DELIVERY OF BACTERIA-DERIVED OUTER MEMBRANE VESICLES

Abstract

Pharmaceutical compositions and preparation including outer membrane vesicles (OMVs) and at least one pharmaceutically acceptable carrier wherein said OMVs are native OMVs produced by the Gram-negative commensal bacteria Bacteroides.

Claims

1: A pharmaceutical preparation including outer membrane vesicles (OMVs) and at least one pharmaceutically acceptable carrier wherein said OMVs are native OMVs produced by the Gram-negative commensal bacteria Bacteroides.

2: A pharmaceutical preparation according to claim 1 wherein the pharmaceutically acceptable carrier is buffered saline.

3: A pharmaceutical preparation according to claim 1 wherein the pharmaceutical preparation primes, activates and/or boosts immune responses to prevent infection, tissue inflammation, tumor growth and/or other injury.

4: A pharmaceutical preparation according to claim 3 wherein said OMVs include and/or deliver biologically active peptides, proteins and/or biologics.

5: A pharmaceutical preparation according to claim 4 wherein said pharmaceutical preparation and/or said OMVs are for delivery of said peptides, proteins or biologics to the gastrointestinal (GI) and/or respiratory tract.

6: A pharmaceutical preparation according to claim 5 wherein 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.

7: A pharmaceutical preparation according to claim 1 wherein the native OMVs are delivered to one or more mucosal sites to induce, activate or boost an immune response.

8: A pharmaceutical preparation according to claim 1 wherein the pharmaceutical preparation includes at least one pharmaceutically active component or one further pharmaceutically active component to the OMVs.

9: A pharmaceutical preparation according to claim 8 wherein the at least one pharmaceutically active component includes a vaccine agent such as antigens and/or pathogen components.

10: A pharmaceutical preparation according to claim 9 wherein the OMVs is an adjuvant to reduce the number of doses and/or amount of vaccine agent or component required to elicit a protective immune response.

11: A pharmaceutical preparation according to claim 1 wherein the outer membrane vesicle (OMV) composition is for use as an adjuvant which is delivered before, at substantially the same time or after a vaccine or one or more vaccine agents.

12-13. (canceled)

14: A pharmaceutical preparation according to 4 wherein the peptide or protein is within or on the outer membrane of OMVs.

15: A pharmaceutical preparation according to any preceding claim wherein the species of Bacteroides producing the OMVs includes 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.

16. (canceled)

17: A pharmaceutical preparation according to claim 15 wherein a mannan-inducible expression system is used in any Bacteroides.

18: A pharmaceutical preparation according to claim 1 wherein the OMVs are provided by B. thetaiotaomicron (Bt) (VPI-5482), and strains thereof, including GH193, GH359 or GH364.

19: A pharmaceutical preparation according to claim 8 wherein the pharmaceutical preparation includes Salmonella typhimurium-derived vaccine antigens.

20. (canceled)

21: A pharmaceutical preparation according to claim 8 wherein the pharmaceutical preparation includes influenza A virus (IAV) derived vaccine antigens.

22-23. (canceled)

24: A pharmaceutical preparation according to claim 8 wherein immunisation is with OMVs containing the core stalk region of the IAV H5 hemagglutinin from an H5N1 strain, inducing heterotypic protection.

25. (canceled)

26: A pharmaceutical preparation according to claim 1 wherein said composition includes a heterologous peptide, protein or biologic.

27. (canceled)

28: A pharmaceutical preparation according to claim 1 wherein the composition is for use in a method for the treatment of typhoid.

29: A pharmaceutical preparation according to claim 1 wherein the composition is for use in a method for the treatment of colitis.

30: A pharmaceutical preparation according to claim 1 wherein the native OMVs are delivered intranasally.

31: A pharmaceutical preparation according to claim 1 wherein administration of said OMVs provides anti-tumour properties.

