Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0216—Bacteriodetes, e.g. Bacteroides, Ornithobacter, Porphyromonas
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55588—Adjuvants of undefined constitution
  • A61K2039/55594—Adjuvants of undefined constitution from bacteria

Definitions

• the invention to which this application relates is the provision of commensal bacteria-derived outer membrane vesicles (OMVs) in a pharmaceutical or pharmacological preparation.
• OMVs are native and/or suitable for delivery of biologies, in particular delivery to the gastrointestinal and respiratory tract in mammals and humans.
• the production of vesicles derived from the outer membrane of commensal bacteria using recombinant bacteria is disclosed in the applicant’s co-pending application PCT/GB201 7/051 1 99 herein incorporated by reference.
• OMVs have been associated with pathogenesis and the storage and transportation of virulence factors produced by enteric Gram-negative pathogens including Helicobacter pylori (VacA), Shigella dysenteriae (Shiga toxin) and enterohemorrhagic Escherichia coli (ClyA) .
• enteric Gram-negative pathogens including Helicobacter pylori (VacA), Shigella dysenteriae (Shiga toxin) and enterohemorrhagic Escherichia coli (ClyA) .
• This paradigm for OMV function has been questioned due to new evidence demonstrating a non-pathogenic, mutualistic role for the OMVs produced by commensal gut bacteria.
• Members of the genus Bacteroides exclusively package carbohydrate and protein hydrolases in OMVs that perform a ‘social function’ by providing substrates for utilization by other bacteria and contributing to microbiota homeostasis.
• OMVs can contain adhesins, sulfatases and proteases which facilitate their interaction with host epithelial cells, allowing them to enter these cells through numerous routes, including micropinocytosis, lipid raft- and clathrin-dependent endocytosis.
• OMVs containing polysaccharide A are detected by dendritic cells via Toll Like Receptor (TLR) 2 leading to enhanced T regulatory cell activity and production of anti-inflammatory cytokines (IL-10) that protect the host from experimental colitis.
• TLR Toll Like Receptor
• IL-10 anti-inflammatory cytokines
• IEC mammalian intestinal epithelial cell
• Minpp is a homologue of a mammalian inositol phosphate polyphosphatase cell-signalling enzyme.
• a pharmaceutical preparation including OMVs and at least one pharmaceutically acceptable carrier wherein said OMVs are native OMVs produced by the Gram-negative commensal bacteria Bacteroides.
• the pharmaceutically acceptable carrier is phosphate-buffered saline (PBS) .
• the pharmaceutical preparation is to prime, activate and/or boost immune responses to prevent infection, tissue inflammation, tumor growth and/or other injury.
• OMVs include and/or deliver biologically active peptides, proteins and/or biologies.
• said pharmaceutical preparation and/or said OMVs are for delivery of said peptides, proteins or biologies to the gastrointestinal (GI) and/or respiratory tract.
• the preparation is to prevent infection, tumour growth, tissue inflammation and/or injury to the gastrointestinal (GI) and/or respiratory tract and other organ systems.
• the native OMVs are delivered to one or more mucosal sites to induce, activate or boost an immune response.
• the pharmaceutical preparation includes at least one pharmaceutically active component.
• the at least one pharmaceutically active component includes a vaccine agent such as antigens and/or pathogen components .
• the OMVs reduce the number of doses and/or amount of vaccine agent or component required to elicit a protective immune response. Further typically the OMV is an adjuvant.
• an outer membrane vesicle for use as an adjuvant.
• OMV adjuvant is delivered before a vaccine or one or more vaccine agents .
• the OMV adjuvant may be delivered at substantially the same time or after the one or more vaccine agents.
• the OMV is a native OMVs produced by the Gram-negative commensal bacteria Bacteroides.
• the peptide or protein is within or on the outer membrane of OMVs.
• Species of Bacteroides include B. acidifaciens, B. caccae, B. coprocola, B. coprosuis, B. eggerthii, B. finegoldii, B. fragilis, B. helcogenes, B. intestinalis, B. massiliensis, B. nordii, B. ovatus, B. stercoris, B. thetaiotaomicron, B. vulgatus, B. plebeius, B. uniformis, B. salyersai, B. pyogenes, B. goldsteinii, B. dorei and B. johnsonii and B. xylanisolvens.
• the related Parabacteroides genera includes P.
• mannan-inducible expression system in accordance with the invention may be used in any Bacteroides.
