Ostrovitjanova str. 1. 2
Pharmbacter LLC, Moscow 127018, Skladochnaya ul., 1 – 1.
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*Corresponding author. Phone/Fax: +7 495 4341677, e-mail: [email protected]
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Abstract
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Bifidobacteria constitute a significant part of healthy intestinal microbiota in adults and
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infants and present a promising platform for construction of genetically modified probiotic agents
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for treatment of gastrointestinal disorders. In this study three strains of Bifidobacterium longum
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were constructed that express and secrete biologically active single-chain antibodies against human
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TNF-α and Clostridium difficile exotoxin A. Anti-TNF-α scFv antibody D2E7 was produced at the
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level of 25 μg/L in broth culture and was mostly retained in the cytoplasm, while VHH-type
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antibodies A20.1 and A26.8 against C. difficile exotoxin A were produced at the levels of 0.3 – 1
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mg/L and secreted very efficiently. The biological activity of both antibody types was demonstrated
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in the mammalian cell-based assays. Expression of A20.1 and A26.8 was also observed in vivo after
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intragastric administration of transformed B. longum strains to (C57/BL6 x DBA/2)F1 mice. The
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obtained B. longum strains may serve as prototypes for construction of novel probiotic medications
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against inflammatory bowel disease (IBD) and C. difficile associated disease (CDAD).
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Keywords: Bifidobacterium, transformation, vector, antibody, scFv, VHH.
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Running Title: Production of antibody fragments in Bifidobacterium longum
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1
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Introduction
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The presumed ability of certain microorganisms ('probiotics') to exert health promoting effects
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on human gastrointestinal tract (GIT) after oral administration underlies the rapidly growing
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probiotic products industry (de Vrese and Schrezenmeir, 2008; Figueroa-González et al., 2011;
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Kechagia et al., 2013; Kumar et al., 2014). Although the efficacy of probiotics is still debated and a
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plausible evidence for significant clinical effects is still missing (Guandalini, 2014; Ghouri et al.,
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2014; Dimidi et al., 2014), the safety of many of probiotic preparations has been confirmed by a
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long history of their production and consumption (AlFaleh and Anabrees, 2013; Urben et al., 2014).
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The latter fact leads to the idea that the food grade category microorganisms used in probiotic
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preparations (e.g. lactic acid bacteria like Lactococcus sp. and Lactobacillus sp., or Bifidobacterium
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sp., and some others) could be used to construct a new generation of the so called 'designer
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probiotics' (Paton et al., 2006; Paton et al., 2010) which are genetically engineered to express
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certain therapeutic molecule (e.g. a protein with proven efficacy) in situ in human GIT upon oral
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ingestion. The in situ delivery of therapeutic proteins using probiotic bacteria for treatment of GIT
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disorders may reduce the severity of adverse side effects seen after systemic delivery, may increase
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local effective concentration, ease drug usage, and reduce treatment costs due to simple
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manufacturing procedures of the probiotic drugs. Currently one line of 'designer probiotics' based
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on Lactococcus lactis has entered clinical trials (Braat et al., 2006; Villatoro-Hernandez et al., 2011;
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Limaye et al., 2013).
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Another promising candidate for construction of 'designer probiotics' is genus
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Bifidobacterium which comprises gram-positive, strictly anaerobic, non-sporing irregular rods.
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Representatives of this genus (B. longum, B. breve, B. bifidum, B. pseudocatenulatum and some
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others) constitute an obligatory part of distal GIT microbiota in healthy adults and dominate this
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microbial community during early infancy (Yatsunenko et al., 2012; Koenig et al., 2011; Turroni et
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al., 2010; Turroni et al., 2012). The indigenous intestinal bifidobacteria are believed to contribute to
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maintenance of intestinal homeostasis and host well-being by digestion and bioconversion of
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dietary compounds, production of vitamins, competition with pathogens, inhibition of
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carcinogenesis and modulation of local and systemic immune responses (Deguchi et al., 1985; Kim
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et al., 2007; Pompei et al., 2007; de Vrese and Schrezenmeir, 2008; Fukuda et al., 2011). Until
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recently functional genomics and genetic engineering studies of genus Bifidobacterium have been
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hampered because of their fastidious nature and high resistance to genetic transformation
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(Guglielmetti et al., 2013; Dominguez and O’Sullivan, 2013; Brancaccio et al., 2013).
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We believe that genetically modified lactococci, although proven to be effective in murine GIT diseases models (Steidler et al., 2000; Vandenbroucke et al., 2010), possess several 2
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disadvantages compared to bifidobacteria, including inability to efficiently colonize human GIT
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(Bolotin et al., 2001) and involvement into some cases of opportunistic infections (Aguirre and
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Collins, 1993). A number of reports which describe the construction of bifidobacterial strains
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expressing therapeutic proteins have been already published (Cronin et al., 2009; Ma et al., 2012;
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Yin et al., 2012; Yu et al., 2012; Losurdo et al., 2013).
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In our opinion one of the most interesting type of therapeutic molecules to be delivered by the
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future 'designer probiotics' are single-chain antibody fragments which include scFv (Ahmad et al.,
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2012) and VHH (Harmsen and De Haard, 2007). These molecules can be expressed efficiently in
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bacteria, including E. coli and L. lactis (Guglielmi and Martineaum, 2009; Vandenbroucke et al.,
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2010), however, to the best of our knowledge, no reports have been published to date describing the
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successful expression of either scFv or VHH in Bifidobacterium. The antibody-producing
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bifidobacteria could be designed to express various types of antibodies that opsonize food-borne
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pathogens, neutralize viruses, toxins, and virulence factors, and down-regulate pro-inflammatory
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cytokines.
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In this study we sought to determine whether scFv-type and VHH-type antibody molecules
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can be expressed and secreted in biologically active form in Bifidobacterium longum. As a model
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we used a well described TNFα neutralizing low-KD human scFv antibody D2E7 (Rajpal et al.,
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2005) and a pair of neutralizing VHH-type antibodies (A20.1 and A26.8) against Clostridium
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difficile exotoxin A (Hussack et al., 2012).
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Materials and Methods
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Bacterial and Animal Cell Cultures
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Bifidobacterium longum strains 44B (Shkoporov et al., 2013) and NCC2705 (Nestlé Culture
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Collection, Nestlé Research Centre, Switzerland), and Bifidobacterium breve UCC2003 (personal
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gift of Prof. D. Van Sinderen, University College Cork, Ireland) were propagated anaerobically in
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RCM (Lab M, UK) broth and agar media supplemented with erythromycin (2.5 μg/ml) when
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needed. Clostridium difficile ATCC 43255 was propagated anaerobically on Columbia broth and
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agar (BD). Strains of Escherichia coli XL-1Blue and Rosetta DE3 [pLysS] were propagated in LB
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medium supplemented with 100 μg/ml ampicillin or 50 μg/ml kanamycin. All incubations were
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done at 37°C, and broth cultures of E. coli were shaken at 200 rpm.
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For preparation of conditioned media (CM) overnight broth cultures of B. longum strains were
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centrifuged at 3,000 g for 10 min, washed with DMEM (Biolot, Russia) and resuspended in DMEM
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with 20 mM HEPES pH 7.0. Cultures were incubated anaerobically for 20 h at 37°C. Cells were
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removed by centrifugation and pH of supernatant was adjusted to pH 7.4 with 1M NaOH followed 3
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by 0.22 μm filter sterilization. Protein fraction from СM was buffer exchanged into fresh DMEM
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and simultaneously concentrated 20-fold by ultrafiltration using Pierce protein concentrators, 9K
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MWCO (Thermo Fisher Scientific, Rockford, IL). Soluble cytoplasmic fractions (SCF) were
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prepared from PBS-washed cells pelleted from 5 ml of broth by vigorous vortexing with 200 mg of
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acid-washed glass beads (0.1 mm diameter) and 0.25 ml PBS with subsequent centrifugation at 12
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000 g for 15 min and collection of supernatant.
