Oral delivery of Escherichia coli persistently infected with M2e-displaying bacteriophages partially protects against influenza A virus

Journal of Controlled Release 264 (2017) 55–65

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Oral delivery of Escherichia coli persistently infected with M2e-displaying bacteriophages partially protects against influenza A virus

MARK

Lei Denga,b,1, Kenny Roosea,b, Emma R. Joba,b, Riet De Ryckeb,c, Evelien Van Hammec, Amanda Gonçalvesc, Eef Parthoensc, Laetitia Cicchelerod, Niek Sandersd, Walter Fiersa,b,⁎, Xavier Saelensa,b,⁎ a

VIB-UGent Center for Medical Biotechnology, VIB, Technologiepark, 927, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, Ghent, Belgium c Inflammation Research Center, VIB, Technologiepark 927, Ghent, Belgium d Laboratory of Gene Therapy, Faculty of Veterinary Sciences, Ghent University, Merelbeke, Belgium b

A R T I C L E I N F O

A B S T R A C T

Keywords: Live oral vaccine Influenza Escherichia coli Bacteriophage

We describe a novel live oral vaccine type. Conceptually, this vaccine is based on a non-lytic, recombinant filamentous bacteriophage that displays an antigen of interest. To provide proof of concept we used the aminoterminal part of a conserved influenza A virus epitope, i.e. matrix protein 2 ectodomain (M2e) residues 2 to 16, as the antigen of interest. Rather than using the phages as purified virus-like particles as a vaccine, these phages were delivered to intestinal Peyer's patches as a live bacterium-phage combination that comprises Escherichia coli cells that conditionally express invasin derived from Yersinia pseudotuberculosis. Invasin-expressing E. coli cells were internalized by mammalian Hep-2 cells in vitro and adhered to mouse intestinal microfold (M) cells ex vivo. Invasin-expressing E. coli cells were permissive for recombinant filamentous bacteriophage f88 that displays M2e and became persistently infected. Oral administration of the live engineered E. coli-invasin-phage combination to mice induced M2e-specific serum IgG antibodies. Mice that had been immunized with invasin-expressing E. coli cells that carried M2e2-16 displaying fd phages seroconverted to M2e and showed partial protection against challenge with influenza A virus. Oral delivery of a live vaccine comprising a bacterial host that is targeted to Peyer's patches and is persistently infected with an antigen-displaying phage, can thus be exploited as an oral vaccine.

1. Introduction Most vaccines are administered by needle injection. This route of vaccination can induce strong systemic B and T cell immunity but is poor at generating an adaptive immune response at mucosal sites such as the respiratory and intestinal epithelium. On the other hand, pathogens such as influenza viruses, noroviruses and rotaviruses enter the host via the mucosal route [1–4]. Killed or subunit vaccines that are administered at intestinal mucosal surfaces are poorly immunogenic whereas live attenuated microbes perform much better in inducing mucosal immunity. Examples of such vaccines are live attenuated influenza and oral polio vaccines [5,6]. How is an adaptive immune response induced by an oral vaccine? In mammals such a response starts from the luminal side of the gut through a set of anatomically defined lymphoid compartments such as

⁎

1

Peyer's patches (PP), mesenteric lymph nodes, the appendix and solitary follicles, collectively known as the gut-associated lymphoid tissue (GALT). This compartmentalized system helps explain why mucosal immunity can operate locally and independently from the systemic immune system. In human, for example, oral immunization may induce substantial antibody levels in the small intestine, the ascending colon, mammary and salivary glands, but antibody responses at distal segments, such as the large intestine, tonsils, genital tract or respiratory tract mucosa are much weaker [7]. A relatively well established route of antigen sampling by the GALT is through Microfold (M) cells. In mice, M cells are differentiated from the cycling Lgr5+ stem cells within dome-associated crypts that surround follicle-associated epithelia (FAE) and distribute sparsely within FAE [8–10]. M cells are an important portal for the entry of macromolecules, commensals and pathogens from the intestinal lumen into

Corresponding authors at: VIB-UGent Center for Medical Biotechnology, VIB, Technologiepark 927, Ghent, Belgium. E-mail addresses: walter.fi[email protected] (W. Fiers), [email protected] (X. Saelens). Present address: Department of Microbiology and Immunology, School of Medicine, Emory University, GA 30322 Atlanta, USA.

http://dx.doi.org/10.1016/j.jconrel.2017.08.020 Received 23 May 2017; Received in revised form 16 August 2017; Accepted 18 August 2017 Available online 24 August 2017 0168-3659/ © 2017 Published by Elsevier B.V.

Journal of Controlled Release 264 (2017) 55–65

L. Deng et al.

with the major coat protein p8, and f88ctr phages have been described [25]. Filamentous f88 phage-producing E. coli cells that also express invasin were generated by transformation of BL21pICA2 cells with pLT32h-inv or pLT32h-invmutD911T vector together with f88M2e2-16 or f88ctr bacteriophage replicative forms (RF). We thus obtained E. coli BL21pICA2/pLT32h-inv/f88M2e, BL21pICA2/pLT32h-invmutD911T/ f88M2e, BL21pICA2/pLT32h-inv/f88ctr and BL21pICA2/pLT32h-invmutD911T/f88ctr, which were maintained by selection on LB medium with 50 μg/ml kanamycin sulfate salt, 50 μg/ml ampicillin sodium salt and 25 μg/ml tetracycline (87128-Sigma, U.S.).

the PP where an immune responses or tolerance may be initiated in the aggregated B and T cell zones [11,12]. An array of “immunosurveillance” apical surface receptors such as Glycoprotein 2, Annexin A5 and complement receptor C5a, allow M cells to sample antigen [8,11,13,14]. Some pathogens have evolved to exploit the uptake mechanisms of M cells to invade the mammalian host [15]. Yersinia pseudotuberculosis, for example, expresses invasin, a large outer membrane anchored surface adhesion protein [16,17]. The crystal structure of invasin has revealed an extended makeup that comprises four bacterial immunoglobulin-like domains followed by a C-type lectin-like domain that is most distal from the bacterial cell [17]. The extracellular domain of this invasin extends approximately 18 nm above the bacterial surface and is important for binding of Y. pseudotuberculosis to β1 integrins that are expressed on the apical surface of M cells [18]. This binding results in clustering of the integrins, leading to uptake of the bacteria [19]. Expression of Y. pseudotuberculosis invasin by E. coli is sufficient for this bacterium to acquire M cell-tropism. Such invasin-expressing E. coli cells have been used as oral vaccine delivery systems in the mouse model, for example to deliver the model antigen ovalbumin (OVA). This oral vaccination protected the mice against a lethal challenge with B16 tumor cells that express OVA, which correlated with the induction of OVA-specific T cells [20]. Matrix protein 2 of influenza A is a tetrameric type III membrane protein that functions as a proton channel. The matrix protein 2 ectodomain (M2e) is 23 amino acid residues in length and is highly conserved among influenza A viruses [21]. M2e-specific immune responses elicited by seasonal vaccines or natural infection are very weak. Nevertheless, vaccines that comprise M2e fused to a carrier can induce strong M2e-specific immune responses and confer protection against various influenza A viruses [22–25]. We previously demonstrated that fd phages with the N-terminal part of M2e (M2e2-16) incorporated as a fusion with the major coat protein p8, could elicit broadly protective immunity in laboratory mice against challenge with different influenza A virus subtypes [25]. Our aim was to develop an economical live oral M2e-based influenza A vaccine. Such a vaccine could be applied on a large scale in livestock animals, such as chickens and swine, that are vulnerable to infection with influenza A viruses. Furthermore, this approach would overcome needle injection and dramatically reduce personnel efforts. We generated invasin-expressing E. coli cells and established a persistent infection of these cells with f88M2e2-16 phages that display M2e amino acid residues 2 to 16 on their surface. We found that oral administration of E. coli cells that express invasin and carry f88M2e2-16 phages could partially protect against influenza A virus challenge in mice.