32-36. (canceled)

Description

[0054] Specific embodiment of the invention are now described with reference to the figures, wherein:

[0055] FIG. 1: Appearance, size, structure and stability of Bt OMVs. (a) Electron microscopy (EM) of Bt cells showing vesicles budding from their surface before release into the milieu (lines in left panel), and EM image of OMVs extracted from cell culture supernatants (right panel). (b) Immuno detection of naïve Bt OMVs using colloidal gold anti-rabbit Ig to detect binding of rabbit anti-Bt OmpA antisera (right panel). Left panel shows absence of staining of OMVs produced by an OmpA deletion mutant of Bt. (c) Size distribution of OMVs produced by Bt determined by nanoparticle tracking analysis. (d) Thermostability of OMVs at day 0 (OMV D.sub.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 naïve 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).

[0056] FIG. 2: Expression of heterologous proteins in Bt OMVs. (a) 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. (b and c) Determination of protein location after treatment with proteinase K (PK). Immunoblotting of StOmpA (b) and KGF-2 (c) with and without pre-treatment of OMV suspensions with proteinase K. NT: not treated; PK: +Proteinase K; B: PK buffer alone. (d) KGF-2 quantification within OMVs. Comparison of recombinant KGF-2 (1-100 ng) with 10 μl of 1 ml OMV suspension (S).

[0057] FIG. 3: Intrinsic adjuvanticity of Bt OMVs. (a) Mice (n=5) were intranasally adminstered PBS alone native Bt OMVs (OMV) in PBS and days later heads and thoracic tissue was processed for immunohistology to visualise immune cell activation and formation of organised lymphoid tissue containing CD45R.sup.+ B cells (B220), CD3.sup.+ T cells (CD3) and macrophages/dendritic cells (Iba-1) in the nasal associated lymphoid tissue (a) the lung parenchyma (b) and mediastinal adipose tissues (c). 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. NC: nasal cavity, NS: nasal septum, P: hard palate.

[0058] FIG. 4: Bt OMV-elicited systemic and mucosal antibody responses. (a) Mice (n=5-6/grp) were administered Bt OMVs expressing 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. Naïve OMVs (nOMV) and PBS were administrated to mice (n=5-6/grp) as control groups. At autopsy, serum (b) and brochoalveolar lavage fluid (BAL) (c) were analysed for anti-OmpA and anti-SseB IgG and IgA antibody titres, respectively, by ELISA. The boxplots identify the mean and upper and lower quartile values for data sets obtained from animals within each treatment group. Analysis of variance for multiple comparisons of means between independent samples (ANOVA) was followed by a Tukey's test. *P<0.05; **P<0.01; ***P<0.001; ns, not significant. Total IgA levels were also determined in salivary gland tissue homogenates (d) and in BAL (e) samples from each group of animals by ELISA using IgA standards as described in the Materials and Methods section.

[0059] FIG. 5: Bt OMVs expressing IAV H5F protein confer a level of protection to virus infection in mice. (a) Mice were immunised intranasally with H5F-OMVs in PBS; controls were administered intranasally with naïve OMVs or PBS alone (mock) at the indicated time-points; after 28 days all were challenged intranasally with a 10-fold lethal dose of IAV strain A/PR/8/34 (PR8, H1N1). At 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 H5 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. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison tests (panels b, c, d, e, g) or two-way ANOVA with Bonferroni post-tests. ns, not significant; *P<0.05; **P<0.01; ***P<0.001.

[0060] FIG. 6: OMVs containing KGF-2 ameliorate DSS-induced colitis in mice. (a) Groups of mice were provided with drinking water with or without 2.5% (w/v) DSS for 7 days. On days 1, 3 and 5 mice were orally gavaged with either PBS, naïve OMVs or OMVs containing KGF-2. (b) Percent weight loss at day 7. (c) Colon length at day 7. (d) Disease Activity Index (DAI) at day 7. (e) Representative images of colons. Data expressed as mean±SD (n=5). 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.

[0061] FIG. 7: OMVs containing KGF-2 protect and restore goblet cells in mice with DSS-induced colitis. (a) Histological score of colon tissue as determined by microscopy of H&E stained sections obtained at necropsy. (b) Number of Alcian Blue stained goblet cells per mm.sup.2 of epithelial area. (c) Microscope images of goblet cell distribution in representative colon sections stained with Alcian Blue. Data expressed as mean±SD (n=5). Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison tests. *P<0.05.