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Mammalian cell lines HT-29 and CHO-K1 were cultured in DMEM supplemented with 10%
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fetal calf serum (HyClone, USA), penicillin G, 100 U/ml, and streptomycin, 100 μg/ml. Cultures
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were incubated at 37°C in atmosphere of 5% CO2. For TcdA toxin and TNFα neutralization
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experiments cells were seeded into 24-well (HT-29) or 96-well (CHO-K1) plates, grown to 70%
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confluency, washed once with Hank's balanced salt solution overlaid with a 9:1 mixture of fresh
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complete DMEM medium and bacterial CM. After 1 h pre-incubation with CM, cells were treated
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with human recombinant TNFα, 15 ng/ml final concentration, or TcdA toxin (prepared as described
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below), 5.5 – 1400 ng/ml. The HT-29 cells were lysed and collected for qRT-PCR after 4 h
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incubation. Culture supernatants for ELISA assay were collected 18 hours after treatment. Cell
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morphology of CHO-K1 was assessed after 24 h incubation.
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Construction of Recombinant Strains
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Genes coding for D2E7 scFv and A20.1 and A26.8 VHH-type antibody fragment were
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synthesized at Evrogen JSC (Russia) with codon usage optimized for the expression in B. longum.
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Synthetic fragments of 795 bp (D2E7), 416 bp (A20.1), and 419 bp (A26.8) were cloned into NdeI
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and HindIII restriction sites of the pESH100 E. coli – Bifidobacterium shuttle expression/secretion
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vector (Khokhlova et al., 2010) according to standard procedures. The E. coli strain XL-1Blue was
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used as а cloning host. The same fragments were cloned into pET-22b and pET-28b (Merck,
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Germany) for the production of control proteins for western blots. Plasmid pESH100/D2E7 was
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introduced into B. longum NCC2705 by electroporation as described previously (Shkoporov et al.,
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2008). Plasmids pESH100/VHH-A20.1 and pESH100/VHH-A26.8 were introduced into the strains
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B. longum 44B and B. breve UCC2003 in a similar way. Plasmids pET22b/D2E7, pET28b/VHH-
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A20.1, and pET28b/VHH-A26.8 were transferred to E. coli Rosetta DE3 [pLysS] for expression.
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Animals
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Male (C57/BL6 x DBA/2)F1 hybrid mice, 10-12 weeks of age were divided into four groups
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(n = 5). Fresh overnight PBS-washed cultures (2 x 10 9 cfu in 0.2 ml of PBS) of transformed B.
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longum (NCC2705 [pESH100/D2E7], 44B [pESH100/VHH-A20.1], and 44B [pESH100/VHH4
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A26.8]), as well as the untransformed control strain B. longum 44B were administered
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intragastrically once a day for three consecutive days. On day 5 after the first injection mice were
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sacrificed and medial portions of small intestine, large intestine, and cecum, containing fecal masses
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were excised. Feces were weighed and resuspended 1:1 in PBS. After 5 min centrifugation at
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10,000 g supernatants were collected and subjected to ELISA.
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Protein Expression and Purification
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For purification of TcdA toxin 2 L 48 h broth culture of Clostridium difficile ATCC 43255 was
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centrifuged at 3,000 g for 60 min, supernatant was collected and proteins were precipitated by
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addition of ammonium sulphate to final concentration of 2 M. After 48 h incubation at 4°C
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precipitate was collected by centrifugation at 4,000 g for 120 min. Pellets were dissolved in 30 ml
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of PBS and centrifuged again at 4,000 g for 60 min twice to remove insoluble material. Supernatant
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was filtered through 0.22 μm membrane and applied onto the 2.5x20 cm Sephadex G-75 column
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equilibrated with 25 mM Tris-HCl pH 8.0 at 1 ml/min speed. Void volume fractions were collected,
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pooled, and applied onto 5 ml Bio-Scale Mini UNOsphere Q cartridge (Bio-Rad, CA) at 5 ml/min.
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Bound TcdA was eluted using 50 mM NaCl and concentrated on Pierce concentrator, 9K MWCO
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(Fig. 1).
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The E. coli Rosetta DE3 [pLysS] derivative strains containing recombinant plasmids were
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grown in 50 ml of LB broth at 37°C and 250 rpm. After the OD600 of the cultures reached 0.6–0.8,
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the expression was induced by addition of 1 mM IPTG. The cells were cultured post-induction at
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26–28 °C for 3–5 h and His-Spin Protein Miniprep kit (Zymo Research, CA) was used for protein
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purification under denaturing conditions (Fig. 1).
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Quantitative RT-PCR
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Total RNA was isolated from HT-29 cells by using ZR RNA MiniPrep kit (Zymo Research,
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USA). First strand cDNA was synthesized using RevertAid Premium reverse transcriptase
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(Fermentas, Lithuania) and oligo-dT primer. Real-time PCR was set up in CFX-96 thermal cycler
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(Bio-Rad) using a pre-made mastermix containing EvaGreen (Syntol) and an appropriate primer
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pair at concentration of 200 nM (Table 1). Reactions were performed using the following program:
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95°C 5 min, followed by 40 cycles of 94°C 20 s, 60°C 20 s, 72°C 20 s. Quantification of target
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transcripts was done by the ΔΔC(t) method using GAPDH as a normalizing house-keeping gene.
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Western Blotting, Dot Blotting, and ELISA
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CM and SCF samples for Western blotting were mixed 1:1 with gel-loading dye and heated at 5
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100°C for 3 min. Samples were electrophoresed in 12% SDS-PAGE gel followed by a transfer onto
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Hybond P membrane (GE-Amersham, NJ, USA) using a semi-dry transfer apparatus at 2 mA/cm2
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for 30 min. Membranes were blocked in 0.2% Tween-20/PBS (PBS-T) and stained using primary
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(monoclonal anti-c-Myc cat. no. 626802, BioLegend, CA) and secondary (HRP goat anti-mouse
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IgG cat. no. 405306, BioLegend, CA) antibodies for 60 min each at room temperature with
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concomitant PBS-T washes (3 x 5 min). Blots were developed using ECL Select reagent (GE-
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Amersham) according to the manufacturer’s instructions. Chemiluminescent detection was
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performed using ChemiDoc XRS+ System (Bio-Rad).
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In vitro antigen binding by A20.1 and A26.8 VHH antibodies was studied using the following
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procedure: antigens (Tcd and BSA) were immobilized on Hybond P membrane and stained using
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the 1:10 dilutions of CM samples from transformed bifidobacteria, membranes were washed with
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PBS-T (3 x 5 min) and then processed as described above for western blots.
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Concentrations of IL-8 in cell culture supernatants were assayed using human IL-8 ELISA kit (Cytokine, Russia) according to the manufacturer’s instructions. In vivo expression of A20.1 and A26.8 antibodies in mice after intragastric administration of
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transformed B. longum strains was assayed using semiquantitative ELISA. The TcdA toxin was
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diluted in 50 mM Na2CO3 (pH 9.7) to final concentration of 30 μg/ml and immobilized on 96-well
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high-binding ELISA plates (Greiner, Germany) by overnight incubation. Wells were washed with
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PBS, blocked with 3% BSA, and filled with 0.1 ml murine fecal supernatants diluted 1:10 in PBS-T.
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After 2 hour incubation wells were washed six times with PBS-T and stained essentially as
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described above for Western blotting. Reactions were developed using TMB and stop solutions
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(Cytokine) and OD450 was recorded using iMark microplate reader (Bio-Rad).
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Results and Discussion
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To clone the genes coding for scFv- and VHH-type single chain antibodies in bifidobacteria
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we used previously described E. coli – Bifidobacterium shuttle expression/secretion vector
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(Khokhlova et al., 2010). The synthetic D2E7 scFv, and A20.1 and A26.8 VHH antibody genes
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were optimized to match the codon usage of B. longum and contained the c-Myc tag-coding
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sequence at the C-termini. When cloned into pESH100 the genes were fused in frame to the
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bifidobacterial AmyB signal peptide (SP) and placed under the control of a relatively strong
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constitutive promoter Pgap from B. longum. The plasmid designated as pESH100/D2E7 was
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introduced into B. longum NCC2705, while the plasmids pESH100/VHH-A20.1 and
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pESH100/VHH-A26.8 were used to transform both, B. breve UCC2003 and B. longum 44B. 6
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The expression of anti-TNFα scFv antibody D2E7 in B. longum NCC2705 was monitored
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using western blot with anti-C-myc tag antibodies. The purified D2E7 (30.7 kDa) produced in E.