2.2. Generation of rabbit antiserum against Y. pseudotuberculosis invasin An HPLC-purified synthetic peptide (CTSLVSSLEASRQSQGS) corresponding to amino acid residues 895-910 of Y. pseudotuberculosis invasin, modified with one additional cysteine residue at the N-terminus, was conjugated to Keyhole limpet hemocyanin (KLH) according to the manual provided with the Imject® Maleimide Activated Carrier Protein Spin Kits (77666, Thermo Scientific, U.S.). The invasin peptide-KLH conjugate (KLH-inv) was used to immunize a rabbit (outsourced to Cer Groupe; http://www.cergroupe.be). After collecting pre-immune serum, the rabbit was primed with KLH-inv emulsified in complete Freund's adjuvant at day 0, and boosted three times with KLH-inv adjuvanted with incomplete Freund's adjuvant at days 14, 28 and 56. Immune sera were collected at days 38 and 66 and a final bleeding was performed at day 80. 2.3. Western blot analysis of invasin expression in bacterial extracts Y. pseudotuberculosis was cultured in 3 ml LB medium at 28 °C for 16 h. E. coli BL21pICA2/pLT32h-inv/f88M2e, BL21pICA2/pLT32hinvmutD911T/f88M2e, BL21pICA2/pLT32h-inv/f88ctr and BL21pICA2/ pLT32h-invmutD911T/f88ctr were grown in LB in the presence or absence of 1 mM IPTG. Bacteria were harvested by centrifugation and resuspended in 0.5 ml lysis buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA and 1 mM azide). Cell extracts were prepared by sonication with an ultrasonic processor (Vibra-CellTM, Sonics & Materials, Inc.). Cell debris was removed by centrifugation and the cleared supernatant mixed with a 20% volume of 6× sample buffer (375 mM Tris-HCl pH 6.8, 6% SDS, 48% glycerol, 9% 2Mercaptoethanol and 0.03% bromophenol blue). The obtained samples were incubated at 100 °C for 5 min before separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred from the gel to a nitrocellulose blotting membrane and the blots were probed with rabbit KLH-inv antiserum (1:2000) followed by horseradish peroxidase-linked secondary anti-rabbit IgG antibody (GE Healthcare UK Ltd.). Immunoreactive proteins were visualized by incubation with ECL western blot substrate (Thermo Fisher Scientific) and exposure to a photographic film.

2. Materials and methods 2.1. E. coli-expressing invasin derived from Y. pseudotuberculosis and f88 phages

2.4. Fluorescent labeling of bacteria and uptake The coding information of wild type and D911T mutant invasin derived from Y. pseudotuberculosis was cloned in the pLT32h expression vector [26]. In the resulting constructs, pLT32h-inv and pLT32h-invmutD911T, transcription of invasin is controlled by the PL promoter. The co-transformed plasmid pICA2 encodes the thermosensitive λ phage repressor gene (cl857) under the PM promoter, which in turn is under the mediation of the bacteriophage P22 anti-repressor that is repressed by lacl and can be de-repressed by the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG) [26,27]. The transformants were maintained by growth in lysogeny broth (LB) with 50 μg/ml kanamycin sulfate salt (K1377-Sigma, U.S.) and 50 μg/ml ampicillin sodium salt (A0166-Sigma, U.S.). The expression of invasin protein in E. coli BL21pICA2/pLT32h-inv or BL21pICA2/pLT32h-invmutD911T was induced by the addition of 1 mM IPTG to the medium at 28 °C for 4 h. F88M2e2-16 phages, which express influenza A M2e2-16 as a fusion

The fluorescent membrane dye PKH26 (Sigma-Aldrich, excitation maximum 551 nm and emission maximum at 567 nm) was used to stain the E. coli membrane. Bacteria from a 500 μM IPTG-induced saturated E. coli culture were pelleted at 1000 g for 10 min at room temperature. The pelleted E. coli cells were washed with PBS and resuspended in 100 μl Diluent C (Sigma-Aldrich). The cell suspension was immediately added to 100 μl of pre-warmed Diluent C (Sigma-Aldrich) with 2 μl PKH26 at 37 °C, thoroughly mixed by pipetting, and incubated at 37 °C for 15 min. Then an equal volume (200 μl) of 1% BSA in PBS was added to the cells followed by further incubation for 1 min. The stained bacteria samples were washed 3 times with PBS before use. For fluorescent microscope imaging, HEp2 cells were first stained with CellTraceTM Oregon Green 488 (C34555, Thermo Fisher Scientific, U.S.), and then inoculated with PKH26-stained bacteria. 56

Journal of Controlled Release 264 (2017) 55–65

L. Deng et al.

approved by the Institutional ethics committee of Ghent University (Eth. Com. No. 2012–032). All efforts were made to minimize the suffering of the animals. Specific-pathogen-free female BALB/c mice were obtained from Charles River (France) and immunized at the age of 8 weeks. The animals were housed in individually ventilated cages under specific pathogen-free conditions in a temperature-controlled room (biosafety level 2) with 14/10-h light/dark cycles.

2.5. Bacteria uptake assay The expression of invasin protein by E. coli BL21pICA2/pLT32h-inv, BL21pICA2/pLT32h-invmutD911T, BL21pICA2/pLT32h-inv/f88M2e, BL21pICA2/pLT32h-invmutD911T/f88M2e, BL21pICA2/pLT32h-inv/ f88ctr and BL21pICA2/pLT32h-invmutD911T/f88ctr was induced by adding 1 mM IPTG at 28 °C for 4 h. IPTG-induced cultures of BL21pICA2 and BL21pICA2/f88M2e were used as negative controls. 5 ml of IPTGinduced bacterial cultures (OD600 = 6) were centrifuged at 1000g, for 10 min at room temperature and then resuspended in 2 ml sterile Dulbecco's phosphate-buffered saline (DPBS; Gibco) before use. Uptake of bacteria by mammalian HEp2 (human epidermoid larynx carcinoma cell type) cells was performed as described [16]. Briefly, one day before co-culture, HEp2 cells were seeded at 5 × 104 cells/well in 24-wellplate in complete medium (Dulbecco's Modified Eagle's Medium (DMEM; Gibco), 10% Fetal Calf Serum (FCS; Gibco), 2 mM L-Glutamine (L-Gln; Gibco), 100 μg/ml streptomycin, 100 units/ml penicillin (S/P; Gibco)). The cell monolayer was washed three times with DPBS. Part of the last wash (S1) was used as a negative control sample and plated on LB agar containing 50 μg/ml kanamycin sulfate. 100 μl (~ 108 cells) E. coli cells was added to the washed cells and the co-cultures were incubated at 37 °C, 5% CO2, > 95% humidity incubator for 30, 90 or 150 min. HEp2 cells were washed 3 times with DPBS. 20 μl of the last wash step (S2) was used for plating out on LB agar plate containing 50 μg/ml kanamycin sulfate. If the HEp2 cells were incubated with BL21pICA2/pLT32h-inv/f88M2e and BL21pICA2/pLT32h-invmutD911T/ f88M2e, LB agar plate also contained 25 μg/ml tetracycline. The HEp2 cells were then treated with DMEM containing 2 mM L-Gln and 50 μg/ ml gentamycin at 37 °C, 5% CO2, > 95% humidity incubator for 30 min to kill extracellular bacteria. Afterwards, the cell cultures were washed 3 times with DPBS and 20 μl of the last wash step (S3) was plated out. Finally, the HEp2 cells were lysed by adding 0.2 ml filtersterilized 1% Triton X-100 to each well followed by 5 min incubation at 37 °C. 20 μl of the lysate (S4) was plated out on LB agar plates to quantify the number of internalized bacteria.