• B. thetaiotaomicron (VPI- 5482), and strains thereof, including GH193, GH359 and GH364, for example.
• the pharmaceutical preparation includes Salmonella Typhimurium-deive vaccine antigens.
• the OMV is an adjuvant of Salmonella Typhimurium- derived vaccine antigens.
• the pharmaceutical preparation includes influenza A virus (IAV) derived vaccine antigens.
• the OMV is an adjuvant of influenza A virus (IAV) derived vaccine antigens.
• IAV influenza A virus
• these antigens are in a form capable of eliciting antigen- specific immune and antibody responses in mucosal tissues and/or systemically.
• immunisation with OMVs containing the core stalk region of the IAV H5 hemagglutinin from an H5N 1 strain induces heterotypic protection.
• the heterotypic protection includes an unrelated subtype HI N l of IAV.
• the pharmaceutical preparation includes OMVs containing plague derived antigens.
• the heterologous peptide, protein or biologic includes the human therapeutic protein keratinocyte growth factor-2 (KGF-2) .
• KGF-2 OMV reduces disease severity and promoted intestinal epithelial repair and recovery from colitis.
• a composition including a native outer membrane vesicle (OMV) for use in a method for the treatment of typhoid wherein said OMV is produced by a native Gram-negative commensal bacteria.
• OMV outer membrane vesicle
• the native bacteria is Bacteroides.
• the native OMVs are from Bacteroides thetaiotaomicron (Bt) .
• composition including an outer membrane vesicle (OMV) for use in a method for the treatment of influenza wherein said OMV is produced by the a native Gram-negative commensal bacteria.
• OMV outer membrane vesicle
• the native bacteria is Bacteroides.
• the native OMVs are from Bacteroides thetaiotaomicron (Bt) .
• composition comprising an outer membrane vesicle (OMV) said OMV for use in a method for the treatment of colitis wherein said OMV is produced by a native Gram-negative commensal bacteria.
• OMV outer membrane vesicle
• the native bacteria are Bacteroides.
• the native OMVs are from B. thetaiotaomicron (Bt)
• the human therapeutic protein is a keratinocyte growth factor.
• the native OMVs are delivered intranasally.
• Bt includes the Bt derivative strains below in Table 1 .
• OMVs typically administration of OMVs reduces the size of established cancers or tumours . Further typically the tumours are breast cancer tumours.
• the OMV anti-tumour effect is dependent on dose.
• the optimal dose range is between substantially 10 7 and 10 9 . Further typically substantially below or above this range, OMVs are ineffective.
• the native OMVs are Bacteroides OMVs.
• a method for producing OMVs including the step of growing a bacterial cells and filtering the same.
• the method further includes any one or any combination of; centrifugation, filter sterilisation, ultrafiltration and/or size exclusion chromatography.
• OMVs were isolated following a method adapted from Stentz et ah , [Stentz et al. 201 5] .
• Cultures of Bt 500 mL were grown under anaerobic conditions at 37°C in an anaerobic cabinet.
• Bacterial starter- cultures were grown overnight in 20 ml ‘Brain Heart Infusion’ (BHI) medium (Oxoid) supplemented with 15 mM haemin (Sigma-Aldrich) (BHIH) .
• BHI Brain Heart Infusion
• 0.5 ml of the starter-culture was used to inoculate 500 ml BHI supplemented with 0.75 mM haemin and the culture was constantly homogeneised using a magnetic stir plate.
• Cells were harvested after 16 h at an approximate OD600 nm of 4.0, which corresponds to early stationary phase.
• the cells were centrifuged at 5500 g for 45 min at 4°C and the supernatants filtered through polyethersulfone (PES) membranes (0.22 pm pore-size) (Sartorius) to remove debris and cells .
• PES polyethersulfone
• Supernatants were concentrated by ultrafiltration (100 kDa molecular weight cut-off, Vivaspin 50R, Sartorius), the retentate was rinsed once with 500 mL of PBS (pH 7.4) and concentrated to 0.5 mL.