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coli was used as a positive control. According to Fig. 2a both the cytoplasmic fraction and the spent
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culture media from pESH100/D2E7-transformed strain contained the D2E7 antibody. The observed
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MW of D2E7 secreted into the media was very close to the calculated MW (28.5 kDa) of the mature
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protein after cleavage of the AmyB SP. However, most of the produced D2E7 antibody was retained
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in the cytoplasm of NCC2705 cells and had the MW (33.5 kDa) of the non-processed pre-protein
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containing the SP. The semi-quantitative western blot analysis indicated that the overall amount of
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D2E7 produced in B. longum NCC2705 culture was quite low and reached approximately 25 μg/L
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(Fig. 2b).
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The indirect ELISA analysis confirmed the ability of D2E7 antibody produced in B. longum
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NCC2705 to bind the surface-immobilized recombinant human TNFα (data not shown). The
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biological activity of the D2E7 scFv antibodies produced in B. longum was determined in an in vitro
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TNFα activity assay on human colon adenocarcinoma HT-29 cell line (Khokhlova et al., 2012).
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Cells were preconditioned with the B. longum NCC2705 [pESH100/D2E7] supernatant, culture
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supernatant from untransformed strain, or control medium, and challenged with human TNFα. The
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inflammatory response in HT-29 cells was assessed by the level of IL-8, IκBα, and CCL20
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transcripts (Fig. 3a). Four hours after TNFα challenge IL-8 and CCL20 expression was induced
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more than 18-fold and 14-fold respectively, while the IκBα expression was up-regulated less
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strongly. Co-incubation of cells with B. longum NCC2705 [pESH100/D2E7] supernatant, but not
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with control strain supernatant, before TNFα stimulation reduced the IL-8 and CCL20 expression
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by 6-fold (P < 0.01) and 2.5-fold (P < 0.05) respectively. Additionally, the IL-8 levels in culture
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media were assayed using ELISA 18 hours after TNFα treatment. It was found that B. longum
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NCC2705 [pESH100/D2E7] supernatant but not control supernatant dramatically reduced the
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amount of secreted IL-8 (Fig. 3b).
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The obtained data suggest that the D2E7 anti-TNFα scFv antibody was relatively efficiently
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expressed in transformed B. longum NCC2705 cells. Although most of the produced antibodies
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were retained in the cytoplasm, the secreted fraction was properly folded and could neutralize the
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TNFα activity in vitro.
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The expression of single-domain VHH-type antibodies A20.1 and A26.8 against C. difficile
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toxin TcdA was investigated upon introduction of the synthetic A20.1 and A26.8 genes into B. breve
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UCC2003 and B. longum 44B. Proteins were detected using western blot with anti-C-myc tag
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antibody and the A20.1 protein (17 kDa) purified from E. coli as a positive control. The A20.1 and
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A26.8 antibodies were expressed and secreted into the culture medium in both B. breve UCC2003 7
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and B. longum 44B (Fig. 4a). However, the expression levels of both antibodies in B. longum 44B
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was at least 10-fold higher than in B. breve UCC2003. For this reason further work was done with
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the strain B. longum 44B only. The apparent MW of the A26.8 secreted into the supernatant was
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close enough to the predicted mass of the protein after SP cleavage (15.3 kDa). However the
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apparent weight of the A20.1 protein (~12 kDa) was significantly smaller than calculated from its
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amino acid sequence (15 kDa) which could be a result of either improper signal peptide cleavage,
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protein degradation, or anomalous electrophoretic mobility. The antibodies A20.1 and A26.8
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produced by B. longum 44B were completely secreted into the milieu which was confirmed by the
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absence of either mature forms or pre-proteins (20 kDa) from the cytoplasmic fractions of the
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transformed strains (Fig. 4b).
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In contrast to the D2E7 scFv antibody, the antibodies A20.1 and A26.8 showed relatively high
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expression levels. According to the semi-quantitative dot-blot (data not shown) the A20.1 level in B.
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longum 44B supernatant was 0.3-0.4 mg/L while the A26.8 was expressed at an even higher rate of
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0.6-1.0 mg/L. The achieved expression level of VHH antibodies in B. longum was comparable to
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that reported by Vandenbroucke et al. (2010) for MT1 anti-TNFα VHH-antibody expressed in L.
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lactis (1.0 – 1.3 mg/L). Moreover, to the best of our knowledge such high expression level of
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heterologous proteins was never demonstrated in bifidobacteria before. Despite the constitutive
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over-expression of the A20.1 and A26.8 antibodies in B. longum 44B, neither the growth
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characteristics nor the other phenotypic traits of the host strain were changed.
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The ability of A20.1 and A26.8 antibodies produced by the transformed B. longum 44B strains
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to bind the TcdA toxin was tested in a dot-blot assay with immobilized TcdA. According to Fig. 5a
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both antibodies concentrated from culture supernatants of B. longum 44B derivatives were able to
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bind to TcdA, but not to control protein BSA, in a dose-dependent manner. The biological activity
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of the B. longum -produced VHH antibodies was investigated using the standard TcdA in vitro
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toxicity assay (Rothman, 1986; Keel and Songer, 2007). The CHO-K1 cells were challenged with a
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purified TcdA (5.5 – 1400 ng/ml) in the presence or absence of A20.1- or A26.8-containing
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bifidobacterial supernatants and after counting of rounded cells percentage the dose-effect curves
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were plotted. The data presented in Fig 5b indicate that the antibody-containing supernatants from
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both, B. longum 44B [pESH100/VHH-A20.1] and B. longum 44B [pESH100/VHH-A26.8], were
262
able to neutralize the toxic effect of TcdA in concentrations of up to 350 ng/ml.
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The above results suggest that the two VHH antibodies against C. difficile TcdA toxin were
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efficiently expressed by transformed B. longum 44B strain. The expressed proteins were completely
265
secreted from the cells into the milieu and retained proper antigen binding and toxin neutralization
266
activity. Although the A20.1 antibody showed elevated electrophoretic mobility it was found to be 8
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almost as effective in the in vitro neutralization test as the A26.8 antibody, which possessed the
268
electrophoretic mobility corresponding to its calculated MW.
269
An attempt was made using semi-quantitative ELISA to detect the in vivo expression of D2E7
270
antibody in feces of (C57/BL6 x DBA/2)F1 mice after intragastric administration of transformed B.
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longum NCC2705. However, no signal could be detected in fecal material taken from small
272
intestine, large intestine, or cecum. In contrast, intragastric administration of B. longum 44B strain
273
transformed with either [pESH100/VHH-A20.1] or [pESH100/VHH-A26.8] resulted in detectable
274
expression of antibodies in all three intestinal segments, with the highest signal levels being
275
observed from cecal samples (Fig. 5c).
276
In summary, we have demonstrated that both, scFv single chain two-domain antibodies and
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VHH-type single chain single-domain antibodies could be effectively expressed and secreted by
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Bifidobacterium longum. The three model antibodies, D2E7, A20.1, and A26.8, were produced in B.
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longum with broadly varied yields (25 μg/L to 1 mg/L of culture), but all showing biological
280
activity in the in vitro cell-culture-based assays. In a preliminary in vivo experiment expression of
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A20.1, and A26.8, but not of D2E7, has been detected in the large bowels of (C57/BL6 x DBA/2)F1
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mice. We conclude that the probiotic bacterium B. longum may be employed as a delivery vehicle
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for in situ production of therapeutic antibodies for treatment of GIT disorders. However, extensive
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studies of the efficiency and safety of such genetically-modified therapeutic strains should be
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conducted in vivo using animal models of human GIT diseases.
286 287
Acknowledgements
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Authors thank Andrei Chaplin (Dept. of Microbiology and Virology, Pirogov Russian
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National Research Medical University, Moscow) for his technical help with protein expression and
290
purification.
291 292
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Kechagia M, Basoulis D, Konstantopoulou S, et al. Health benefits of probiotics: a review. ISRN Nutr 2013, DOI: 10.5402/2013/481651.
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biologically active human interleukin-10 in Bifidobacterium breve. Arch Microbiol 2010;192:769-
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Khokhlova EV, Smeianov VV, Efimov BA, et al. Anti-inflammatory properties of intestinal Bifidobacterium strains isolated from healthy infants. Microbiol Immunol 2012;56:27-39.
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Kim JE, Kim JY, Lee KW, Lee HJ. Cancer chemopreventive effects of lactic acid bacteria. J Microbiol Biotechnol 2007;17:1227-35.