2.8. M cells staining A lectin staining procedure was used for the detection of mouse M cells [30]. The Peyer's patches (PP) from eight-week-old naïve BALB/c mice were excised from ileum fragment and fixed in 4% PFA for 1 h at room temperature. After removal of excess fixative by washing three times using PBS, excised PP tissues were embedded in warm 4% agarose in distilled water in six-well plate. Once the agarose medium solidified at room temperature, the block containing PP was trimmed to remove excessive agarose and cross-sectioned in 100 μm thick slices using a vibrating microtome (vibratome; Leica Biosystems, Germany). Tissues were then incubated in 10% FBS (Gibco, U.S.) in PBS containing 0.1% glycine (Biosolve, France). The sections were stained with 20 μg/ml fluorescein isothiocyanate conjugated Ulex europaeus Agglutinin 1 (FITC-UEA-1; FL-1061, Vector Laboratories) for 2 h and 1 μg/ml Hoechst DNA staining for 15 min. After a PBS rinse, samples were stored in a Tris-buffered solution, pH 8.0, containing 30% glycerol and 0.1% NaN3 (Sigma, U.S.). The specimens were imaged with a laser scanning confocal microscope (LSM780, Zeiss, Germany). 2.9. Characterization of bacteria invasin into Peyer's patches

HEp2 cells were fixed and prepared for Electron microscopy as previously described [28,29]. Briefly, HEp2 cells were seeded on glass cover slips and fixed in freshly prepared 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at room temperature for 3 h. The fixation solution was replaced and the cells were incubated for another 3 h at 4 °C. Samples were then washed 3 times with 0.1 M sodium cacodylate buffer at 4 °C, and incubated in 1% OsO4 with 1.5% K3Fe(CN)6 in 0.1 M sodium cacodylate buffer at room temperature, overnight. Next, the samples were washed 4 times in 0.1 M sodium cacodylate buffer at 4 °C, and subsequently dehydrated by adding ascending concentrations of ethanol (7%, 15%, 30%, 50%, 70%, 95% and finally 100%) at 4 °C, 10 min for each step. After dehydration the samples were kept in 1% uranyl acetate for 2 h in the dark for bulk staining and subsequently embedded in Spurr's resin. Ultrathin sections of a gold interference colour were made with an ultramicrotome (EM UC6, Leica, Germany), followed by post-staining at 20 °C for 40 min in a Leica EM AC20 in uranyl acetate and for 10 min in lead stain. Sections were collected on Formvar-coated copper slot grids. Grids were observed with a JEM1010 transmission electron microscope (JEOL) operating at 80 kV with Image Plate Technology from Ditabis.

A standard method for characterization of bacteria invasin into PP was used with minor modifications [31]. Eight-week-old female BALB/ c mice were used for the analysis. Mice were anesthetized with 5% (v/ v) vaporized isoflurane mixed with air (flow rate: 200 ml/min). A 1 cm incision was made in the abdomen and part of the small intestine containing PP was taken out. The ileum was ligated at two ends at 1–1.5 cm distance interval with sewing yarn, initially at one loose side. Next, 50 μl of 107 PKH26-stained bacteria (BL21pICA2/pLT32h-inv or BL21pICA2/pLT32h-invmutD911T) suspension were injected into the exposed ileum section and the mouse abdomen was closed with a clip. For comparison, a mouse from which the ileum had been inoculated with BL21pICA2/pLT32h-invmutD911T was also taken out at 90 min post inoculation. Immediately after the procedure, the anesthetized mice were euthanized by cervical dislocation. From one mouse three randomly selected ileum fragments containing PP that had been inoculated with BL21pICA2/pLT32h-inv were taken out at 90 min post inoculation. The excised PP was flushed with PBS using a pipet and whole mount stained with 20 μg/ml FITC-UEA-1 and wheat germ agglutinin (WGA) conjugated with Alexa Fluor® 350. The tissue fragments were directly imaged using a Macroscope (Nikon AZ100M Stereo Microscope). To image bacteria uptake by M cells in one PP, the PP loop was removed at 90 min post inoculation. The tissues were fixed with 4% PFA, blocked with 10% FBS and stained with FITC-UEA-1. M cells were also stained with rat anti-mouse GP2 antibody and subsequently goat anti-rat secondary antibody conjugated with Alexa Fluor 633. The specimens were imaged using laser scanning confocal microscope (LSM780, Zeiss).

2.7. Mice

2.10. In vivo optical imaging

Specific pathogen free female BALB/c mice (eight-week-old) were used for oral immunization studies. All mouse experiments were conducted according to the national (Belgian Law 14/08/1986 and 22/12/ 2003, Belgian Royal Decree 06/04/2010) and European (EU Directives 2010/63/EU, 86/609/EEG) animal regulations. Animal protocols were

In vivo imaging was carried out using an IVIS ® Lumina II system (Perkin-Elmer, Zaventem, Belgium) equipped with an anesthesia device. During imaging, the mice were anesthetized with 5% (v/v) vaporized isoflurane (05260-05, IsoFlo®, Abbott Animal Health, U.S.) mixed with gas anesthesia medical oxygen (flow rate: 200 ml/min)

2.6. Transmission electron microscopy

57

Journal of Controlled Release 264 (2017) 55–65

L. Deng et al.

2.14. Infection with influenza A virus

using a rodent anesthesia device MatrxTM, VIP 3000 (Midmark, U.S.). SRfluor680® NHS Ester (SR-1005, Molecular Targeting Technologies, Inc., U.S.; crystalline dark green powder; spectral properties in DMSO: Absorbance Max = 650 nm and Emission Max = 678 nm) is advised to stain E. coli for in vivo imaging [32] and company website (http://www. mtarget.com). As the standard rodent food (V1534-300, Ssniff, Soest, Germany) [33] has a strong autofluorescence, BALB/c mice were fed with a special rodent diet (T.2018., Harlan Laboratories, The Netherlands) for one week before imaging. This substantially decreased the fecal autofluorescence. Images were taken before feeding with fluorescent SRfluor680 stained bacteria BL21pICA2, and time-points, 0 min (start feeding), 30 min, 60 min and 180 min after feeding by oral gavage.

Four weeks after the last immunization, mice were anesthetized by i.p. injection of 44 mg/kg Ketamine and 5 mg/kg Xylazine in 100 μl DPBS and subsequently intranasally infected with influenza A virus in 50 μl dose. For morbidity monitoring we used 0.2 LD50 (lethal dose 50%) of mouse adapted X47 (H3N2) [34]. One LD50 of X47 virus corresponds to 24 plaque forming units (pfu).

2.15. Lung viral titer determination Three mice from each immunization group were euthanized on day 6 after sublethal 0.2 LD50 infection and lungs were removed aseptically and homogenized in DPBS at 10% (w:v) using a RZR 2020 homogenizer (Heidolph Instruments, Schwabach, Germany). The homogenates were centrifuged (5 min, 400g and 4 °C) to remove cellular debris before storage at −80 °C. Titers of infectious virus were determined in triplicate by titration on MDCK cells in serum-free 2 μg/ml TPCK-treated trypsin-containing DMEM medium. Endpoint virus titers were determined by measuring chicken red blood cell agglutination activity in the cell supernatant 7 days after infection and using the calculation of Reed and Muench [35].

2.11. Immunization of mice Eight-week-old specific pathogen free female BALB/c mice were intragastrically immunized by gavage using a reusable blunt-end feeding needle. Before immunization, all mice (n = 25) were bled to obtain pre-immune serum. Four groups of mice (n = 4 per group) were orally administered 2 doses (109E. coli cells in 100 μl 5% sodium bicarbonate per dose) each day for 3 consecutive days. 21 days after the last immunization, mice were bled from a tail vein and fecal samples were collected. As a positive control, mice in one group were intraperitoneally (i.p.) primed with 1010 purified f88M2e2-16 bacteriophages [25] adjuvanted with an equal volume of Incomplete Freund's adjuvant in a total dose of 200 μl. These mice were bled from a tail vein 21 days after the immunization.