• Size distribution of vesicles was performed on 1 ml of OMV suspensions diluted 100 times with PBS. Videos were generated using a Nanosight nanoparticle instrument (NanoSight Ltd) to count the number of OMVs in each sample. A 1 -min AVI file was recorded and analysed using NTA (Version 2.3 Build 001 1 RC, Nanosight) software to calculate size distributions and vesicle concentrations using the following settings: calibration: 166 nm/pixel; blur: auto; detection threshold: 10, minimum track length: auto, temperature: 21.9C, viscosity: 0.96 cP. The accuracy of the measurement was confirmed using 100 nm silver nanoparticles (Sigma- Aldrich) . Healthy donors and patients
• PBMCs Peripheral blood mononuclear cells
• PBMC peripheral blood mononuclear cells
• PBMC peripheral blood mononuclear cells
• PBMC peripheral blood mononuclear cells
• FIG. 1 Appearance, size, structure and stability of Bt OMVs.
• EM Electron microscopy
• OMV D 0 Thermostability of OMVs at day 0 (OMV D 0 ) and after storage of OMV suspensions at 4°C or 40°C for 30 days as measured using immunoblotting to detect OmpA in extracts of naive OMVs (OMV) or OMVs of ompA deletion mutants (OMV-), and of neat (1) or ten-times concentrated (10) OMV storage buffer (SB) (PBS was the storage buffer) .
• FIG. 2 Expression of heterologous proteins in Bt OMVs.
• SP BtompA Schematic of cloning procedure for the export of proteins of interest into the lumen or at the surface membrane of OMVs. The secretion peptide of Bt OmpA (SP BtompA) is indicated in yellow and fused at the N-terminus of the gene of interest
• PK proteinase K
• NT not treated
• PK + Proteinase K
• B PK buffer alone.
• FIG. 3 Intrinsic adjuvanticity of Bt OMVs.
• B220 CD45R + B cells
• CD3 + T cells CD3 + T cells
• Iba-1 macrophages/dendritic cells
• Red arrows define nasal-associated lympoid tissue (NALT), bronchus-associated lymphoid tissue (BALT) and fat- associated lymphoid tissue (FALC) in a, b and c respectively.
• NALT nasal-associated lympoid tissue
• BALT bronchus-associated lymphoid tissue
• FALC fat- associated lymphoid tissue
• FIG. 4 Bt OMV-elicited systemic and mucosal antibody responses
• Bt OMVs expres sing the Salmonella OmpA or SseB proteins via the oral (OG), intranasal (IN) or intraperitoneal (IP) routes according to the dosing regimen described in the Material and Methods section.
• Arrows indicate time of immunization.
• necropsy serum (b) and brochoalveolar lavage fluid (BAL) (c, d) were analysed for IAV IgG and IgA antibodies by ELISA using UV-inactivated PR8 virus. BAL samples were also analysed for F15 HA specific IgA antibodies (e) using recombinant H5 HA as the target antigen. Immune serum and BAL from PR8 IAV-infected mice (PR8) were used as reference samples . (f) The weight of individual animals in each group was assessed daily. (g) Lung homogenates were assessed for viral load (PFU/g lung tissue) at necropsy.
• FIG. 6 OMVs containing KGF-2 ameliorate DSS-induced colitis in mice.
• DAI Disease Activity Index
• Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison tests. Mice gavaged with PBS and receiving regular drinking water were considered as the reference group for statistical analysis. ns, not significant; *P ⁇ 0.05; **P ⁇ 0.01 ; ***P ⁇ 0.001.
• FIG. 7 OMVs containing KGF-2 protect and restore goblet cells in mice with DSS-induced colitis
• a Histological score of colon tis sue as determined by microscopy of H&E stained sections obtained at necropsy
• b Number of Alcian Blue stained goblet cells per mm 2 of epithelial area.
• Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison tests . *P ⁇ 0.05.
• DAI Disease Activity Index
• FIG. 9 Stimulation of dendritic cells by OMVs .
• OMV outer membrane vesicles
• PBMC peripheral blood mononuclear cells
• Figure 9A and B As a positive control, PBMC were exposed to heat-killed Bt.
• the amounts of IL-6 and IL-10 in total supernatants of the PBMCs were measured ( Figure 9A and B) .
• FIG. 10 Acquisition of fluorescent labelled Bt OMVs by dendritic cells and subsequent trafficking to lymph nodes after intranasal adminstration.
• OMVs were fluorescently-labelled with DiO and administered intranasally to mice.
• NALT nasal-associated lymphoid tissue
• CLN cervical lymph nodes
• MN mediastinal lymph nodes
• FIG 11 The impact of orally administered Bt OMVs on the recipients’ intestinal microbiota.
• results are expressed in the logarithm of the CFU normalized to the weight of individual faecal samples for each day and growth medium. Data are expressed as means ⁇ SD. Statistically significant differences were evaluated using a Dunnett bilateral post- hoc test to compare days after OMV administration vs. the control day 0. *P ⁇ 0.05; **P ⁇ 0.01.