[0062] FIG. 8. Treatment of DSS-induced colitis with Bt OMVs. A. Groups of mice were provided drinking water with or without 2.25% (w/v) DSS for 5 days. On days 5, 7 and 9 mice were orally gavaged (OG) with either PBS or native OMVs B. Disease Activity Index (DAI) at day 11. C. Percentage weight loss at day 11. ***P<0.001; *P=0.017. OG.

[0063] FIG. 9. Stimulation of dendritic cells by OMVs. To test whether the outer membrane vesicles (OMV) isolated from Bt elicit an immune response, whole peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated for 20 h with descending concentrations of OMV purified by size-exclusion (FIGS. 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 (FIGS. 9A and B). The response of DCs to Bt or OMV was also measured with PBMC from patients with Crohn's disease (n=3) (FIGS. 9C and D).

Supplementary Data

[0064] FIG. 10: Acquisition of fluorescent labelled Bt OMVs by dendritic cells and subsequent trafficking to lymph nodes after intranasal administration. OMVs were fluorescently-labelled with DiO and administered intranasally to mice. At dl and d5 post-inoculation mice were sacrificed. Cells from nasal-associated lymphoid tissue (NALT), cervical lymph nodes (CLN) and mediastinal lymph nodes (MLN) were separated and analysed by FACS using antibodies to dendritic cells (CD11c) and antibodies to define dendritic cell subsets (CD11b and CD103). (a) Representative gating strategy showing from left to right gating out debris, for single cells, for total dendritic cells (DC, CD11c.sup.+), for the subset of DC that have taken up OMV (DiO.sup.+, CD11c.sup.+) and the for the subsets of OMV DC that express either CD11b or CD103. (b) Day 1 post-administration. Graphs show the percentage of cells in NALT and CLN that are DCs, and the percentage of DC that are labelled or labelled and CD11b.sup.+. (c) Day 5 post-administration. Graphs show the percentage of cells in CLN and MLN that are DCs, and the percentage of DC that are labelled or labelled and CD11b.sup.+ or CD103.sup.+.

[0065] FIG. 11: The impact of orally administered Bt OMVs on the recipients' intestinal microbiota. Mice (n=5) were orally gavaged with native OMVs on day 3, 5 and 7, and faeces was collected on day 0, 4, 7 and 8 to evaluate the impact of OMVs on the host microbiota. Faeces were weighed, homogenized in PBS and serially diluted prior to plating on different agar media to select for: Total aerobes (on nutrient media); Total anaerobes (on Wilkins-Chalgren [W-G] or Brain Heart Infusion [BHI] media); or Bacteroides spp. (on BHI media supplemented with gentamicin and amikacin). The 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.

[0066] FIG. 12: Colonization of OMV-StOmpA immunised mice after Salmonella challenge. Mice (n=5-6/grp) immunised with either naïve 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.sup.8 CFU Salmonella enterica ser. Typhimurium SL1344. 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 μg/ml streptomycin. The boxplots identify the mean and upper and lower quartile values for data sets obtained from animals within each treatment group.

[0067] FIG. 13: Evaluating the biological activity of KGF-2 contained in Bt OMVs using an epithelial wound-healing assay. (a) Representative micrographs of healing of a scratch wound in a confluent monolayer of Caco2 cells after exposure to PBS, naïve OMVs, KGF-2 OMVs or recombinant KGF-2 for 72 h. Red dotted lines demarcate the wound margin. (b) Graphical representation of cell growth across the wound area after 72 h as determined by pixel.sup.2.

BT OMVS HAVE INHERENT ADJUVANTICITY

[0068] Many conventional vaccines rely on the inclusion of adjuvants to enhance their immunogenicity and to reduce the number of doses and amount of antigen (or pathogen component) required to elicit a protective immune response, particularly in immunocompromised individuals [2, 39]. To formally evaluate the adjuvant properties of Bt OMVs, 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. 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). Of note, OMVs (native) were also effective at eliciting the formation of lymphoid clusters in mediastinal adipose tissue (fat-associated lymphoid clusters or FALC) (FIG. 3c).