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Koenig JE, Spor A, Scalfone N, et al. Succession of microbial consortia in the developing infant gut microbiome. Proc Natl Acad Sci U S A 2011;108:4578-85.
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Kumar H, Salminen S, Verhagen H, et al. Novel probiotics and prebiotics: road to the market. 11
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Curr Opin Biotechnol 2014, DOI: 10.1016/j.copbio.2014.11.021.
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Limaye SA, Haddad RI, Cilli F, et al. Phase 1b, multicenter, single blinded, placebo-
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controlled, sequential dose escalation study to assess the safety and tolerability of topically applied
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AG013 in subjects with locally advanced head and neck cancer receiving induction chemotherapy.
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Cancer 2013;119:4268-76.
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Losurdo L, Quintieri L, Caputo L, et al. Cloning and expression of synthetic genes encoding
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angiotensin-I converting enzyme (ACE)-inhibitory bioactive peptides in Bifidobacterium
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pseudocatenulatum. FEMS Microbiol Lett 2013;340:24-32.
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Ma Y, Luo Y, Huang X, et al. Construction of Bifidobacterium infantis as a live oral vaccine
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that expresses antigens of the major fimbrial subunit (CfaB) and the B subunit of heat-labile
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enterotoxin (LTB) from enterotoxigenic Escherichia coli. Microbiology 2012;158:498-504.
383 384 385
Paton AW, Morona R, Paton JC. Designer probiotics for prevention of enteric infections. Nat Rev Microbiol 2006;4:193-200.
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Paton AW, Morona R, Paton JC. Bioengineered bugs expressing oligosaccharide receptor
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mimics: toxin-binding probiotics for treatment and prevention of enteric infections. Bioeng Bugs
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2010;1:172-7.
390 391 392
Pompei A, Cordisco L, Amaretti A, et al. Folate production by bifidobacteria as a potential probiotic property. Appl Environ Microbiol 2007;73:179-85.
393 394 395
Rajpal A, Beyaz N, Haber L, et al. A general method for greatly improving the affinity of antibodies by using combinatorial libraries. Proc Natl Acad Sci U S A 2005;102:8466-71.
396 397 398
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Shkoporov AN, Efimov BA, Khokhlova EV, et al. Characterization of plasmids from human
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infant Bifidobacterium strains: sequence analysis and construction of E. coli-Bifidobacterium
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shuttle vectors. Plasmid 2008;60:136-48. 12
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Shkoporov AN, Efimov BA, Khokhlova EV, et al. Draft Genome Sequences of Two Pairs of
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Human Intestinal Bifidobacterium longum subsp. longum Strains, 44B and 1-6B and 35B and 2-2B,
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Consecutively Isolated from Two Children after a 5-Year Time Period. Genome Announc 2013,
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reveals metabolic pathways for host-derived glycan foraging. Proc Natl Acad Sci U S A
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415 416 417
Turroni F, Peano C, Pass DA, et al. Diversity of bifidobacteria within the infant gut microbiota. PLoS One 2012, DOI: 10.1371/journal.pone.0036957.
418 419 420
Urben LM, Wiedmar J, Boettcher E, et al. Bugs or drugs: are probiotics safe for use in the critically ill? Curr Gastroenterol Rep 2014;16:388.
421 422 423
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424 425
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428 429 430
de Vrese M, Schrezenmeir J. Probiotics, prebiotics, and synbiotics. Adv Biochem Eng Biotechnol 2008;111:1-66.
431 432 433
Yatsunenko T, Rey FE, Manary MJ, et al. Human gut microbiome viewed across age and geography. Nature 2012;486:222-7.
434 435
Yin Y, Kou L, Wang JJ, et al. Therapeutic efficacy of Bifidobacterium longum-mediated
436
human interleukin-2 with endostatin or TRAIL in transplanted tumors in mice. Exp Ther Med 13
437
2012;3:481-486.
438 439
Yu Z, Huang Z, Sao C, et al. Bifidobacterium as an oral delivery carrier of interleukin-12 for
440
the treatment of Coxsackie virus B3-induced myocarditis in the Balb/c mice. Int Immunopharmacol
441
2012;12:125-30.
442
14
442 443
Table 1. PCR primers used in the study. Primer
Nucleotide sequence, 5'-3'
Amplicon size, bp
IL8-F1
CAAGGAAAACTGGGTGCAGA
163
IL8-R1
CCTACAACAGACCCACACAA
GAPDH-F1
CTTTGACGCTGGGGCTGGCATT
GAPDH-R1
TTGTGCTCTTGCTGGGGCTGGT
IKBA-F1
CAAGCACCCGGATACAGCAG
IKBA-R1
ACAAGTCCATGTTCTTTCAGCCC
CCL20-F1 CCL20-R1
ACAGACTTGGGTGAAATATATTGTGCGTC GCACAAATTAGATAAGCACTAAACCCTCCA
444 445
15
161
204
174
445 446 447
Figure 1. Purified proteins used in the study. 12% PAAG electrophoresis. Lane 1, A20.1
448
VHH antibody produced in E. coli Rosetta DE3 [pLysS]; lane 2, A26.8 VHH antibody produced in
449
the same strain; lane 3, TcdA toxin purified from Clostridium difficile ATCC 43255 culture
450
supernatant.
451
16
451 452
Figure 2. Western blot analysis of D2E7 scFv antibody expression in B. longum
453
NCC2705. a, Conditioned media (lanes 1-3) and soluble cytoplasmic fractions (lanes 4-6) of
454
transformed and control strains. Lanes 1, 2, 4, and 5, B. longum NCC2705 [pESH100/D2E7]; lanes
455
3 and 6, untransformed B. longum NCC2705. b, Semi-quantitative analysis of D2E7 expression in
456
the soluble cytoplasmic fraction of B. longum NCC2705 [pESH100/D2E7]. Lane 1, Tenfold
457
concentrated cytoplasmic fraction of B. longum NCC2705 [pESH100/D2E7]; lanes 2-6, twofold
458
dilutions (0.0625, 0.125, 0.25, 0.5, and 1 μg/ml) of recombinant D2E7 produced in E. coli Rosetta
459
DE3 [pLysS].
460
17
460 461
Figure 3. In vitro neutralization of the human TNFα activity by the D2E7 scFv antibody
462
produced in B. longum NCC2705. a, qRT-PCR analysis of IL-8, IκBα, and CCL20 transcripts in
463
total RNA samples from HT-29 cells four hours after treatment with human TNFα (15 ng/ml) and B.
464
longum conditioned media. Expression levels were normalized to that of the housekeeping gene
465
GAPDH. b, ELISA analysis of IL-8 concentrations in HT-29 culture supernatants 18 hours after 18
466
treatment with human TNFα (15 ng/ml) and B. longum conditioned media. The data shown are
467
mean values and standard errors obtained from at least three independent experiments. TNFα, cells
468
treated with TNFα (positive control); BL, cells treated with TNFα in the presence of conditioned
469
medium from untransformed B. longum NCC2705; BL-D2E7, cells treated with TNFα in the
470
presence of conditioned medium from B. longum NCC2705 [pESH100/D2E7]; control, untreated
471
negative control cells. * P < 0.05, ** P < 0.01 versus positive control.
472
19
472 473
Figure 4. Western blot analysis of A20.1 and A26.8 VHH antibodies expression in B.
474
longum 44B and B. breve UCC2003. a, Conditioned media of transformed and control B. longum
475
44B and B. breve UCC2003. Lanes 1-2, B. longum 44B [pESH100/VHH-A20.1]; lanes 3-4, B.
476
longum 44B [pESH100/VHH-A26.8]; lanes 5-6, B. breve UCC2003 [pESH100/VHH-A20.1]; lanes
477
7-8, B. breve UCC2003 [pESH100/VHH-A26.8]; lane 9, untransformed B. longum 44B; lane 10,
478
untransformed B. breve UCC2003; lanes 11-14, twofold dilutions (0.5, 0.25, 0.125, and 0.0625 20
479
μg/ml) of recombinant VHH A20.1 produced in E. coli Rosetta DE3 [pLysS]. b, Conditioned media
480
and soluble cytoplasmic fractions of transformed B. longum 44B. Lanes 1 and 2 conditioned media
481
from B. longum 44B [pESH100/VHH-A20.1] and B. longum 44B [pESH100/VHH-A26.8]; lanes 3
482
and 4, cytoplasmic fraction of B. longum 44B [pESH100/VHH-A20.1]; lanes 5 and 6, cytoplasmic
483
fraction of B. longum 44B [pESH100/VHH-A26.8].