2.16. Statistical analysis Two-way ANOVA statistical analyses of multiple groups in the morbidity monitoring after X47 infection was performed using the Prism statistical software package (version 6; GraphPad Software, Inc., San Diego California; http://www.graphpad.com). To determine the statistical significance between values from two different groups, a twotailed Student's t-test was used. The significance levels are indicated with asterisks (single-asterisk, P < 0.05; double-asterisks, P < 0.01; triple-asterisks, P < 0.005).

2.12. Fecal immunoglobulin extraction A single fecal sample was obtained from the spontaneous defecation from each mouse and stored in pre-weighed 1.5 ml eppendorf tubes at − 80 °C. Fecal immunoglobulin extraction buffer (DPBS, 0.5% Tween 20 (Sigma), and 0.05% sodium azide) was added to each tube at 10% ratio (1 g wet weight in 10 ml extraction buffer). Samples were thoroughly homogenized by a combination of manual shaking and mechanical homogenization on a vortex mixer. The fecal suspensions were centrifuged at 1500g, 4 °C for 20 min and the supernatant was used in ELISA.

3. Results 3.1. Generation of invasin-expressing E. coli cells To target E. coli cells to M cells in PPs, we transformed E. coli with an expression vector for invasin derived from Y. pseudotuberculosis. It has been shown that the aspartic acid residue at position 911 (D911) in invasin is critical for its binding to β1 integrins expressed on the mammalian cell surface [36,37]. Therefore, we also generated E. coli cells that express the invasin D911T mutant, which was used as negative control throughout the experiments. We chose an expression system in which invasin expression was controlled by a constitutively expressed cI repressor and an IPTG-inducible P22 anti-repressor. Western blot analysis revealed that wild type and D911T mutant invasin were expressed to comparable amounts following IPTG induction (Fig. 1A). We previously described that a truncated version of M2e could be displayed on the surface of fd phages when fused to p8, the major coat protein of these phages [25]. These recombinant phages were viable and, when purified and used as a vaccine, induced M2e-specific serum IgG antibodies that protected mice against an otherwise lethal challenge with influenza A virus [25]. Fd phages cause a persistence infection in E. coli that does not kill the bacterial host. Therefore, we wondered whether invasin expressing E. coli cells were still susceptible to fd phage infection and whether this would affect the expression of invasin and M2e. As shown in Fig. 1A, inducible invasin expression was compatible with f88-ctr/f88-M2e phage infection. In addition, M2e-p8 expression was evident in f88-M2e infected E. coli cells that were grown in the presence of IPTG (Fig. 1B).

2.13. Serological analysis by enzyme-linked immunosorbent assay Blood was collected from the lateral tail vein before vaccination, 21 days and 42 days after the last vaccination and 15 days after challenge. Titers of total M2e-specific serum IgG and IgA, f88-specific serum IgG and IgA, and invasin-specific serum IgG and IgA were determined by enzyme-linked immunosorbent assay (ELISA) using 96-well Maxisorp immuno-plates (Thermo, Nunc, U.S.) coated overnight with human consensus M2e2-24 (SLLTEVETPIRNEWGCRCNDSSD), a cysteine-modified invasin895-910 peptide (CTSLVSSLEASRQSQGS) or purified f88ctr phages. All peptides were HPLC-purified and used at 2 μg/ml in carbonate buffer (3.39 g/l Na2CO3; 5.71 g/l NaHCO3; pH 9.6) at 50 μl/well at 37 °C. After coating, plates were washed twice with PBS + 0.1% Tween 20 and blocked with 1% bovine serum albumin (Sigma-Aldrich, U.S.) in PBS. The specific antibody titers in mouse serum were determined by incubating 1/3 serial dilutions in coated plates, starting with 1/33 dilution, for 1 h. The wells were then washed and incubated with sheep anti-mouse IgG serum conjugated with horseradish peroxidase (HRP; GE Healthcare UK Ltd.), or HRPconjugated goat anti-mouse IgA (Thermo Fisher Scientific, U.S.) for 45 min at room temperature. Finally, plates were washed and incubated with tetramethylbenzidine substrate (Sigma-Aldrich, U.S.) for 5 min and the reaction was stopped by adding 50 μl of 1 M·H2SO4. 58

Journal of Controlled Release 264 (2017) 55–65

L. Deng et al.

the HEp2 cells and adherence was allowed before thorough washing away of excess bacteria. The last wash step contained a comparable load of bacteria in the 3 settings (S2 in Fig. 2B). Subsequently, gentamycin was added to the cell cultures to kill the residual E.coli cells that resided outside of the Hep2 cells (S3 in Fig. 2B). Finally, the HEP2 cells were lysed. Viable E. coli cells were only retrieved in the detergent solubilized HEp2 cells fraction (S4 in Fig. 2B) in the setting with BL21pICA2/pLT32h-inv bacteria, providing further evidence that heterologous invasin expression facilitates uptake of E. coli by the HEp2 cells (Fig. 2B). Next, we investigated if infection of the invasin-expressing bacteria with f88M2e2-16 phages would still allow uptake by the HEp2 cells. Therefore, we incubated, BL21pICA2/f88M2e, BL21pICA2/pLT32h-inv/ f88M2e and BL21pICA2/pLT32h-invmutD911T/f88M2e with HEp2 cells and visualized internalized bacteria by confocal microscopy. 90 mins after co-incubation, BL21pICA2/pLT32h-inv/f88M2e bacteria started to invade HEp2 cells, whereas only a few BL21pICA2/pLT32hinvmutD911T/f88M2e bacteria attached to the HEp2 cells (Fig. 3A). BL21pICA2/f88M2e bacteria did not bind to and were not internalized by the HEp2 cells. To test if the internalized bacteria were viable, cells were lysed and the number of colony forming units (CFU) was determined by plating these lysates on selection plates. CFUs of BL21pICA2/pLT32h-inv/f88M2e bacteria were significantly higher than the counts of BL21pICA2/f88M2e and BL21pICA2/pLT32h-invmutD911T/ f88M2e (P < 0.005) (Fig. 3B). Therefore, we can conclude that the presence of fd bacteriophages did not abolish the ability of the BL21pICA2-invasin bacteria to invade HEp2 cells. 3.3. Expression of invasin in E. coli allows targeting to the Peyer's patches and M cells In a next set of experiments, we addressed the question if invasinexpressing E. coli cells would preferentially associate with intestinal PPs. M cells form the barrier between the intestinal lumen and the underlying immune cells in the PPs. Invasin-expressing E. coli bacteria therefore have to pass this barrier in order to invade the PP. Characterization of M cells showed their sparse distribution on the surface of PPs (Fig. S1). We next applied bacteria that expressed invasin or invasinD911T and were fluorescently labelled with the lipophilic dye PKH26, ex vivo to a mouse ileum loop. PPs in this loop were stained with GP2- and UEA-1 markers to visualize mature M cells [11,39]. Wild type invasin but not D911T mutant invasin expressing E. coli bacteria co-localized with mature M cells within the PPs (Fig. 4). This PP targeting feature of invasin expressing bacteria was concordant with the results of the ileum section imaging using a macroscope (Fig. S2). To further document the potential M-cell targeting specificity of our oral vaccine, we administered invasin-expressing and f88M2e phageinfected bacteria to mice by oral gavage. 3 h later the PPs and ileum samples were isolated, washed and homogenized, and the number of live bacteria present in the homogenates was determined by plating. On average 5360 ( ± 1780) CFUs of BL21pICA2/pLT32h-inv/f88M2e were found in PPs (Fig. 5). Plating of samples retrieved from the PP or ileum homogenates derived from mice that had been inoculated with empty vector (plus f88M2e phages) transformed E. coli cells did not contain viable E. coli cells. An average of 840 ( ± 397) CFUs were found in the PP homogenates derived from mice that had been inoculated with bacteria expressing D911T mutant invasin (Fig. 5). Taken together these results suggest that the binding to β1-integrins of invasin expressed in E. coli remains functional after oral administration of these bacteria, carrying f88M2e phages, to laboratory mice.