• FIG 12 Colonization of OMV-StOmpA immunised mice after Salmonella challenge.
• Mice (n 5-6/grp) immunised with either naive Bt OMVs or Bt StOmpA-OMVs via the oral or parental (intraperitoneal; IP.) route (see Materials and Methods for immunisation protocol) were subsequently challenged with an oral dose of 10 8 CFU Salmonella enterica ser. Typhimurium SL1344.
• mice Five days later animals were euthanised and the bacterial load in the ileum and colonic contents; homogenates of mesenteric lymph nodes (MLN); ileum tissue; spleen; and liver were determined by plating serial dilutions onto xylose lysine deoxycholate agar plates supplemented with 50 mg/ml streptomycin.
• the boxplots identify the mean and upper and lower quartile values for data sets obtained from animals within each treatment group.
• Figure 13 Evaluating the biological activity of KGF-2 contained in Bt OMVs using an epithelial wound-healing assay.
• Bt OMVs have inherent adjuvanticity
• mice were administered a single dose of native OMVs in PBS via the intranasal route and 5 days later head and thoracic organs were removed en bloc and analysed by immunohistology for the presence of organised lymphoid structures and follicles indicative of an active immune response. Large organised lymphoid follicles were present in both the nasal cavity (nasal-associated lymphoid tissue or NALT (Fig.
• Fig. 3a and the lungs (bronchus-associated lymphoid tissue or BALT) (Fig. 3b) which contained dendritic cells, T cells and large numbers of B cells. These structures were absent in mice administered PBS alone (Fig. 3a, b) .
• OMVs native were also effective at eliciting the formation of lymphoid clusters in mediastinal adipose tissue (fat- associated lymphoid clusters or FALC) (Fig. 3c) .
• Intranasal OMV form ulation protect against pulmonary IA V infection During infection the weight of all infected animals declined with the greatest weight los s seen in the control (PBS administered) animals that lost almost 20% of their body weight (Fig. 5f) . Animals immunised with H5F-OMVs displayed a more gradual decline in weight los s after infection, as did those immunized with native OMVs.
• Intranasally-administered OMVs increased global IgA levels in in both the salivary glands (Fig. 4d) and BAL (Fig. 4e), which is consistent with the adjuvant properties of Bt OMVs and their ability to activate the immune system in both of these sites and generate organised lymphoid follicles and tissues containing large numbers of B cells (Fig. 3) .
• Bt and its derivative strains are grown under anaerobic conditions at 37°C in ‘Brain Heart Infusion’ (BHI) medium (Oxoid) supplemented with 1 5 mM haemin (Sigma-Aldrich) (BHIH) for bacterial pre-culture or 0.75 mM haemin for OMV preparations.
• BHI Brain Heart Infusion
• Antibiotic- resistance markers in Bt were selected using erythromycin (5 mg/ml) and tetracycline (1 mg/ml) .
• Escherichia coli strains were grown in Luria-Bertani (LB) medium at 37 °C with ampicillin 100 mg/ml (or 200 mg/ml trimethoprim for strain J 53 [pR751]) .
• Lactococcus lactis strain UKLc 10 and its derivative strains were grown in Ml 7 medium (Oxoid) supplemented with 5 g/1 glucose at 30°C.
• Antibiotics were added as selection agents when appropriate: ampicillin 200 mg/ml, erythromycin 5 mg/ml and chloramphenicol 10 mg/ml.
• the E. coli strain J 53 /R751 was supplemented with trimethoprim 200 mg/ml when grown for 1 8 h.
• a 101 8 bp chromosomal DNA fragment upstream from BT_3852 and including the first 1 8 nucleotides of its 5'-end region was amplified by PCR using the primer pair f-5'ompA_SpHI , r-5'ompA_SalI. This product was then cloned into the SpHI/Sall sites of the E. coli- Bacteroides suicide shuttle vector pGH014 [20] .
• the resulting plasmid containing the DBT_3852: :tetQ construct was mobilized from E. coli strain GC10 into Bt by triparental filter mating [1 9] , using E. coli E1B 101 (pRK2013) as the helper strain.