[0069] Consistent with the immune response priming ability of Bt OMVs, within 24 h of intranasal administration of fluorescent labelled native OMVs it was possible to detect their uptake in the NALT and draining cervical lymph nodes (CLN) (Supplementary FIG. 10). At day 5, there was evidence of trafficking of OMVs to both the cervical and mesenteric lymph nodes which was almost exclusively mediated by CD11c.sup.+, CD11b.sup.+ CD103.sup.− dendritic cells (Supplementary FIG. 10).

[0070] From a biosafety perspective, neither orally nor intranasally administered native OMVs (or vaccine antigen formulated OMVs) had any adverse health effects with no tissue pathology evident in treated animals at post mortem (data not shown). Orally administered OMVs also had no or a minor and/or transient effect on intestinal microbes as determined from culturing faecal samples on selective media (Supplementary FIG. 11).

Intranasal OMV Formulation Protect Against Pulmonary IAV Infection

[0071] During infection the weight of all infected animals declined with the greatest weight loss 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 loss after infection, as did those immunized with native OMVs.

Bt OMV-Elicit Mucosal Antibody Responses

[0072] 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).

Further Method

[0073] Bt and its derivative strains (Table 1) are grown under anaerobic conditions at 37° C. in ‘Brain Heart Infusion’ (BHI) medium (Oxoid) supplemented with 15 μM haemin (Sigma-Aldrich) (BHIH) for bacterial pre-culture or 0.75 μM haemin for OMV preparations. Antibiotic-resistance markers in Bt were selected using erythromycin (5 μg/ml) and tetracycline (1 μg/ml).

[0074] Escherichia coli strains were grown in Luria-Bertani (LB) medium at 37° C. with ampicillin 100 μg/ml (or 200 μg/ml trimethoprim for strain J53 [pR751]). Lactococcus lactis strain UKLc10 and its derivative strains were grown in M17 medium (Oxoid) supplemented with 5 g/l glucose at 30° C. Antibiotics were added as selection agents when appropriate: ampicillin 200 μg/ml, erythromycin 5 μg/ml and chloramphenicol 10 μg/ml. The E. coli strain J53/R751 was supplemented with trimethoprim 200 μg/ml when grown for 18 h. The E. coli strain GC10 and the L. lactis strain UKLc10 were transformed by electroporation using a Gene Pulser II (Bio-Rad). For constructs relating to pUK200, the host L. lactis strain UKLc10 was used.

[0075] Construction of other plasmids described below was achieved using E. coli strain GC10 as the host. Plasmids were mobilized from the E. coli into the Bt following a triparental filter mating protocol [19] using the helper strain J53/R751. All primers used are detailed in Table 2.

TABLE-US-00002 TABLE 2 Primer sequences used in this stdy Primer Sequence (5′.fwdarw.3′)* f-5′ompA_SphI ATCTGCATGCTTTCGAGGAAGAACCGATGGTTGC r-5′ompA_SalI ATACGTCGACAATATAGCGGACTGCAATCC f-3′ompA-BamHI ACTTGGATCCTTCTGAATCGTGTGGTATTGG r-3′ompA_SacI ACTAGAGCTCATCTGTAGAGAAGAAACGGG SPBTOmpA-fwd CATGTTGCTGGCTTTTGCCGGCGTTGCGTCTGT CGCTTCTGCGCAGCAACCGTGACTGTAACTGAA TACGAGGTTATTCATATGTGACG SPBTOmpA-rev AATTCGTCACATATGAATAACCTCGTATTCAGT TACAGTCACGGTTTGCTGCGCAGAAGCCGACAG ACGCAACGCCGGCAAAAGCCAGCAA OmpAST-fwd TGACCATATGGCTCCGAAAGATAACACC OmpAST_rev GTCAGAATTCTTAAGCCTGCGGCTGAGTTA SseB_fwd TGACCATATGTCTTCAGGAAACATCTT SseB_rev TGACGAATTCATGAGTACGACGTTTTCTGCG XhoI_STOmpA_rev ATATCTCGAGGAAACTTAAGCCTGCGG XhoI_SseB_rev ATATCTCGAGATGAGTACGTTTTCTGCG