484
21
484 22
485
Figure 5. In vitro neutralizing activity and in vivo expression of A20.1 and A26.8 VHH
486
antibodies. a, Dot blot analysis of B. longum-produced A20.1 and A26.8 VHH antibodies binding
487
to TcdA immobilized on a PVDF membrane in quantities from 35 to 140 ng. Bovine serum albumin
488
(BSA) immobilized in quantities from 50 to 200 ng was used as a control. Blots were stained with
489
conditioned media from B. longum 44B [pESH100/VHH-A20.1] (BL-A20.1) and B. longum 44B
490
[pESH100/VHH-A26.8] (BL-A20.1), and then developed as described in Materials and Methods
491
section. b, Dose-effect curve of TcdA toxicity on CHO-K1 cell line in the presence or absence of
492
conditioned media from B. longum 44B [pESH100/VHH-A20.1] (BL-A20.1) and B. longum 44B
493
[pESH100/VHH-A26.8] (BL-A20.1). Conditioned media were applied on the CHO-K1 cells prior
494
to the treatment with TcdA toxin (5.5 to 1400 ng/ml). 24 hours after treatment the percentage of
495
rounded cells was manually calculated using phase contrast microscopy. c, In vivo expression of
496
A20.1 and A26.8 antibodies in (C57/BL6 x DBA/2)F1 mice after intragastric administration of
497
transformed B. longum strains (BL-D2E7, BL-A20.1, and BL-A26.8) and untransformed B. longum
498
44B (BL) assayed using semiquantitative ELISA (Materials and Methods section for details);
499
control +, culture supernatant from strain BL-A26.8; control -, empty medium. * P < 0.05, ** P
1
Production of biologically active scFv and VHH antibody fragments in Bifidobacterium
2
longum
3 4
Shkoporov A.N.1,2*, Khokhlova E.V.1, Savochkin K.A.2, Kafarskaya L.I. 1, and Efimov B.A. 1
5
1
6 7
Department of Microbiology and Virology, Pirogov Russian National Research Medical
University, Moscow 117997, Ostrovitjanova str. 1. 2
Pharmbacter LLC, Moscow 127018, Skladochnaya ul., 1 – 1.
8 9
*Corresponding author. Phone/Fax: +7 495 4341677, e-mail: [email protected]
10 11
Abstract
12
Bifidobacteria constitute a significant part of healthy intestinal microbiota in adults and
13
infants and present a promising platform for construction of genetically modified probiotic agents
14
for treatment of gastrointestinal disorders. In this study three strains of Bifidobacterium longum
15
were constructed that express and secrete biologically active single-chain antibodies against human
16
TNF-α and Clostridium difficile exotoxin A. Anti-TNF-α scFv antibody D2E7 was produced at the
17
level of 25 μg/L in broth culture and was mostly retained in the cytoplasm, while VHH-type
18
antibodies A20.1 and A26.8 against C. difficile exotoxin A were produced at the levels of 0.3 – 1
19
mg/L and secreted very efficiently. The biological activity of both antibody types was demonstrated
20
in the mammalian cell-based assays. Expression of A20.1 and A26.8 was also observed in vivo after
21
intragastric administration of transformed B. longum strains to (C57/BL6 x DBA/2)F1 mice. The
22
obtained B. longum strains may serve as prototypes for construction of novel probiotic medications
23
against inflammatory bowel disease (IBD) and C. difficile associated disease (CDAD).
24 25
Keywords: Bifidobacterium, transformation, vector, antibody, scFv, VHH.
26 27
Running Title: Production of antibody fragments in Bifidobacterium longum
28
1
29
Introduction
30
The presumed ability of certain microorganisms ('probiotics') to exert health promoting effects
31
on human gastrointestinal tract (GIT) after oral administration underlies the rapidly growing
32
probiotic products industry (de Vrese and Schrezenmeir, 2008; Figueroa-González et al., 2011;
33
Kechagia et al., 2013; Kumar et al., 2014). Although the efficacy of probiotics is still debated and a
34
plausible evidence for significant clinical effects is still missing (Guandalini, 2014; Ghouri et al.,
35
2014; Dimidi et al., 2014), the safety of many of probiotic preparations has been confirmed by a
36
long history of their production and consumption (AlFaleh and Anabrees, 2013; Urben et al., 2014).
37
The latter fact leads to the idea that the food grade category microorganisms used in probiotic
38
preparations (e.g. lactic acid bacteria like Lactococcus sp. and Lactobacillus sp., or Bifidobacterium
39
sp., and some others) could be used to construct a new generation of the so called 'designer
40
probiotics' (Paton et al., 2006; Paton et al., 2010) which are genetically engineered to express
41
certain therapeutic molecule (e.g. a protein with proven efficacy) in situ in human GIT upon oral
42
ingestion. The in situ delivery of therapeutic proteins using probiotic bacteria for treatment of GIT
43
disorders may reduce the severity of adverse side effects seen after systemic delivery, may increase
44
local effective concentration, ease drug usage, and reduce treatment costs due to simple
45
manufacturing procedures of the probiotic drugs. Currently one line of 'designer probiotics' based
46
on Lactococcus lactis has entered clinical trials (Braat et al., 2006; Villatoro-Hernandez et al., 2011;
47
Limaye et al., 2013).
48
Another promising candidate for construction of 'designer probiotics' is genus
49
Bifidobacterium which comprises gram-positive, strictly anaerobic, non-sporing irregular rods.
50
Representatives of this genus (B. longum, B. breve, B. bifidum, B. pseudocatenulatum and some
51
others) constitute an obligatory part of distal GIT microbiota in healthy adults and dominate this
52
microbial community during early infancy (Yatsunenko et al., 2012; Koenig et al., 2011; Turroni et
53
al., 2010; Turroni et al., 2012). The indigenous intestinal bifidobacteria are believed to contribute to
54
maintenance of intestinal homeostasis and host well-being by digestion and bioconversion of
55
dietary compounds, production of vitamins, competition with pathogens, inhibition of
56
carcinogenesis and modulation of local and systemic immune responses (Deguchi et al., 1985; Kim
57
et al., 2007; Pompei et al., 2007; de Vrese and Schrezenmeir, 2008; Fukuda et al., 2011). Until
58
recently functional genomics and genetic engineering studies of genus Bifidobacterium have been
59
hampered because of their fastidious nature and high resistance to genetic transformation
60
(Guglielmetti et al., 2013; Dominguez and O’Sullivan, 2013; Brancaccio et al., 2013).
61 62
We believe that genetically modified lactococci, although proven to be effective in murine GIT diseases models (Steidler et al., 2000; Vandenbroucke et al., 2010), possess several 2
63
disadvantages compared to bifidobacteria, including inability to efficiently colonize human GIT
64
(Bolotin et al., 2001) and involvement into some cases of opportunistic infections (Aguirre and
65
Collins, 1993). A number of reports which describe the construction of bifidobacterial strains
66
expressing therapeutic proteins have been already published (Cronin et al., 2009; Ma et al., 2012;
67
Yin et al., 2012; Yu et al., 2012; Losurdo et al., 2013).
68
In our opinion one of the most interesting type of therapeutic molecules to be delivered by the
69
future 'designer probiotics' are single-chain antibody fragments which include scFv (Ahmad et al.,
70
2012) and VHH (Harmsen and De Haard, 2007). These molecules can be expressed efficiently in
71
bacteria, including E. coli and L. lactis (Guglielmi and Martineaum, 2009; Vandenbroucke et al.,
72
2010), however, to the best of our knowledge, no reports have been published to date describing the
73
successful expression of either scFv or VHH in Bifidobacterium. The antibody-producing
74
bifidobacteria could be designed to express various types of antibodies that opsonize food-borne
75
pathogens, neutralize viruses, toxins, and virulence factors, and down-regulate pro-inflammatory
76
cytokines.
77
In this study we sought to determine whether scFv-type and VHH-type antibody molecules
78
can be expressed and secreted in biologically active form in Bifidobacterium longum. As a model
79
we used a well described TNFα neutralizing low-KD human scFv antibody D2E7 (Rajpal et al.,
80
2005) and a pair of neutralizing VHH-type antibodies (A20.1 and A26.8) against Clostridium
81
difficile exotoxin A (Hussack et al., 2012).