• Transconjugants were selected on BElI-haemin agar containing gentamicin (200 mg/L) and tetracycline (1 mg/L) . Determination of susceptibility to either tetracycline or erythromycin was done to identify recombinants that were tetracycline resistant and erythromycin susceptible after re-streaking transconjuguant bacteria on LB-agar containing tetracycline or both antibiotics. PCR analysis and sequencing were used to confirm allelic exchange. A transconjugant, GE1290, containing the DBT_3852: :tetQ construct inserted into the Bt chromosome was selected for further studies. Generation of recombinant Bt strains
• Bt Salmonella OmpA/SseB The Bacteroides expression vector pGH090 [21] was first digested with Ndel to remove this site by Klenow treatment and to create a blunt-ended fragment that was then religated.
• Signal peptide prediction was obtained by SignalP (http: / /www. cbs. dtu. dk/ services/SignalP/) . After annealing of the oligonucleotides the resulting double-strand DNA contained EcoRI and SpHI 5' overhangs at each end. This linker was cloned into the EcoRI/SpHI sites of the Ndel deleted version of pGH090, resulting in the pGH202 plasmid. The 1 131 bp Salmonella ompA (without signal peptide) and the 591 bp sseB coding region were amplified by PCR from S.
• the resulting fragments were digested with Ndel and EcoRI and cloned into Ndel/EcoRI-digested pGE1202, yielding plasmids pGE11 82 and pGE11 83, respectively.
• the latter plasmid was then trans formed into E. coli-competent cells (GC 10) by electroporation using a Gene Pulser II (Bio-Rad) . Success ful cloning was confirmed by sequencing.
• the plasmid was mobilized from E. coli to Bt using a triparental mating procedure [19] , together with E. coli J 53 (pR751) ; the correct structure of the Bt carrying pGH182 (GE1484) was confirmed by sequencing.
• Bt IAV A 635bp synthetic gene construct encoding a synthetic influenza (H5F; from IAV strain H5N 1 [VN/04:A/VietNam/ 1203/04]) pre-fusion headless HA mini-stem N-terminally fused to the OmpA signal peptide of Bt was created in silico and its codon usage was optimised for expression in the same species.
• the resulting gene cassette was obtained by gene synthesis and subsequently cloned into the E. coli plasmid pEX-K1 68 (Eurofins) .
• the cassette contained BspHI and EcoRI restriction sites at its 5' and 3' ends, respectively, allowing for the translational fusion of the gene to the start codon in the Bacteroides expression vector pGH090 [21] .
• the gene was excised from pEX-K168 using BspHI and EcoRI and ligated into the NcoI/EcoRI-restricted pGH090 expression vector, resulting in pGH184. Finally the sequence integrity of the cloned fragment was confirmed by sequencing.
• the plasmid was mobilized from E. coli into Bt through a triparental mating procedure.
• Bt KGF-2 A 581 bp synthetic gene construct encoding the human fibroblast growth factor-10/keratinocyte growth factor-2 (KGF-2) N- terminally fused to the OmpA signal peptide of Bt was created in silico and its codon usage was optimised for expression in the same species.
• the resulting gene cas sette was obtained by gene synthesis and subsequently cloned into the E. coli plasmid pEX-A2 (Eurofins) as described for the IAV constructs.
• the cas sette contained BspHI and EcoRI restriction sites at its 5' and 3' ends, respectively, allowing for the translational fusion of the gene to the start codon in the Bacteroides expres sion vector pGH 0902.
• the gene was excised from pEX-A2 using Eco53KI and EcoRI and ligated into pUK200 [22] , which had been restricted with Smal and EcoRI, resulting in plasmid pUK200_KGF-2.
• the KGF-2 cas sette was excised from pUK200_KGF-2 through restriction with BspITI and EcoRI and subsequently ligated into the NcoI/EcoRI-restricted pGH090 expression vector, resulting in pGH173.
• sequence integrity of the cloned fragment was confirmed by sequencing.
• the plasmid was mobilized from E. coli into Bt using a triparental mating procedure.
• StOmpA was cloned into His 6-tag expression vector pET- 1 5b (Novagen) . Briefly, PCR fragments incorporating the coding sequences of ompA and s seB genes were cloned into the Ndel/Xhol restriction sites of pET-1 5b and the resulting plasmids pGH1 65 and pGH201 transformed into Rosetta2 (DE3) pLysS cells (Table 1) . Cultures of the resulting strains were induced at an of OD600 nm of 0.6 by adding I mM IPTG for 5 h after which time cells were harvested by centrifugation (5500 g for 20 min) . The pellet was stored at -20°C for future use. StOmpA and StSseB proteins were purified under native conditions using protocols adapted from the QIAexpres s Ni-NTA Fast Start Handbook (Qiagen) with the amount of protein recovered determined using the Bio-Rad Protein Assay.