Construction of a BT 3852 Deletion Mutant

[0076] A 1018 bp chromosomal DNA fragment upstream from BT_3852 and including the first 18 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/SalI sites of the E. coli-Bacteroides suicide shuttle vector pGH014 [20]. A 761 bp chromosomal DNA fragment downstream from BT_3852, including the last 46 nucleotides of the 3′-end region, was amplified by PCR using the primer pair f-3′ompA_BamHI, r-3′ompA_SacI and was cloned into the BamHI/SacI sites of the pGH014-based plasmid. The resulting plasmid containing the ΔBT_3852::tetQ construct, was mobilized from E. coli strain GC10 into Bt by triparental filter mating [19], using E. coli HB101(pRK2013) as the helper strain. Transconjugants were selected on BHI-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, GH290, containing the ΔBT_3852::tetQ construct inserted into the Bt chromosome was selected for further studies.

Generation of Recombinant Bt Strains

[0077] Bt Salmonella OmpA/SseB: The Bacteroides expression vector pGH090 [21] was first digested with NdeI to remove this site by Klenow treatment and to create a blunt-ended fragment that was then religated. A sequence containing 90 bp of the BT_3852 gene 5′ end (encoding a major outer membrane protein, OmpA) corresponding to the signal peptide sequence (SpOmpA) of the protein obtained from the microbial genome database (http://mbgd.genome.ad.jp/) was used to design the complementary oligonucleotide pair SPBTOmpA_fwd and SPBTOmpA_rev. 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 NdeI deleted version of pGH090, resulting in the pGH202 plasmid. The 1131 bp Salmonella ompA (without signal peptide) and the 591 bp sseB coding region were amplified by PCR from S. Typhimurium serovar enterica SL 1334 genomic DNA using the primer pairs OmpAST_fwd with OmpAST_rev, and SseB_fwd with SseB_rev, respectively. The resulting fragments were digested with NdeI and EcoRI and cloned into NdeI/EcoRI-digested pGH202, yielding plasmids pGH182 and pGH183, respectively. The latter plasmid was then transformed into E. coli-competent cells (GC10) by electroporation using a Gene Pulser II (Bio-Rad). Successful cloning was confirmed by sequencing. The plasmid was mobilized from E. coli to Bt using a triparental mating procedure [19], together with E. coli J53 (pR751); the correct structure of the Bt carrying pGH182 (GH484) was confirmed by sequencing.

[0078] Bt IAV: A 635 bp synthetic gene construct encoding a synthetic influenza (HSF; from IAV strain H5N1 [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-K168 (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.

[0079] 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 cassette was obtained by gene synthesis and subsequently cloned into the E. coli plasmid pEX-A2 (Eurofins) as described for the IAV constructs. 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 pGH0902. The gene was excised from pEX-A2 using Eco53KI and EcoRI and ligated into pUK200 [22], which had been restricted with SmaI and EcoRI, resulting in plasmid pUK200_KGF-2. Next the KGF-2 cassette was excised from pUK200_KGF-2 through restriction with BspHI and EcoRI and subsequently ligated into the NcoI/EcoRI-restricted pGH090 expression vector, resulting in pGH173. Finally the sequence integrity of the cloned fragment was confirmed by sequencing. The plasmid was mobilized from E. coli into Bt using a triparental mating procedure.

[0080] Expression and purification of recombinant StOmpA and StSseB StOmpA was cloned into His6-tag expression vector pET-15b (Novagen). Briefly, PCR fragments incorporating the coding sequences of ompA and sseB genes were cloned into the NdeI/XhoI restriction sites of pET-15b and the resulting plasmids pGH165 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 1 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 QIAexpress Ni-NTA Fast Start Handbook (Qiagen) with the amount of protein recovered determined using the Bio-Rad Protein Assay.

OMV Isolation and Characterisation

[0081] OMVs were isolated following a method adapted from Stentz et al., [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 μm 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 μg/ml total protein). The final OMV suspensions were filter sterilized (0.22 μm pore size). The protein content of the final OMV suspensions was determined using the Bio-Rad Protein Assay.

[0082] The distribution of heterologous proteins within Bt OMVs was established in a Proteinase K accessibility/protection assay [20]. Briefly, a suspension of 250 μg 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.