82 83
Materials and Methods
84
Bacterial and Animal Cell Cultures
85
Bifidobacterium longum strains 44B (Shkoporov et al., 2013) and NCC2705 (Nestlé Culture
86
Collection, Nestlé Research Centre, Switzerland), and Bifidobacterium breve UCC2003 (personal
87
gift of Prof. D. Van Sinderen, University College Cork, Ireland) were propagated anaerobically in
88
RCM (Lab M, UK) broth and agar media supplemented with erythromycin (2.5 μg/ml) when
89
needed. Clostridium difficile ATCC 43255 was propagated anaerobically on Columbia broth and
90
agar (BD). Strains of Escherichia coli XL-1Blue and Rosetta DE3 [pLysS] were propagated in LB
91
medium supplemented with 100 μg/ml ampicillin or 50 μg/ml kanamycin. All incubations were
92
done at 37°C, and broth cultures of E. coli were shaken at 200 rpm.
93
For preparation of conditioned media (CM) overnight broth cultures of B. longum strains were
94
centrifuged at 3,000 g for 10 min, washed with DMEM (Biolot, Russia) and resuspended in DMEM
95
with 20 mM HEPES pH 7.0. Cultures were incubated anaerobically for 20 h at 37°C. Cells were
96
removed by centrifugation and pH of supernatant was adjusted to pH 7.4 with 1M NaOH followed 3
97
by 0.22 μm filter sterilization. Protein fraction from СM was buffer exchanged into fresh DMEM
98
and simultaneously concentrated 20-fold by ultrafiltration using Pierce protein concentrators, 9K
99
MWCO (Thermo Fisher Scientific, Rockford, IL). Soluble cytoplasmic fractions (SCF) were
100
prepared from PBS-washed cells pelleted from 5 ml of broth by vigorous vortexing with 200 mg of
101
acid-washed glass beads (0.1 mm diameter) and 0.25 ml PBS with subsequent centrifugation at 12
102
000 g for 15 min and collection of supernatant.
103
Mammalian cell lines HT-29 and CHO-K1 were cultured in DMEM supplemented with 10%
104
fetal calf serum (HyClone, USA), penicillin G, 100 U/ml, and streptomycin, 100 μg/ml. Cultures
105
were incubated at 37°C in atmosphere of 5% CO2. For TcdA toxin and TNFα neutralization
106
experiments cells were seeded into 24-well (HT-29) or 96-well (CHO-K1) plates, grown to 70%
107
confluency, washed once with Hank's balanced salt solution overlaid with a 9:1 mixture of fresh
108
complete DMEM medium and bacterial CM. After 1 h pre-incubation with CM, cells were treated
109
with human recombinant TNFα, 15 ng/ml final concentration, or TcdA toxin (prepared as described
110
below), 5.5 – 1400 ng/ml. The HT-29 cells were lysed and collected for qRT-PCR after 4 h
111
incubation. Culture supernatants for ELISA assay were collected 18 hours after treatment. Cell
112
morphology of CHO-K1 was assessed after 24 h incubation.
113 114
Construction of Recombinant Strains
115
Genes coding for D2E7 scFv and A20.1 and A26.8 VHH-type antibody fragment were
116
synthesized at Evrogen JSC (Russia) with codon usage optimized for the expression in B. longum.
117
Synthetic fragments of 795 bp (D2E7), 416 bp (A20.1), and 419 bp (A26.8) were cloned into NdeI
118
and HindIII restriction sites of the pESH100 E. coli – Bifidobacterium shuttle expression/secretion
119
vector (Khokhlova et al., 2010) according to standard procedures. The E. coli strain XL-1Blue was
120
used as а cloning host. The same fragments were cloned into pET-22b and pET-28b (Merck,
121
Germany) for the production of control proteins for western blots. Plasmid pESH100/D2E7 was
122
introduced into B. longum NCC2705 by electroporation as described previously (Shkoporov et al.,
123
2008). Plasmids pESH100/VHH-A20.1 and pESH100/VHH-A26.8 were introduced into the strains
124
B. longum 44B and B. breve UCC2003 in a similar way. Plasmids pET22b/D2E7, pET28b/VHH-
125
A20.1, and pET28b/VHH-A26.8 were transferred to E. coli Rosetta DE3 [pLysS] for expression.
126 127
Animals
128
Male (C57/BL6 x DBA/2)F1 hybrid mice, 10-12 weeks of age were divided into four groups
129
(n = 5). Fresh overnight PBS-washed cultures (2 x 10 9 cfu in 0.2 ml of PBS) of transformed B.
130
longum (NCC2705 [pESH100/D2E7], 44B [pESH100/VHH-A20.1], and 44B [pESH100/VHH4
131
A26.8]), as well as the untransformed control strain B. longum 44B were administered
132
intragastrically once a day for three consecutive days. On day 5 after the first injection mice were
133
sacrificed and medial portions of small intestine, large intestine, and cecum, containing fecal masses
134
were excised. Feces were weighed and resuspended 1:1 in PBS. After 5 min centrifugation at
135
10,000 g supernatants were collected and subjected to ELISA.
136 137
Protein Expression and Purification
138
For purification of TcdA toxin 2 L 48 h broth culture of Clostridium difficile ATCC 43255 was
139
centrifuged at 3,000 g for 60 min, supernatant was collected and proteins were precipitated by
140
addition of ammonium sulphate to final concentration of 2 M. After 48 h incubation at 4°C
141
precipitate was collected by centrifugation at 4,000 g for 120 min. Pellets were dissolved in 30 ml
142
of PBS and centrifuged again at 4,000 g for 60 min twice to remove insoluble material. Supernatant
143
was filtered through 0.22 μm membrane and applied onto the 2.5x20 cm Sephadex G-75 column
144
equilibrated with 25 mM Tris-HCl pH 8.0 at 1 ml/min speed. Void volume fractions were collected,
145
pooled, and applied onto 5 ml Bio-Scale Mini UNOsphere Q cartridge (Bio-Rad, CA) at 5 ml/min.
146
Bound TcdA was eluted using 50 mM NaCl and concentrated on Pierce concentrator, 9K MWCO
147
(Fig. 1).
148
The E. coli Rosetta DE3 [pLysS] derivative strains containing recombinant plasmids were
149
grown in 50 ml of LB broth at 37°C and 250 rpm. After the OD600 of the cultures reached 0.6–0.8,
150
the expression was induced by addition of 1 mM IPTG. The cells were cultured post-induction at
151
26–28 °C for 3–5 h and His-Spin Protein Miniprep kit (Zymo Research, CA) was used for protein
152
purification under denaturing conditions (Fig. 1).
153 154
Quantitative RT-PCR
155
Total RNA was isolated from HT-29 cells by using ZR RNA MiniPrep kit (Zymo Research,
156
USA). First strand cDNA was synthesized using RevertAid Premium reverse transcriptase
157
(Fermentas, Lithuania) and oligo-dT primer. Real-time PCR was set up in CFX-96 thermal cycler
158
(Bio-Rad) using a pre-made mastermix containing EvaGreen (Syntol) and an appropriate primer
159
pair at concentration of 200 nM (Table 1). Reactions were performed using the following program:
160
95°C 5 min, followed by 40 cycles of 94°C 20 s, 60°C 20 s, 72°C 20 s. Quantification of target
161
transcripts was done by the ΔΔC(t) method using GAPDH as a normalizing house-keeping gene.
162 163
Western Blotting, Dot Blotting, and ELISA
164
CM and SCF samples for Western blotting were mixed 1:1 with gel-loading dye and heated at 5
165
100°C for 3 min. Samples were electrophoresed in 12% SDS-PAGE gel followed by a transfer onto
166
Hybond P membrane (GE-Amersham, NJ, USA) using a semi-dry transfer apparatus at 2 mA/cm2
167
for 30 min. Membranes were blocked in 0.2% Tween-20/PBS (PBS-T) and stained using primary
168
(monoclonal anti-c-Myc cat. no. 626802, BioLegend, CA) and secondary (HRP goat anti-mouse
169
IgG cat. no. 405306, BioLegend, CA) antibodies for 60 min each at room temperature with
170
concomitant PBS-T washes (3 x 5 min). Blots were developed using ECL Select reagent (GE-
171
Amersham) according to the manufacturer’s instructions. Chemiluminescent detection was
172
performed using ChemiDoc XRS+ System (Bio-Rad).