• OMVs were isolated following a method adapted from Stentz et ah , [20] . Briefly, cultures of Bt (500 mL) were centrifuged at 5500 g for 45 min at 4°C and the supernatants filtered through polyethersulfone (PES) membranes (0.22 pm pore-size) (Sartorius) to remove debris and cells. Supernatants were concentrated by ultrafiltration (100 kDa molecular weight cut-off, Vivaspin 50R, Sartorius), the retentate was rinsed once with 500 mL of PBS (pH 7.4) and concentrated to 1 mL (approx. 700 mg/ml total protein) . The final OMV suspensions were filter sterilized (0.22 pm pore size) . The protein content of the final OMV suspensions was determined using the Bio-Rad Protein Assay.
• PES polyethersulfone
• the distribution of heterologous proteins within Bt OMVs was established in a Proteinase K acces sibility/protection assay [20] . Briefly, a suspension of 250 pg of OMVs in 0.1 M phosphate/ 1 mM EDTA buffer (pH 7.0) was incubated for 1 h at 37°C in the presence of 100 mg/L proteinase K (Sigma-Aldrich) . Proteinase K activity was stopped by addition of 1 mM phenylmethanesulfonyl fluoride (PMSF) and samples analysed by immunoblotting. The Sseb content of Bt OMVs was determined by targeted proteomics done by the Proteomics Facility, University Bristol, UK.
• PMSF phenylmethanesulfonyl fluoride
• Electron microscopy Suspensions of OMVs were fixed for 1 h using 25% glutaraldehyde then centrifuged at 13,000g for 10 min.
• the OMV pellets were mixed 1 : 1 with molten 2% low gelling temperature agarose (TypeVII, Sigma), which was solidified by chilling and then cut into ⁇ l mm3 cubes.
• sample pieces were then further fixed in 2.5% glutaraldehyde in 0.1 M PIPES buffer for 1 6 h at 4°C after which time they were washed three times in 0.1 M PIPES buffer and dehydrated through a series of ethanol solutions (30, 50, 70, 80, 90%, and 3 times in 100%) after which the ethanol was replaced with a 1 : 1 mix of 100% ethanol:LR White medium grade resin and put on a rotator for 1 h. This was followed by a 1 :2 and then a 1 : 3 mix of 100%eethanol:LR White resin mix and finally 100% resin, with at least 1 h between each change. The resin was changed twice more with fresh 100% resin with 8 h between changes.
• the sample pieces were each trans ferred into BEEM embedding capsules with fresh resin and polymerised for 1 6 h at 60°C. Sections of ⁇ 90 nm thick were cut using an ultramicrotome (Ultracut E, Reichert-Jung) with a glass knife and collected on film/carbon coated gold grids.
• a modified version of the Aurion Immunogold labelling (IGL) protocol http: / /www. aurion.nl/the_aurion_method/Post_embedding_conv ) was used with l h antibody incubations and detergent (0.1 % TWEEN) .
• the primary anti-Bt OmpA antisera was obtained by immunising rabbits with the peptide GGPREDGSYKQRWDYMN (Cambrige Research Biochemical), and was used at a dilution of 1 /500.
• the secondary anti rabbit Ig (GAR-10, Agar Scientific) was used at a dilution of 1 /50. After antibody labelling, sections were stained with 2% uranyl acetate for 40 min and imaged in a FEI Tecnai G2 20 Twin transmission electron microscope at 200kV.
• mmuno blotting Bt cell and OMV extracts were obtained by sonication and the supernatants added to SDS Page loading buffer (NuPage) containing dithiothreitol (Invitrogen) .
• SDS Page loading buffer NuPage
• dithiothreitol Invitrogen
• Approximately 7 mg of the total protein was loaded onto 12% precast Tris-Glycine gels (Novex) and separated by electrophoresis at 180 volts for 40 min. The gel was then trans ferred onto a polyvinylidene difluoride (PVDF) membrane at 25 volts for 2 h in a solution containing Tris-Glycine Trans fer Buffer (Novex) .