Electron Microscopy

[0083] Suspensions of OMVs were fixed for 1 h using 25% glutaraldehyde then centrifuged at 13,000 g 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 ˜1 mm3 cubes. The sample pieces were then further fixed in 2.5% glutaraldehyde in 0.1M PIPES buffer for 16 h at 4° C. after which time they were washed three times in 0.1M 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% ethanol: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 transferred into BEEM embedding capsules with fresh resin and polymerised for 16 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 1 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 200 kV.

Immunoblotting

[0084] Bt cell and OMV extracts were obtained by sonication and the supernatants added to SDS Page loading buffer (NuPage) containing dithiothreitol (Invitrogen). Approximately 7 μg 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 transferred onto a polyvinylidene difluoride (PVDF) membrane at 25 volts for 2 h in a solution containing Tris-Glycine Transfer Buffer (Novex). 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 16-18 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). After washing with TBS-Tween, membranes were incubated in 5% BSA in TBS-Tween containing HRP-conjugated goat anti rabbit IgG (1:1000 dilution, ThermoFisher) for 1 oh at 20 C. After 3 washes with TBS-Tween, SuperSignal West Pico chemiluminescent Substrate (ThermoFisher) was used to detect bound antibody.

Mammalian Cell Culture

[0085] The human colonic epithelial cell line Caco-2 (ECACC 86010202) was cultured at 37° C. and 5% CO2 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).

Epithelial Cell Scratch Assay

[0086] 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 μ-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. The remaining cells were incubated for 72 h in 1% FBS medium supplemented with heparin (300 μg/mL grade I-A, >180USP 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 Zeiss) 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.

Animal Experiments

[0087] All animal experiments were done using 6 to 8 week old C57BL/6 male mice that were bred and maintained in animal facilities either at the University of East Anglia (UK) or the University of Liverpool. Mice were housed in individually ventilated cages and exposed to a 12 h light/dark cycle with free access to a standard laboratory chow diet. Animal experiments were conducted in full accordance with the Animal Scientific Procedures Act 1986 under UK Home Office (HMO) approval and HMO project license 70/8232 (UEA) and 70/8599 (UoL).

[0088] OMV vaccines and vaccination: To evaluate oral Salmonella OMV vaccine formulations, groups of mice (n=5-6/group) were gavaged with either 100 μl of StOmpA-OMVs or native OMVs in PBS. Prior to each immunization food was removed for approximately 4 h to decrease stomach acidity. Booster oral immunisations were given 1 and 2 months later. An additional control group of animals were immunised with StOmpA-OMVs via the intraperitoneal route. To assess intranasal immunisation with Salmonella and influenza virus OMV vaccine formulations, groups of mice (n=5-10/group 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. For infectious challenge with Salmonella, 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. For infectious challenge with IAV, H5F-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) H1N1 strain of IAV in 50 μl 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 tissue was used to determine virus titre. For in vivo OMV trafficking studies, mice were intranasally administered with DiO-labelled H5F-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.

[0089] 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 1016 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% CO2. The media was aspirated and replaced with overlay media containing 2.4% Avicel. Plates were incubated at 37° C., 5% CO2 for 72 hours. Avicel was aspirated, plates were washed and cells were fixed in acetone:methanol (60:40) for 10 min. Cells were allowed to air dry prior to staining with crystal violet for 10 minutes, washed and air dried. Plaques were counted and then multiplied by the dilution factor and the volume of virus plated to give viral titre (PFU/ml).

[0090] Acute colitis: The dextran sulphate sodium (DSS) induced mouse model of acute colitis was used to test the therapeutic potential of KGF-containing OMVs. Mice were divided into six groups (n=5/grp) and administered with either PBS, native OMVs, KGF-OMVs, DSS+PBS, DSS+native OMVs or, DSS+KGF-OMVs for 7 days. Experimental colitis was induced in the appropriate treatment groups of mice by administration of 2.5% w/v DSS (36,000-50,000 Da, MP Biomedicals, USA) in drinking water ad libitum for 7 days. The other groups of mice received fresh water alone throughout the duration of the experiment. PBS and OMVs were administered by oral gavage (100 μL) 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 51) comprising daily body weights, stool consistency and rectal bleeding assessments. 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. Tissue sections (5 μm) were prepared from each block, stained with hematoxylin (Mayer's hemalum, Merk, Germany) and eosin (Y-solution 0.5% aqueous, Merck, Germany) (H&E), and with Alcian blue (Sigma-Aldrich, USA) and Nuclear Red (Sigma-Aldrich, USA) to visualise goblet cells. Sections were observed under a DMI 3000B microscope at 40× magnification (Leica, Germany) and assessed in a blinded fashion. The histological changes were scored (Table S2) and goblet cells were enumerated using ImageJ software (USA).