173
In vitro antigen binding by A20.1 and A26.8 VHH antibodies was studied using the following
174
procedure: antigens (Tcd and BSA) were immobilized on Hybond P membrane and stained using
175
the 1:10 dilutions of CM samples from transformed bifidobacteria, membranes were washed with
176
PBS-T (3 x 5 min) and then processed as described above for western blots.
177 178 179
Concentrations of IL-8 in cell culture supernatants were assayed using human IL-8 ELISA kit (Cytokine, Russia) according to the manufacturer’s instructions. In vivo expression of A20.1 and A26.8 antibodies in mice after intragastric administration of
180
transformed B. longum strains was assayed using semiquantitative ELISA. The TcdA toxin was
181
diluted in 50 mM Na2CO3 (pH 9.7) to final concentration of 30 μg/ml and immobilized on 96-well
182
high-binding ELISA plates (Greiner, Germany) by overnight incubation. Wells were washed with
183
PBS, blocked with 3% BSA, and filled with 0.1 ml murine fecal supernatants diluted 1:10 in PBS-T.
184
After 2 hour incubation wells were washed six times with PBS-T and stained essentially as
185
described above for Western blotting. Reactions were developed using TMB and stop solutions
186
(Cytokine) and OD450 was recorded using iMark microplate reader (Bio-Rad).
187 188 189
Results and Discussion
190
To clone the genes coding for scFv- and VHH-type single chain antibodies in bifidobacteria
191
we used previously described E. coli – Bifidobacterium shuttle expression/secretion vector
192
(Khokhlova et al., 2010). The synthetic D2E7 scFv, and A20.1 and A26.8 VHH antibody genes
193
were optimized to match the codon usage of B. longum and contained the c-Myc tag-coding
194
sequence at the C-termini. When cloned into pESH100 the genes were fused in frame to the
195
bifidobacterial AmyB signal peptide (SP) and placed under the control of a relatively strong
196
constitutive promoter Pgap from B. longum. The plasmid designated as pESH100/D2E7 was
197
introduced into B. longum NCC2705, while the plasmids pESH100/VHH-A20.1 and
198
pESH100/VHH-A26.8 were used to transform both, B. breve UCC2003 and B. longum 44B. 6
199
The expression of anti-TNFα scFv antibody D2E7 in B. longum NCC2705 was monitored
200
using western blot with anti-C-myc tag antibodies. The purified D2E7 (30.7 kDa) produced in E.
201
coli was used as a positive control. According to Fig. 2a both the cytoplasmic fraction and the spent
202
culture media from pESH100/D2E7-transformed strain contained the D2E7 antibody. The observed
203
MW of D2E7 secreted into the media was very close to the calculated MW (28.5 kDa) of the mature
204
protein after cleavage of the AmyB SP. However, most of the produced D2E7 antibody was retained
205
in the cytoplasm of NCC2705 cells and had the MW (33.5 kDa) of the non-processed pre-protein
206
containing the SP. The semi-quantitative western blot analysis indicated that the overall amount of
207
D2E7 produced in B. longum NCC2705 culture was quite low and reached approximately 25 μg/L
208
(Fig. 2b).
209
The indirect ELISA analysis confirmed the ability of D2E7 antibody produced in B. longum
210
NCC2705 to bind the surface-immobilized recombinant human TNFα (data not shown). The
211
biological activity of the D2E7 scFv antibodies produced in B. longum was determined in an in vitro
212
TNFα activity assay on human colon adenocarcinoma HT-29 cell line (Khokhlova et al., 2012).
213
Cells were preconditioned with the B. longum NCC2705 [pESH100/D2E7] supernatant, culture
214
supernatant from untransformed strain, or control medium, and challenged with human TNFα. The
215
inflammatory response in HT-29 cells was assessed by the level of IL-8, IκBα, and CCL20
216
transcripts (Fig. 3a). Four hours after TNFα challenge IL-8 and CCL20 expression was induced
217
more than 18-fold and 14-fold respectively, while the IκBα expression was up-regulated less
218
strongly. Co-incubation of cells with B. longum NCC2705 [pESH100/D2E7] supernatant, but not
219
with control strain supernatant, before TNFα stimulation reduced the IL-8 and CCL20 expression
220
by 6-fold (P < 0.01) and 2.5-fold (P < 0.05) respectively. Additionally, the IL-8 levels in culture
221
media were assayed using ELISA 18 hours after TNFα treatment. It was found that B. longum
222
NCC2705 [pESH100/D2E7] supernatant but not control supernatant dramatically reduced the
223
amount of secreted IL-8 (Fig. 3b).
224
The obtained data suggest that the D2E7 anti-TNFα scFv antibody was relatively efficiently
225
expressed in transformed B. longum NCC2705 cells. Although most of the produced antibodies
226
were retained in the cytoplasm, the secreted fraction was properly folded and could neutralize the
227
TNFα activity in vitro.
228
The expression of single-domain VHH-type antibodies A20.1 and A26.8 against C. difficile
229
toxin TcdA was investigated upon introduction of the synthetic A20.1 and A26.8 genes into B. breve
230
UCC2003 and B. longum 44B. Proteins were detected using western blot with anti-C-myc tag
231
antibody and the A20.1 protein (17 kDa) purified from E. coli as a positive control. The A20.1 and
232
A26.8 antibodies were expressed and secreted into the culture medium in both B. breve UCC2003 7
233
and B. longum 44B (Fig. 4a). However, the expression levels of both antibodies in B. longum 44B
234
was at least 10-fold higher than in B. breve UCC2003. For this reason further work was done with
235
the strain B. longum 44B only. The apparent MW of the A26.8 secreted into the supernatant was
236
close enough to the predicted mass of the protein after SP cleavage (15.3 kDa). However the
237
apparent weight of the A20.1 protein (~12 kDa) was significantly smaller than calculated from its
238
amino acid sequence (15 kDa) which could be a result of either improper signal peptide cleavage,
239
protein degradation, or anomalous electrophoretic mobility. The antibodies A20.1 and A26.8
240
produced by B. longum 44B were completely secreted into the milieu which was confirmed by the
241
absence of either mature forms or pre-proteins (20 kDa) from the cytoplasmic fractions of the
242
transformed strains (Fig. 4b).
243
In contrast to the D2E7 scFv antibody, the antibodies A20.1 and A26.8 showed relatively high
244
expression levels. According to the semi-quantitative dot-blot (data not shown) the A20.1 level in B.
245
longum 44B supernatant was 0.3-0.4 mg/L while the A26.8 was expressed at an even higher rate of
246
0.6-1.0 mg/L. The achieved expression level of VHH antibodies in B. longum was comparable to
247
that reported by Vandenbroucke et al. (2010) for MT1 anti-TNFα VHH-antibody expressed in L.
248
lactis (1.0 – 1.3 mg/L). Moreover, to the best of our knowledge such high expression level of
249
heterologous proteins was never demonstrated in bifidobacteria before. Despite the constitutive
250
over-expression of the A20.1 and A26.8 antibodies in B. longum 44B, neither the growth
251
characteristics nor the other phenotypic traits of the host strain were changed.
252
The ability of A20.1 and A26.8 antibodies produced by the transformed B. longum 44B strains
253
to bind the TcdA toxin was tested in a dot-blot assay with immobilized TcdA. According to Fig. 5a
254
both antibodies concentrated from culture supernatants of B. longum 44B derivatives were able to
255
bind to TcdA, but not to control protein BSA, in a dose-dependent manner. The biological activity
256
of the B. longum -produced VHH antibodies was investigated using the standard TcdA in vitro
257
toxicity assay (Rothman, 1986; Keel and Songer, 2007). The CHO-K1 cells were challenged with a
258
purified TcdA (5.5 – 1400 ng/ml) in the presence or absence of A20.1- or A26.8-containing
259
bifidobacterial supernatants and after counting of rounded cells percentage the dose-effect curves
260
were plotted. The data presented in Fig 5b indicate that the antibody-containing supernatants from
261
both, B. longum 44B [pESH100/VHH-A20.1] and B. longum 44B [pESH100/VHH-A26.8], were
262
able to neutralize the toxic effect of TcdA in concentrations of up to 350 ng/ml.