• PVDF polyvinylidene difluoride
• the membrane was blocked with 10 % BSA in TBS-Tween (TBS [50 mM Tris-HCl; 150 mM NaCl; pH 7.5] with 0.05 % Tween) by shaking for 30 min at 20°C. The blocking solution was then discarded and the membrane incubated for 1 6- 18 h at 4°C in TBS-Tween with 5% BSA containing primary antibody (anti-Salmonella OmpA [Antibody Research Corporation] , -KGF-2 [Peprotech] or -IAV or Anti- polyHisitdine Clone HIS-1 (Sigma-Aldrich) .
• TBS-Tween TBS [50 mM Tris-HCl; 150 mM NaCl; pH 7.5] with 0.05 % Tween
• the human colonic epithelial cell line Caco-2 (ECACC 86010202) was cultured at 37 ° C and 5 % C02 in Dulbecco's Modified Eagle Medium (DMEM) with 4.5 g/L glucose and L-glutamine (Lonza, Switzerland) supplemented with 5% foetal bovine serum (FBS, Lonza) .
• DMEM Dulbecco's Modified Eagle Medium
• FBS foetal bovine serum
• Caco-2 cells were grown in T25 flasks until they reached 90% confluency. Cells were digested using trypsin EDTA (200 mg/L, 170,000 U Trypsin/L, Lonza) and seeded onto 8-well m-slides (Ibidi) . Cells were grown until they formed a 90% confluent monolayer and then serum-starved for 8 h. A scratch was made on the monolayer using a sterile tip and cells were washed with PBS to remove cell debris.
• trypsin EDTA 200 mg/L, 170,000 U Trypsin/L, Lonza
• Ibidi 8-well m-slides
• the remaining cells were incubated for 72 h in 1 % FBS medium supplemented with heparin (300 pg/mL grade I-A, > 1 80USP units/ml; Sigma-Aldrich) in the presence of PBS, native OMVs, KGF-2 OMVs or recombinant KGF-2 (500 ng/mL, PeproTech) .
• Wound healing was monitored by taking images immediately after scratching (time 0 control) and every 24 hours using an Invertoskop ID03 inverted microscope (Carl Zeis s) and a Sony Xperia Z5 compact digital camera (Sony) .
• the measurements of the recovered scratch area (pixel2) at each time point were analysed using ImageJ software. The experiment was performed in triplicate.
• mice were anaesthetized then intranasally dosed with either StOmpA OMVs, StSseB OMVs, H5F OMVs, native OMVs ( ⁇ 0.5 ug) or PBS and 7 and 14 days later received booster immunizations .
• StOmpA-OMV orally or ip immunised mice were orally administered 108 CFU of S. Typhimurium SL1344 on day 28 and 5 days later the bacterial load in different tissues was determined.
• mice For infectious challenge with IAV, F15F-OMV immunised mice were anaesthetised on day 28 with ketamine via the intra-muscular route and inoculated intranasally with 103 PFU A/PR/8/34 (PR8) H1 N 1 strain of IAV in 50 ml sterile PBS, which is equivalent to a 10-fold lethal dose. Weights of each animal were recorded from the day of challenge up until the end point at day 33 when the mice were euthanised. At necropsy, blood/serum and bronchoalveolar lavage fluid were taken for antibody and cytokine analyses and lung tis sue was used to determine virus titre.
• mice were intranasally administered with DiO-labelled F15F-OMVs and 1 and 5 days later OMV acquisition and uptake was determined using flow cytometry in: the macrophage and dendritic cells of the BAL; nasal associated lymphoid tissue (NALT); and cervical and mediastinal lymph nodes.
• IAV quantification Plaque assays were performed on homogenates of lung tissue from PR8-infected mice as described previously [23] . Briefly, viral samples from lungs were titrated in a 10-fold serial dilution from 101 6 to 10 in DMEM supplemented with TPCK-trypsin. Each dilution was incubated with MDCK cells in individual wells of a 24 well plate for 1 hour at 37°C, 5% C02. The media was aspirated and replaced with overlay media containing 2.4% Avicel. Plates were incubated at 37 °C, 5% C02 for 72 hours. Avicel was aspirated, plates were washed and cells were fixed in acetone:methanol (60: 40) for 10 min.
• DSS dextran sulphate sodium
• mice were administered by oral gavage (100 mL) on days 1 , 3 and 5 and on day 7 mice were euthanized.
• Fresh faecal pellets were collected daily by placing individual mice in an empty cage without bedding material for 5- 15 min.
• the extent of colitis was evaluated using a disease activity index (Table SI) comprising daily body weights, stool consistency and rectal bleeding as sessments .