[0091] 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. Groups of male C57BL/6 mice of 8-11 weeks of age were divided into 4 groups (n=5-10/grp), 2 of them were administered 2.25% w/v DSS (36,000-50,000 MW, MP Biomedicals, USA) in drinking water for 5 days. 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 μL) on days 5, 7 and 9 and on day 11 mice were euthanized. The extent of colitis was evaluated using a disease activity index comprising daily body weights, colon and caecum appearance, stool consistency and rectal bleeding assessments.

[0092] Antibody ELISA: ELISA plates were coated with target antigens (UV inactivated IAV [PR8] virus or H5 (H5N1) (A/Vietnam/1203/2004) Recombinant Protein (P5060, 2B Scientific Ltd), Salmonella OmpA or SseB proteins) in 0.1M NaHCO.sub.3 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. 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 HRP-anti-mouse IgG (1:1000, Thermo-Fisher) or HRP-anti-mouse IgA (1:1000, Life Technologies) for 20 min at 20° C. Plates were again washed six times with PT then incubated in darkness with TMB High Sensitivity substrate solution (BioLegend) for 30 min at 20° C. The reaction was stopped by the addition of 2 N H2SO4 and the optical density was measured at 450 nm using a TECAN infinite f50 spectrophotometer (Männedorf, Switzerland). Abcam's IgA Mouse ELISA Kit was used to determine total IgA in salivary glands and BAL Flow cytometry: Approximately 1×106 tissue-derived cells were incubated in PBS supplemented with 2% FCS (PBS-FCS) for 15 min at 4° C. prior to the addition of fluorochrome-conjugated monoclonal antibodies specific for CD11b (clone M1/70, eBioscience) CD11c (clone N418, eBioscience), or CD103 (clone 2E7, eBioscience) in PBS-FCS and incubated for 30 min at 4° C. in darkness. Cells were then washed in PBS-FCS and fixed in PBS supplemented with 4% paraformaldehyde for 15 min at 20° C. prior to analysis on a MACSQuant Analyzer 10 (Miltenyi Biotech). Data were analysed using FlowJo.

Immunohistology

[0093] 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 (3-5 μm) were prepared and were stained with haematoxylin eosin for histological examination, or subjected to immunohistological staining. Immunohistology (IH) 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).

Statistical Analysis

[0094] Data were subjected to the D'Agostino & Pearson omnibus normality test. One-way ANOVA followed by a Tukey's multiple comparison post hoc tests were made using GraphPad Prism 5 software (USA). Statistically significant differences between two mean values were established by a p-value <0.05. Data are presented as the mean±standard deviation.

Cancer OMV

[0095] The study design involved: [0096] Breast cancer tumour cells injected subcutaneously into adult mice on day 0 [0097] Bt OMVs injected intraperitoneally on days 7, 10, 12 [0098] Tumours harvested on day 14

[0099] Assays: [0100] Tumour volumes measurement. [0101] Whole body imaging of animals that received fluorescently labelled

[0102] OMVs. [0103] Tumours were snap frozen for histology, cytokine analysis, transcript analysis.

[0104] Results: [0105] Tumours excised and imaged (FIG. 14) [0106] Plotted OMV dose versus tumour volume (FIG. 15) [0107] The tumours from the labelled animals were intensely fluorescent consistent with OMVs localising and accessing the tumour.

[0108] Conclusions: [0109] Native Bt OMVs possess anti-tumour properties, being able to significantly reduce the size of established breast cancer tumours. [0110] The OMV anti-tumour effect is dependent on dose. The optimal dose range is between 10.sup.7 and 10.sup.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.