Fig. 1. Expression of invasin and M2e by recombinant BL21 E. coli cells. (A) Western blot detection of heterologous Y. pseudotuberculosis invasin expression by recombinant E.coli BL21 cells. 15 μl of crude cell extract from IPTG-induced (+) or uninduced (−) saturated E. coli culture infected with F88-ctr or F88-M2e phages were loaded on a 10% SDS-PAGE and the corresponding Western blot was probed with an anti-invasin immune serum. One lane of the gel was loaded with a cell extract of Y. pseudotuberculosis bacteria, which served as a positive control. (B) M2e(2-16) expression by BL21 cells expressing wild type or D911T mutant invasin that are persistently infected with f88 phages. Lysates of the indicated BL21 transformants were analyzed by Western blot and detected with anti-f88 phage p8 monoclonal antibody (top panel) or anti-M2e mAb for M2e(2-16) expression (lower panel). Lanes labelled with f88-M2e and f88-ctr were loaded with lysates of the corresponding purified phages.

3.2. E. coli expressing invasin from Y. pseudotuberculosis are engulfed by HEp2 cells To investigate the functionality of the heterologous invasin in E. coli, we performed an in vitro bacterial uptake assay using HEp2 cells, which are known to express β1 integrin [38]. HEp2 cells were incubated with BL21pICA2, BL21pICA2/pLT32h-inv or BL21pICA2/pLT32hinvmutD911T bacteria for different time periods, fixed and analyzed by TEM. BL21pICA2/pLT32h-inv bacteria were internalized by HEp2 cells starting 90 min after co-culture and by 150 min of co-incubation a substantial number of bacteria were visible inside the cells (Fig. 2A). In contrast, empty vector and D911T mutant invasin transformed E. coli cells were not or barely internalized by the mammalian cells, respectively (Fig. 2A). Additionally, a bacterial viability assay was used as a readout for uptake of recombinant E. coli cells by HEp2 cells. The principle of this assay is that only bacteria that are internalized by the mammalian cells are protected from a non-cell membrane permeable antibiotic that is added to the mammalian cell culture medium. No viable bacteria were detectable in wash fraction S1 (before the addition of bacteria to the HEp2 cells) (Fig. 2B). We next added 108E.coli cells to

3.4. Oral administration of invasin-expressing E. coli carrying f88M2e2-16 bacteriophage induces M2e specific serum IgG We hypothesized that invasin-expressing bacteria that are persistently infected with a bacteriophage such as f88M2e, could be exploited 59

Journal of Controlled Release 264 (2017) 55–65

L. Deng et al.

Fig. 2. Invasin-expressing bacteria can bind to and become internalized by HEp2 cells. (A) Transmission electron microscopy analysis of binding and invasion of E. coli BL21pICA2, BL21pICA2/pLT32h-inv or BL21pICA2/pLT32h-invmutD911T to HEp2 cells. HEp2 cell culture samples were co-incubated with the indicated E. coli cells and 30, 90 and 150 min later the cells were washed and fixed with TEM fixative and visualized by transmission electron microscopy (Bars represent 10 μm or 20 μm as indicated). Shown is a representative image of 3 images per setting and time point. (B) Bacteria uptake by HEp2 cells assayed by an antibiotic protection protocol. HEp2 cells were allowed to grow into a monolayer and were then washed (S1). Next, the cells were co-cultured with E. coli BL21pICA2, BL21pICA2/pLT32h-inv or BL21pICA2/pLT32h-invmutD911T for 90 min. The cells were washed three times and the third wash was retained (S2). Then gentamycin was added to the cells for 30 min, which kills extracellular bacteria. Cells were washed again 3 times and the third wash (S3) was retained. Finally, cells were lysed with Triton X-100 containing lysis buffer and this cell lysate was retained (S4). Life bacteria in S1, S2, S3 and S4 were determined by plating 20 μl of these samples on an LB agar plate containing 50 μg/ml kanamycin sulfate and counting the number of colonies the next day (CFU = colony forming units). Bars represent the average of 3 replicates ± standard derivation. N.S. indicates that there is no significance between BL21pICA2/pLT32h-inv and BL21pICA2/pLT32h-invmutD911T groups in S2. p < 0.005 indicates the significant difference between BL21pICA2/pLT32h-inv and BL21pICA2/pLT32h-invmutD911T groups in S4 (two-tailed t-test).

60

Journal of Controlled Release 264 (2017) 55–65

L. Deng et al.

Fig. 3. HEp2 cells engulf E. coli cells expressing wild type invasin. (A) confocal immunofluorescence microscopy images of HEp2 cells that have been pre-incubated with E. coli cells transformed with different expression vectors. HEp2 cells were stained with Celltracker (green) and cocultured with PKH26-stained (red) recombinant E. coli BL21pICA2/pLT32h-inv/f88M2e or BL21pICA2/f88M2e, BL21pICA2/pLT32h-invmutD911T/f88M2e (red) for 90 min. Cell nuclei were stained with Hoechst (blue). Bars represent 30 μm in the upper panels and 10 μm in the lower panels. (B) Quantification of the engulfed bacteria by HEp2 cells. The number of live bacteria that were cell-associated after co-incubation and hence protected from extracellular gentamycin was determined as in Fig. 2B. The presence of life bacteria was determined by plating the S4 fraction (cell lysate) on LB agar plates containing 50 μg/ml kanamycin and 25 μg/ml tetracycline and the number of CFU was determined. Horizontal bars represent the average of 3 replicates ± standard deviation (P < 0.005, two-tailed ttest). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

BL21pICA2/pLT32h-inv/f88M2e or BL21pICA2/pLT32h-inv/f88ctr (Fig. 6B). This humoral immune response correlated with a biologically intact invasin since mice that had received bacteria expressing the D911T invasin mutant did not mount serum IgG specific for f88 structural proteins (Fig. 6B). Likewise, serum IgG specific for an invasin-derived peptide was only observed in mice that had received orally administered BL21 bacteria expressing wild type invasin (Fig. 6C).

as an oral vaccination strategy in order to induce an adaptive immune response directed against a heterologous antigen that is displayed by the “hitchhiking” recombinant phage. To evaluate this possibility, E. coli/invasin/f88M2e2-16 bacteria were administered to BALB/c mice by oral gavage. Three weeks after the last of six oral administrations, serum samples were prepared and analyzed for the presence of IgG directed against M2e, f88ctr or invasin. M2e-specific serum IgG titers were found in mice that had received BL21pICA2/pLT32h-inv/f88M2e but not in those that had received bacteria expressing f88ctr phages or D911T mutant invasin (Fig. 6A). From previous work, it is known that approximately 95% of the major coat protein in f88M2e phages is wild type p8 [25]. Wild type p8 is therefore a much more abundant heterologous antigen than M2e2-16 for the mice that had received bacteria infected with f88M2e phages by oral gavage. In line with this, we observed high f88ctr-specific serum IgG levels in mice that had received

3.5. Protection against challenge with influenza A virus of orally vaccinated mice To evaluate if oral vaccination with live invasin-expressing bacteria carrying f88M2e phages would lead to protection against influenza A virus infection, we challenged the mice with a mouse-adapted X47 61

Journal of Controlled Release 264 (2017) 55–65

L. Deng et al.

Fig. 5. Bacteria co-expressing invasin and f88M2e2-16 bacteriophages can invade Peyer's patches in vivo after oral administration. 3 h after oral administration of 109 recombinant E. coli cells expressing f88M2e2-16 together with wild type invasin (BL21/inv/f88M2e), D911T mutant invasin (BL21/invmutD911T/f88M2e) or without invasin (BL21/f88M2e) to BALB/c mice, an ileum loop was excised. The loop was washed with PBS and tissue fragments (0.5 cm length) containing Peyer's patches (PP) or not (ileum) were homogenized in 100 μl PBS. Live kanamycin-resistant bacteria in the tissue homogenates were plated on LB agar plates containing 50 μg/ml kanamycin. Data points are from 5 randomly selected ileum segments. The horizontal bar represents the average and error bars indicate the standard deviation (P < 0.005, between inv/f88M2e group and invmut/ f88M2e group, two-tailed t-test).