263
The above results suggest that the two VHH antibodies against C. difficile TcdA toxin were
264
efficiently expressed by transformed B. longum 44B strain. The expressed proteins were completely
265
secreted from the cells into the milieu and retained proper antigen binding and toxin neutralization
266
activity. Although the A20.1 antibody showed elevated electrophoretic mobility it was found to be 8
267
almost as effective in the in vitro neutralization test as the A26.8 antibody, which possessed the
268
electrophoretic mobility corresponding to its calculated MW.
269
An attempt was made using semi-quantitative ELISA to detect the in vivo expression of D2E7
270
antibody in feces of (C57/BL6 x DBA/2)F1 mice after intragastric administration of transformed B.
271
longum NCC2705. However, no signal could be detected in fecal material taken from small
272
intestine, large intestine, or cecum. In contrast, intragastric administration of B. longum 44B strain
273
transformed with either [pESH100/VHH-A20.1] or [pESH100/VHH-A26.8] resulted in detectable
274
expression of antibodies in all three intestinal segments, with the highest signal levels being
275
observed from cecal samples (Fig. 5c).
276
In summary, we have demonstrated that both, scFv single chain two-domain antibodies and
277
VHH-type single chain single-domain antibodies could be effectively expressed and secreted by
278
Bifidobacterium longum. The three model antibodies, D2E7, A20.1, and A26.8, were produced in B.
279
longum with broadly varied yields (25 μg/L to 1 mg/L of culture), but all showing biological
280
activity in the in vitro cell-culture-based assays. In a preliminary in vivo experiment expression of
281
A20.1, and A26.8, but not of D2E7, has been detected in the large bowels of (C57/BL6 x DBA/2)F1
282
mice. We conclude that the probiotic bacterium B. longum may be employed as a delivery vehicle
283
for in situ production of therapeutic antibodies for treatment of GIT disorders. However, extensive
284
studies of the efficiency and safety of such genetically-modified therapeutic strains should be
285
conducted in vivo using animal models of human GIT diseases.
286 287
Acknowledgements
288
Authors thank Andrei Chaplin (Dept. of Microbiology and Virology, Pirogov Russian
289
National Research Medical University, Moscow) for his technical help with protein expression and
290
purification.
291 292
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14
442 443
Table 1. PCR primers used in the study. Primer
Nucleotide sequence, 5'-3'
Amplicon size, bp
IL8-F1
CAAGGAAAACTGGGTGCAGA
163
IL8-R1
CCTACAACAGACCCACACAA
GAPDH-F1
CTTTGACGCTGGGGCTGGCATT
GAPDH-R1
TTGTGCTCTTGCTGGGGCTGGT
IKBA-F1
CAAGCACCCGGATACAGCAG
IKBA-R1
ACAAGTCCATGTTCTTTCAGCCC
CCL20-F1 CCL20-R1
ACAGACTTGGGTGAAATATATTGTGCGTC GCACAAATTAGATAAGCACTAAACCCTCCA
444 445
15
161
204
174
445 446 447
Figure 1. Purified proteins used in the study. 12% PAAG electrophoresis. Lane 1, A20.1
448
VHH antibody produced in E. coli Rosetta DE3 [pLysS]; lane 2, A26.8 VHH antibody produced in
449
the same strain; lane 3, TcdA toxin purified from Clostridium difficile ATCC 43255 culture
450
supernatant.
451
16
451 452
Figure 2. Western blot analysis of D2E7 scFv antibody expression in B. longum
453
NCC2705. a, Conditioned media (lanes 1-3) and soluble cytoplasmic fractions (lanes 4-6) of
454
transformed and control strains. Lanes 1, 2, 4, and 5, B. longum NCC2705 [pESH100/D2E7]; lanes
455
3 and 6, untransformed B. longum NCC2705. b, Semi-quantitative analysis of D2E7 expression in
456
the soluble cytoplasmic fraction of B. longum NCC2705 [pESH100/D2E7]. Lane 1, Tenfold
457
concentrated cytoplasmic fraction of B. longum NCC2705 [pESH100/D2E7]; lanes 2-6, twofold
458
dilutions (0.0625, 0.125, 0.25, 0.5, and 1 μg/ml) of recombinant D2E7 produced in E. coli Rosetta
459
DE3 [pLysS].
460
17
460 461
Figure 3. In vitro neutralization of the human TNFα activity by the D2E7 scFv antibody
462
produced in B. longum NCC2705. a, qRT-PCR analysis of IL-8, IκBα, and CCL20 transcripts in
463
total RNA samples from HT-29 cells four hours after treatment with human TNFα (15 ng/ml) and B.
464
longum conditioned media. Expression levels were normalized to that of the housekeeping gene
465
GAPDH. b, ELISA analysis of IL-8 concentrations in HT-29 culture supernatants 18 hours after 18
466
treatment with human TNFα (15 ng/ml) and B. longum conditioned media. The data shown are
467
mean values and standard errors obtained from at least three independent experiments. TNFα, cells
468
treated with TNFα (positive control); BL, cells treated with TNFα in the presence of conditioned
469
medium from untransformed B. longum NCC2705; BL-D2E7, cells treated with TNFα in the
470
presence of conditioned medium from B. longum NCC2705 [pESH100/D2E7]; control, untreated
471
negative control cells. * P < 0.05, ** P < 0.01 versus positive control.
472
19
472 473
Figure 4. Western blot analysis of A20.1 and A26.8 VHH antibodies expression in B.
474
longum 44B and B. breve UCC2003. a, Conditioned media of transformed and control B. longum
475
44B and B. breve UCC2003. Lanes 1-2, B. longum 44B [pESH100/VHH-A20.1]; lanes 3-4, B.
476
longum 44B [pESH100/VHH-A26.8]; lanes 5-6, B. breve UCC2003 [pESH100/VHH-A20.1]; lanes
477
7-8, B. breve UCC2003 [pESH100/VHH-A26.8]; lane 9, untransformed B. longum 44B; lane 10,
478
untransformed B. breve UCC2003; lanes 11-14, twofold dilutions (0.5, 0.25, 0.125, and 0.0625 20
479
μg/ml) of recombinant VHH A20.1 produced in E. coli Rosetta DE3 [pLysS]. b, Conditioned media
480
and soluble cytoplasmic fractions of transformed B. longum 44B. Lanes 1 and 2 conditioned media
481
from B. longum 44B [pESH100/VHH-A20.1] and B. longum 44B [pESH100/VHH-A26.8]; lanes 3
482
and 4, cytoplasmic fraction of B. longum 44B [pESH100/VHH-A20.1]; lanes 5 and 6, cytoplasmic
483
fraction of B. longum 44B [pESH100/VHH-A26.8].
484
21
484 22
485
Figure 5. In vitro neutralizing activity and in vivo expression of A20.1 and A26.8 VHH
486
antibodies. a, Dot blot analysis of B. longum-produced A20.1 and A26.8 VHH antibodies binding
487
to TcdA immobilized on a PVDF membrane in quantities from 35 to 140 ng. Bovine serum albumin
488
(BSA) immobilized in quantities from 50 to 200 ng was used as a control. Blots were stained with
489
conditioned media from B. longum 44B [pESH100/VHH-A20.1] (BL-A20.1) and B. longum 44B
490
[pESH100/VHH-A26.8] (BL-A20.1), and then developed as described in Materials and Methods
491
section. b, Dose-effect curve of TcdA toxicity on CHO-K1 cell line in the presence or absence of
492
conditioned media from B. longum 44B [pESH100/VHH-A20.1] (BL-A20.1) and B. longum 44B
493
[pESH100/VHH-A26.8] (BL-A20.1). Conditioned media were applied on the CHO-K1 cells prior
494
to the treatment with TcdA toxin (5.5 to 1400 ng/ml). 24 hours after treatment the percentage of
495
rounded cells was manually calculated using phase contrast microscopy. c, In vivo expression of
496
A20.1 and A26.8 antibodies in (C57/BL6 x DBA/2)F1 mice after intragastric administration of
497
transformed B. longum strains (BL-D2E7, BL-A20.1, and BL-A26.8) and untransformed B. longum
498
44B (BL) assayed using semiquantitative ELISA (Materials and Methods section for details);
499
control +, culture supernatant from strain BL-A26.8; control -, empty medium. * P < 0.05, ** P