• Table SI disease activity index
• At autopsy the colon was aseptically extracted and photographed, and the contents collected in sterile vials and stored at—80 °C. The colon length was measured, and representative samples (0.5 cm length) were taken from the distal region for histology. Histological samples were fixed in 10% neutral buffered formalin and embedding in paraffin.
• DSS-induced colitis Treatment of DSS-induced colitis with Bt OMVs.
• the dextran sulphate sodium (DSS) induced mouse model of acute colitis was used to test the therapeutic potential of OMVs.
• the control groups of mice received fresh water alone throughout for the duration of the experiment.
• PBS and OMVs were administered by oral gavage (100 mL) on days 5, 7 and 9 and on day 1 1 mice were euthanized.
• the extent o f colitis was evaluated using a disease activity index comprising daily body weights, colon and caecum appearance, stool consistency and rectal bleeding as ses sments.
• Antibody ELISA ELISA plates were coated with target antigens (UV inactivated IAV [PR8] virus or H5 (H5N 1) (A/Vietnam/ 1203/2004) Recombinant Protein (P5060, 2B Scientific Ltd), Salmonella OmpA or SseB proteins) in 0.1 M NaHCO3 and incubated for 12- 16 hours at 4 °C. Plates were washed three times with PBS that had been supplemented with 0.05% Tween 20 (PT), and then incubated with blocking solution (PBS with 2% BSA) for 3 h at 20 °C, and then washed six times with PT.
• target antigens UV inactivated IAV [PR8] virus or H5 (H5N 1) (A/Vietnam/ 1203/2004) Recombinant Protein (P5060, 2B Scientific Ltd), Salmonella OmpA or SseB proteins
• Fecal pellets were homogenized in phosphate- buffered saline (pH , 7.2) with soybean trypsin inhibitor (0.5 mg/mL; Sigma), phenylmethylsulfonyl fluoride (0.25 mg/mL; Sigma), 0.05 M EDTA, and 0.05% Tween 20 (Sigma) .
• the fecal homogenates and bronchoalveolar lavage (BAL) and serum samples were diluted in PBS with 1 % BSA, 0.05% Tween (PBT) and added to the plate wells and incubated for 12- 16 h at 4 °C.
• Immune serum and BAL from PR8 IAV- infected mice were used as reference samples for analysing anti-IAV antibody responses in H5F-OMV-immunised animals. Plates were then washed six times with PT and incubated with PBT containing either H RP-anti-mouse IgG (1 : 1000, Thermo-Fisher) or H RP-anti-mouse IgA (1 : 1000, Life Technologies) for 20 min at 20oC. Plates were again washed six times with PT then incubated in darknes s with TMB High Sensitivity substrate solution (BioLegend) for 30 min at 20oC.
• mice From all mice, the entire, skinned heads were fixed in 10% buffered formalin for 48 h. Subsequently, approximately 2 mm slices were prepared by sagittal sections, using a diamond saw (Exakt Band System 300 CL; EXAKT Technologies Inc.), yielding a total of six sections from the tip of the nose to the foramen occipitale magnum. Sections were gently decalcified for 7 days in RDF Mild Decalcifier (CellPath Ltd) at room temperature. Likewise, thoracic organs (lungs, lymph nodes, heart and thymus) were removed en bloc, fixed for 24 h in 10% buffered formalin and trimmed. Head and organ specimens were then routinely paraffin wax embedded.
• Consecutive sections were prepared and were stained with haematoxylin eosin for histological examination, or subj ected to immunohistological staining.
• Immunohistology was performed using the horseradish peroxidase method as previously described [24, 25] .
• Primary antibodies used were rat anti-mouse CD45R (clone B220, BD Biosciences; B cells), rabbit anti-CD3 (clone SP7; Bioscience; T cells) and rabbit anti-Iba- 1 (Wako; macrophages and dendritic cells) .
• Tumours were snap frozen for histology, cytokine analysis, transcript analysis.
• tumours from the labelled animals were intensely fluorescent consistent with OMVs localising and accessing the tumour.
• Native Bt OMVs posses s anti-tumour properties, being able to significantly reduce the size of established breast cancer tumours.
• the OMV anti-tumour effect is dependent on dose.
• the optimal dose range is between 10 7 and 10 9 . Below and above this range, OMVs are ineffective. This is consistent with findings from in vitro experiments using primary immune cells and assessing the nature of their response to OMVs.

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