(Fig. 6D). Moreover, BL21pICA2/pLT32h-inv/f88M2e immunization significantly reduced lung virus titers at day 6 post infection compared to BL21pICA2/pLT32h-invmutD911T/f88M2e and BL21pICA2/pLT32hinvmutD911T/f88ctr immunization groups (Fig. 6E). Based on this outcome, we conclude that oral immunization of laboratory mice with E. coli cells that express invasin and are infected with f88M2e2-16 phages provides partial protection against body weight loss following H3N2 influenza virus infection. 4. Discussion In this study we developed and characterized a new live vaccine concept that could be used for oral immunization. This concept is based on E. coli cells that are persistently infected with fd phages that display a heterologous antigen of interest on their surface. The E. coli cells in turn express invasin derived from Y. pseudotuberculosis, which is known to facilitate targeting to PPs in the gut. We explored the feasibility and potential of this approach using the conserved influenza A virus M2e epitope. It was previously reported that invasin-expressing bacteria can bind β1 integrins on HEp2 cells or on the apical surface of M cells [16,20,38,40]. We characterized BL21 E. coli cells expressing invasin from Y. pseudotuberculosis for binding to and internalization by HEp2 cells in vitro, and for their propensity to bind to M cells in PPs ex vivo and in vivo. As negative controls, E. coli cells expressing a invasin D911T mutant were used throughout this study. The D911 residue is critical for invasin to bind β1-integrins [36]. Confocal image analysis revealed that PKH26-stained wild type invasin expressing E. coli cells co-localized with GP2-stained cells (Fig. 4). GP2, exclusively expressed on M cells among intestinal epithelial cells, is a marker for functionally mature M cells and serves as a grabbing receptor for a subset of FimH-expressing commensal and pathogenic bacteria [11,41,42]. FimH is a major component of the type I pilus of a group of gram-negative enterobacilli such as pathogenic E. coli and Salmonella enterica. However, we did not observe binding to HEp2 cells or M cells of E. coli BL21 that expressed the invasin D911T mutant or that had been transformed with the empty vector. This is in line with

Fig. 4. Wild type invasin-expressing bacteria can penetrate into mouse Peyer's patches ex vivo. BL21pICA2/pLT32h-inv but not BL21pICA2/pLT32h-invmutD911T bacteria are associated with functionally mature M cells in the vicinity of follicle-associated epithelial cells (FAE). After 90 min incubation with PKH26-stained (red) recombinant BL21pICA2/ pLT32h-inv (left) or BL21pICA2/pLT32h-invmutD911T (right), the excised Peyer's patches from mouse ileum were stained with FITC-UEA (green) and Glycoprotein 2 (GP2, pseudo-colored orange) and then imaged by laser scanning confocal microscope. The bars represent 30 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

virus. We used a sublethal dose of 0.2 LD50 because the observed M2especific serum IgG titers were relatively low. In line with this sublethal challenge dose, all mice experienced transient body weight loss and survived the challenge. Mice that had received BL21pICA2/pLT32h-inv/ f88M2e bacteria by oral gavage experienced statistically significantly less weight loss on days 6, 7 and 8 compared with the control mice 62

Journal of Controlled Release 264 (2017) 55–65

L. Deng et al.

Fig. 6. Oral administration of a recombinant live E. coli invasin-expressing and f88-M2e phage-infected combination induces M2e specific antibody response and is associated with reduced morbidity following challenge with influenza A virus in mice. BALB/c mice (n = 4 per group) received six times 109E. coli cells in 100 μl 5% sodium bicarbonate by oral gavage (two times daily on three consecutive days). 21 days after the last gavage, serum was prepared and assayed for the presence of M2e-, f88- and invasin-specific IgG by ELISA. As a positive control for seroconversion, serum from mice that were immunized by intraperitoneal injection of purified bacteriophage f88M2e2-16 (equivalent of 1010 phages) adjuvanted with incomplete Freund's adjuvant was used. Pooled pre-immune serum was used as general negative control in all groups. (A) End point M2e-specific serum IgG titers deduced from M2e peptide coated wells. “37” is a mouse IgG1 monoclonal specific for M2e and served as a technical control. “Anti-p8 mAb” is a mouse monoclonal antibody directed against the major f88 phage p8 coat protein. (B) Endpoint serum IgG titers specific for f88 structural proteins. Wells of the ELISA plates were coated with purified f88ctr phages. (C) Endpoint serum IgG titers specific for coated invasin peptide 895-910. Serum from mice that had been intranasally-inoculated with 107 recombinant E. coli BL21pICA2/pLT32h-inv/f88M2e was used as a positive control for invasin seroconversion (P < 0.005 between inv/f88M2e group and invmutD911T/f88M2e group in three ELISA assays, t-test). (D) Eight-week-old female BALB/c mice (n = 4 per group) were immunized with 109 recombinant E. coli BL21pICA2/pLT32h-inv/f88M2e (f88M2e/inv), BL21pICA2/pLT32h-inv/f88ctr (f88ctr/inv), BL21pICA2/pLT32h-invmutD911T/ f88M2e (f88M2e/invmut) or BL21pICA2/pLT32h-invmutD911T/f88ctr (f88ctr/invmut) via the oral route. Mice received 6 doses of bacteria within 3 consecutive days. Four weeks after the last gavage all mice were sedated and intranasally infected with 0.2 LD50 of mouse adapted X47 H3N2. Body weights of individual were determined daily for 2 weeks following challenge. The differences in weight loss between the f88M2e2-16/inv group and f88M2e2-16/invmutD911T group on 6, 7 and 8 day post infection are significantly different as indicated by the double-asterisks (**: P < 0.01 between f88M2e2-16/inv group and f88M2e2-16/invmutD911T group, two-way ANOVA). (E) Determination of mouse lung virus titers at day 6 post a sublethal dose infection with X47 (P < 0.05 between inv/f88M2e group and invmutD911T/f88M2e group, P < 0.05 between inv/f88M2e group and invmutD911T/f88ctr group, n.s. indicates no significant difference, t-test).

the reported lack of fimbriae structures on E.coli BL21 cells [43]. Therefore, in the absence of wild type invasin, fimbriae-mediated binding to M cells did not contribute to M cell-binding and the induction of antibody responses after oral vaccination. The main purpose of this study was to evaluate if oral immunization with live E. coli cells that express invasin and are persistently infected with a bacteriophage, could lead to the induction of an immune response against an antigen of interest displayed on the phage. This was indeed the case, as we obtained proof-of-concept using the N-terminal part of the conserved influenza A virus antigen M2e. However, the obtained serum IgG titers against M2e were relatively low (Fig. 6). Nevertheless, we observed partial protection with faster recovery from infection and lower lung virus titers in BL21pICA2/pLT32h-inv/f88M2e

recipient mice following challenge with a sublethal dose of a H3N2 influenza A virus (Fig. 6D). Mucosal immunization typically leads to the induction of IgA responses. However, we did not detect M2e-specific IgA antibodies in serum and stool samples of orally immunized mice (data not shown). This may be due to the relatively low efficiency of the system, e.g. the low abundance of M2e2-16-p8 hybrid proteins compared to wild type p8 in the delivered phages. In addition, it was reported that ovalbumin-expressing E. coli cells can penetrate into PPs after oral immunization by gavage by a process that requires invasin. This immunization strategy resulted in a systemic cellular response against the ovalbumin antigen but mucosal specific immune responses were not reported in this study [20]. Why are the seroconversion rates against M2e (and the major coat

63

Journal of Controlled Release 264 (2017) 55–65

L. Deng et al.

Appendix A. Supplementary data

proteins of the phages) after oral administration of the bacteria-invasinf88M2e2-16 phage combination relatively low? First, the truncated M2e2-16 may be too short to also induce an M2e-specific T helper response [44,45]. Despite this, we previously showed that immunization of mice with purified f88M2e2-16 phages results in robust M2e-specific serum IgG responses [25]. Presumably, T helper epitopes in p8 can contribute to the induction of an efficient B-cell response against M2e that is part of the M2e2-16-p8 fusion. Removal of the membrane-anchored invasin proteins from the E. coli surface by proteases in the gut lumen may also be a limiting factor [46]. The harsh conditions in the gastro-intestinal tract may also explain the relatively low invasion efficacy of BL21pICA2/pLT32h-inv/f88M2e into PP in vivo. Only a low number of live bacteria could be extracted from PP homogenates even though the mice had received 6 doses of 109E. coli cells by gavage (Fig. 5). Based on these recovery data, this means that approximately only 1 out of 105 bacteria penetrated into PP of the ileum (approximately 5000 bacteria per PP and assuming approximately 10 PP/ ileum). Possibly, commensal bacteria represent a physical barrier. In the future, experiments with antibiotic treated mice could be performed to address this surmised interference by gut commensals. To investigate the distribution of E. coli in the gut after oral administration, we tried to track the E. coli strains that had been labelled with SRfluor680® NHS Ester using in vivo imaging. Such fluorescent labelling of the E. coli cells did not significantly reduce their viability and successful labelling of bacteria was determined by confocal imaging (Fig. S3). The in vivo imaging analysis demonstrated that the majority of intragastrically administered bacteria had passed through the whole mouse digestive tract within 3 h following a single gavage (Fig. S4). Therefore, the luminal residing time of orally administered bacteria may be too short to allow a large amount of invasin-expressing bacteria to adhere to and be transcytosed by M cells. There are possibilities to potentially improve the vaccine immunogenicity. The recombinant f88M2e2-16 was viable and grew to high titers 1011/ml in E.coli culture, but incorporated a relatively low ratio of M2e2-16-p8, 5% of total p8 subunits. Adding a short flexible linker between M2e and the N-terminus of p8 may enhance the peptide display rate while avoiding malfolding of the recombinant p8. The hypothesis is that E.coli strain used is highly sensitive to acid in stomach. This could be overcome by selecting E.coli strains that are more resistant. Using tablet formulations is a possibility although this would likely increase the cost of the oral vaccine. In summary, we have described a new, tri-partite live oral vaccination strategy that is very economical and could ultimately be implemented in mass vaccination campaigns.

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2017.08.020. References [1] A.O. Kolawole, M.B. Gonzalez-Hernandez, H. Turula, C. Yu, M.D. Elftman, C.E. Wobus, Oral norovirus infection is blocked in mice lacking Peyer's patches and mature M cells, J. Virol. 90 (2016) 1499–1506. [2] S.D. Trask, S.M. McDonald, J.T. Patton, Structural insights into the coupling of virion assembly and rotavirus replication, Nat. Rev. Microbiol. 10 (2012) 165–177. [3] Y. Shu, C.K. Li, Z. Li, R. Gao, Q. Liang, Y. Zhang, L. Dong, J. Zhou, J. Dong, D. Wang, L. Wen, M. Wang, T. Bai, D. Li, X. Dong, H. Yu, W. Yang, Y. Wang, Z. Feng, A.J. McMichael, X.N. Xu, Avian influenza A(H5N1) viruses can directly infect and replicate in human gut tissues, J. Infect. Dis. 201 (2010) 1173–1177. [4] J.A. Belser, K.M. Gustin, T.R. Maines, M.J. Pantin-Jackwood, J.M. Katz, T.M. Tumpey, Influenza virus respiratory infection and transmission following ocular inoculation in ferrets, PLoS Pathog. 8 (2012) e1002569. [5] T.R. Hird, N.C. Grassly, Systematic review of mucosal immunity induced by oral and inactivated poliovirus vaccines against virus shedding following oral poliovirus challenge, PLoS Pathog. 8 (2012) e1002599. [6] I. Isakova-Sivak, L.M. Chen, Y. Matsuoka, J.T. Voeten, I. Kiseleva, J.G. Heldens, H. den Bosch, A. Klimov, L. Rudenko, N.J. Cox, R.O. Donis, Genetic bases of the temperature-sensitive phenotype of a master donor virus used in live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2), Virology 412 (2011) 297–305. [7] J. Holmgren, C. Czerkinsky, Mucosal immunity and vaccines, Nat. Med. 11 (2005) S45–53. [8] N.A. Mabbott, D.S. Donaldson, H. Ohno, I.R. Williams, A. Mahajan, Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium, Mucosal Immunol. 6 (2013) 666–677. [9] W. de Lau, P. Kujala, K. Schneeberger, S. Middendorp, V.S. Li, N. Barker, A. Martens, F. Hofhuis, R.P. DeKoter, P.J. Peters, E. Nieuwenhuis, H. Clevers, Peyer's patch M cells derived from Lgr5(+) stem cells require SpiB and are induced by RankL in cultured "miniguts", Mol. Cell. Biol. 32 (2012) 3639–3647. [10] H.J. Snippert, L.G. van der Flier, T. Sato, J.H. van Es, M. van den Born, C. KroonVeenboer, N. Barker, A.M. Klein, J. van Rheenen, B.D. Simons, H. Clevers, Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells, Cell 143 (2010) 134–144. [11] K. Hase, K. Kawano, T. Nochi, G.S. Pontes, S. Fukuda, M. Ebisawa, K. Kadokura, T. Tobe, Y. Fujimura, S. Kawano, A. Yabashi, S. Waguri, G. Nakato, S. Kimura, T. Murakami, M. Iimura, K. Hamura, S. Fukuoka, A.W. Lowe, K. Itoh, H. Kiyono, H. Ohno, Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response, Nature 462 (2009) 226–230. [12] S. Sato, S. Kaneto, N. Shibata, Y. Takahashi, H. Okura, Y. Yuki, J. Kunisawa, H. Kiyono, Transcription factor Spi-B-dependent and -independent pathways for the development of Peyer's patch M cells, Mucosal Immunol. 6 (2013) 838–846. [13] P. Verbrugghe, W. Waelput, B. Dieriks, A. Waeytens, J. Vandesompele, C.A. Cuvelier, Murine M cells express annexin V specifically, J. Pathol. 209 (2006) 240–249. [14] S.H. Kim, D.I. Jung, I.Y. Yang, J. Kim, K.Y. Lee, T. Nochi, H. Kiyono, Y.S. Jang, M cells expressing the complement C5a receptor are efficient targets for mucosal vaccine delivery, Eur. J. Immunol. 41 (2011) 3219–3229. [15] V.R. Nair, L.H. Franco, V.M. Zacharia, H.S. Khan, C.E. Stamm, W. You, D.K. Marciano, H. Yagita, B. Levine, M.U. Shiloh, Microfold cells actively translocate Mycobacterium tuberculosis to initiate infection, Cell Rep. 16 (2016) 1253–1258. [16] R.R. Isberg, D.L. Voorhis, S. Falkow, Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells, Cell 50 (1987) 769–778. [17] J.W. Fairman, N. Dautin, D. Wojtowicz, W. Liu, N. Noinaj, T.J. Barnard, E. Udho, T.M. Przytycka, V. Cherezov, S.K. Buchanan, Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis, Structure 20 (2012) 1233–1243. [18] M.S. Francis, The pathogenic Yersiniae—advances in the understanding of physiology and virulence, Front. Cell. Infect. Microbiol. 3 (2013) 51. [19] R.R. Isberg, P. Barnes, Subversion of integrins by enteropathogenic Yersinia, J. Cell Sci. 114 (2001) 21–28. [20] R.J. Critchley-Thorne, A.J. Stagg, G. Vassaux, Recombinant Escherichia coli expressing invasin targets the Peyer's patches: the basis for a bacterial formulation for oral vaccination, Mol. Ther. 14 (2006) 183–191. [21] L. Deng, K.J. Cho, W. Fiers, X. Saelens, M2e-based universal influenza A vaccines, Vaccine 3 (2015) 105–136. [22] M.C. Kim, J.S. Lee, Y.M. Kwon, E. O, Y.J. Lee, J.G. Choi, B.Z. Wang, R.W. Compans, S.M. Kang, Multiple heterologous M2 extracellular domains presented on virus-like particles confer broader and stronger M2 immunity than live influenza A virus infection, Antivir. Res. 99 (2013) 328–335. [23] S. Neirynck, T. Deroo, X. Saelens, P. Vanlandschoot, W.M. Jou, W. Fiers, A universal influenza A vaccine based on the extracellular domain of the M2 protein, Nat. Med. 5 (1999) 1157–1163. [24] K. El Bakkouri, F. Descamps, M. De Filette, A. Smet, E. Festjens, A. Birkett, N. Van Rooijen, S. Verbeek, W. Fiers, X. Saelens, Universal vaccine based on ectodomain of matrix protein 2 of influenza A: Fc receptors and alveolar macrophages mediate protection, J. Immunol. 186 (2011) 1022–1031.

Conflict of interest Walter Fiers holds patent rights on the use of M2e-based influenza vaccines.

Acknowledgments We thank Lars Vereecke and Chris Guerin from the Inflammation Research Center, VIB (Gent, Belgium) for providing technical support and scientific advice. Lei Deng was supported by State Scholarship Fund (File No. 2011674067) from the China Scholarship Council and by IUAP-BELVIR p7/45.

Author contribution Conceived and designed the experiments: LD KR RDR NS WF XS. Performed the experiments: LD RDR EVH AG EP LC. Analyzed the data: LD KR RDR LC NS WF XS. Wrote the paper: LD, ERJ and XS. 64

Journal of Controlled Release 264 (2017) 55–65

L. Deng et al.

[36] J.M. Leong, P.E. Morrissey, A. Marra, R.R. Isberg, An aspartate residue of the Yersinia pseudotuberculosis invasin protein that is critical for integrin binding, EMBO J. 14 (1995) 422–431. [37] M.G. Panthani, T.A. Khan, D.K. Reid, D.J. Hellebusch, M.R. Rasch, J.A. Maynard, B.A. Korgel, In vivo whole animal fluorescence imaging of a microparticle-based oral vaccine containing (CuInSe(x)S(2 − x))/ZnS core/shell quantum dots, Nano Lett. 13 (2013) 4294–4298. [38] R.R. Isberg, J.M. Leong, Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells, Cell 60 (1990) 861–871. [39] J.D. Rouch, A. Scott, N.Y. Lei, R.S. Solorzano-Vargas, J. Wang, E.M. Hanson, M. Kobayashi, M. Lewis, M.G. Stelzner, J.C. Dunn, L. Eckmann, M.G. Martin, Development of functional microfold (M) cells from intestinal stem cells in primary human Enteroids, PLoS One 11 (2016) e0148216. [40] P. Dersch, R.R. Isberg, An immunoglobulin superfamily-like domain unique to the Yersinia pseudotuberculosis invasin protein is required for stimulation of bacterial uptake via integrin receptors, Infect. Immun. 68 (2000) 2930–2938. [41] H. Ohno, K. Hase, Glycoprotein 2 (GP2): grabbing the FimH bacteria into M cells for mucosal immunity, Gut Microbes 1 (2010) 407–410. [42] S. Kimura, M. Yamakami-Kimura, Y. Obata, K. Hase, H. Kitamura, H. Ohno, T. Iwanaga, Visualization of the entire differentiation process of murine M cells: suppression of their maturation in cecal patches, Mucosal Immunol. 8 (2015) 650–660. [43] H. Chart, H.R. Smith, R.M. La Ragione, M.J. Woodward, An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5alpha and EQ1, J. Appl. Microbiol. 89 (2000) 1048–1058. [44] D. Pejoski, W. Zeng, S. Rockman, L.E. Brown, D.C. Jackson, A lipopeptide based on the M2 and HA proteins of influenza A viruses induces protective antibody, Immunol. Cell Biol. 88 (2010) 605–611. [45] D.G. Eliasson, K. El Bakkouri, K. Schon, A. Ramne, E. Festjens, B. Lowenadler, W. Fiers, X. Saelens, N. Lycke, CTA1-M2e-DD: a novel mucosal adjuvant targeted influenza vaccine, Vaccine 26 (2008) 1243–1252. [46] J. Trcek, M.F. Oellerich, K. Niedung, F. Ebel, S. Freund, K. Trulzsch, Gut proteases target Yersinia invasin in vivo, BMC Res. Notes 4 (2011) 129.

[25] L. Deng, L.I. Ibanez, V. Van den Bossche, K. Roose, S.A. Youssef, A. de Bruin, W. Fiers, X. Saelens, Protection against influenza a virus challenge with M2e-displaying filamentous Escherichia coli phages, PLoS One 10 (2015) e0126650. [26] M. De Filette, W. Martens, K. Roose, T. Deroo, F. Vervalle, M. Bentahir, J. Vandekerckhove, W. Fiers, X. Saelens, An influenza A vaccine based on tetrameric ectodomain of matrix protein 2, J. Biol. Chem. 283 (2008) 11382–11387. [27] M. Nico, August, Corneel M., R. Erik, R., C. Walter, F., Regulatory system for inducible expression of genes with lambdoid promoters, in, 1998. [28] F. Osorio, S.J. Tavernier, E. Hoffmann, Y. Saeys, L. Martens, J. Vetters, I. Delrue, R. De Rycke, E. Parthoens, P. Pouliot, T. Iwawaki, S. Janssens, B.N. Lambrecht, The unfolded-protein-response sensor IRE-1alpha regulates the function of CD8alpha + dendritic cells, Nat. Immunol. 15 (2014) 248–257. [29] E. Wirawan, L. Vande Walle, K. Kersse, S. Cornelis, S. Claerhout, I. Vanoverberghe, R. Roelandt, R. De Rycke, J. Verspurten, W. Declercq, P. Agostinis, T. Vanden Berghe, S. Lippens, P. Vandenabeele, Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria, Cell Death Dis. 1 (2010) e18. [30] M.H. Jang, M.N. Kweon, K. Iwatani, M. Yamamoto, K. Terahara, C. Sasakawa, T. Suzuki, T. Nochi, Y. Yokota, P.D. Rennert, T. Hiroi, H. Tamagawa, H. Iijima, J. Kunisawa, Y. Yuki, H. Kiyono, Intestinal villous M cells: an antigen entry site in the mucosal epithelium, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 6110–6115. [31] L.G. Baum, J.C. Paulson, Sialyloligosaccharides of the respiratory epithelium in the selection of human influenza virus receptor specificity, Acta Histochemica Supplementband 40 (1990) 35–38. [32] J.R. Johnson, N. Fu, E. Arunkumar, W.M. Leevy, S.T. Gammon, D. Piwnica-Worms, B.D. Smith, Squaraine rotaxanes: superior substitutes for Cy-5 in molecular probes for near-infrared fluorescence cell imaging, Angew. Chem. Int. Ed. Engl. 46 (2007) 5528–5531. [33] T. Costa, A.J. Chaves, R. Valle, A. Darji, D. van Riel, T. Kuiken, N. Majo, A. Ramis, Distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species, Vet. Res. 43 (2012) 28. [34] J.J. Skehel, D.C. Wiley, Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin, Annu. Rev. Biochem. 69 (2000) 531–569. [35] L.J. Reed, H. Muench, A simple method of estimating fifty per cent endpoints, Am. J. Epidemiol. 27 (1938) 493–497.

65