Immunogenic compositions and uses thereof

Abstract

Compositions capable of enhancing and/or eliciting an immune response in a subject and methods of using the compositions. The compositions are capable of enhancing an IgA immune response and/or an IgG immune response and comprise an agent capable of reducing the level of binding of ATP to a P2X7 receptor to a subject. The compositions are for oral administration.

Claims

1. A method of enhancing and/or eliciting an immune response in a subject, the method comprising administering a composition capable of enhancing an IgA immune response and/or an IgG immune response comprising an ATP-hydrolysing enzyme and an immunogen to the subject, wherein the composition is administered to the subject orally, and wherein the immunogen is a bacterial antigen, a parasitic antigen or a viral antigen capable of eliciting an immune response against a gastrointestinal pathogen or a mucosally transmitted systemic pathogen.

2. The method of claim 1, where the ATP-hydrolysing enzyme is apyrase.

3. The method of claim 2, wherein the apyrase is Shigella flexneri apyrase.

4. The method of claim 1, wherein the immunogen is capable of eliciting an immune response against a pathogen selected from the group consisting of Vibrio cholerae, Clostridium difficile, Clostridium botulinum, Escherichia coli, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella enterica, Salmonella bognori, Ascaris lumbricoides, Giardia lamblia, Entamoeba histolytica, poliovirus, rotavirus, Adenovirus, Hepatitis A and human immunodeficiency virus.

5. The method of claim 1, wherein the composition comprises a recombinant bacterium comprising a nucleic acid encoding the ATP-hydrolysing enzyme.

6. The method of claim 5, wherein the recombinant bacterium further comprises a nucleic acid encoding the immunogen.

7. The method of claim 5, wherein the recombinant bacterium is Escherichia coli or attenuated Salmonella enterica.

8. The method of claim 1, wherein the composition comprises a bacteriophage comprising a nucleic acid encoding the ATP-hydrolyzing enzyme.

9. The method of claim 8, wherein the bacteriophage further comprises a nucleic acid encoding the immunogen.

10. The method of claim 1, wherein the composition comprises a viral vector comprising a nucleic acid encoding the ATP-hydrolyzing enzyme.

11. The method of claim 10, wherein the viral vector further comprises a nucleic acid encoding the immunogen.

12. The method of claim 1, wherein the IgA immune response and/or the IgG immune response is a mucosal response.

13. The method of claim 1, wherein the IgA immune response and/or the IgG immune response is in the gut.

14. The method of claim 1, wherein the composition is formulated for administration in a nanocapsule.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A-1G Bacterial origin of intestinal ATP. (FIG. 1A) ATP concentration in the lumen of ileum from SPF and germ free mice (GF), bile, urine and serum. (FIG. 1B) ATP concentration in culture medium (bars) and cell growth (OD.sub.600) of the indicated bacterial species isolated from the small intestine. (FIG. 1C) ATP concentration in serum from (left to right) portal, jugular and inferior caval veins, and heart. (FIG. 1D) FACS analysis of ileal bacteria either untreated (LB) or treated with VAM (VAM) for membrane damage (DIBAC.sup.+ cells, upper dot plots), cell death (SybrGren.sup.+DAPI.sup.+ cells, lower dot plots). (FIG. 1E) ATP concentrations (upper panel) and ileal bacteria growth (lower panel) in untreated (LB) and VAM-treated cultures. (FIG. 1F) Ileal ATP concentrations from mice either gavaged with PBS (left bar) or VAM (right bar). (FIG. 1G) Analysis by FACS of Annexin V.sup.+ cells within Tfh cells from PPs of WT and p2rx7.sup.−/− mice either untreated (left bar) or orally gavaged with VAM (right bar).

(2) FIGS. 2A-2I Efficient induction of secretory anti-E. coli IgA response by apyrase. (FIG. 2A, FIG. 2B) Schematic representation of ATP secretion by E. coli (OM, outer membrane, PS, periplasmic space, IM, inner membrane, Cyt, cytosol) (FIG. 2A) and impact of apyrase (FIG. 2B); ATP concentrations (bars) in culture medium and bacterial growth (OD.sub.600) over time for pBAD28 (FIG. 2A) or pHND10 transformants (FIG. 2B). (FIG. 2C) FACS analysis for anti-E. coli IgA in intestinal wash from mice immunized with pBAD28 or pHND10 transformants. (FIG. 2D, FIG. 2E) Intestinal anti-E. coli IgA quantification in mice immunized with pBAD28 (FIG. 2D) or pHND10 (FIG. 2E) transfected E. coli and tested either with the respective (FIG. 2D) or reciprocal bacterial strain (FIG. 2E). (FIG. 2F) Fold increase of ileal ATP in mice treated with CA (right bars) or PSV (left bars) before and after (12 h) bacterial gavaging. (FIG. 2G) Quantification of anti-E. coli IgA in response to pBAD28 or (FIG. 2H) pHND10 transformants (different γ scale). (FIG. 2I) FACS analysis for IgA in intestinal fluid of mice gavaged with pBAD28 or pHND10 transfected E. coli on the indicated bacterial species.

(3) FIGS. 3A-3F Efficient induction of secretory anti-E. coli IgA response by apyrase. (FIG. 3A) E. coli staining by IgA in serial dilutions of intestinal washes; (FIG. 3B) the table shows intestinal IgA concentrations and geometric mean of E. coli specific antibodies. (FIG. 3C) Geometric mean of anti-E. coli IgA by FACS plotted against total intestinal IgA concentration and relative function of the fitting curve. (FIG. 3D) Diagram showing time points of E. coli gavaging and ATP measurements in the presence of different antibiotic associations. (FIG. 3E, FIG. 3F) Quantification of anti-E. coli IgA in non-immunized mice (Unim; left bar) or in response to pBAD28 (right bar) and pHND10 (middle bar) transformants in the presence of CA (FIG. 3E) or PSV (FIG. 3F).

(4) FIG. 4 Endoluminal ATP levels in germ-free mice monocolonized with pBAD28 harbouring E. coli (middle bar), pHND10 harbouring E. coli (right bar), and control germ-free mice (GF; left bar).

(5) FIG. 5 Analysis of Tfh cell number (left graph) and germinal centre cell number (right graph) in animals monocolonized with pBAD28 harbouring E. coli (middle bars), pHND10 harbouring E. coli (right bars) and control germ-free mice (GF; left bars).

(6) FIG. 6 Geometric mean of anti-E. coli IgA in intestinal fluid by FACS plotted against total intestinal IgA concentration, from germ-free mice monocolonized with pBAD28 harbouring E. coli, germ-free mice colonized with pHND10 harbouring E. coli and control germ-free mice (GF).

(7) FIGS. 7A-7B ATP concentrations (bars) in culture medium and bacterial growth (OD.sub.600) over time for pBAD28 harbouring (FIG. 7A) or pHND10 harbouring (FIG. 7B) avirulent S. typhimurium.

(8) FIG. 8 Geometric mean of anti-S. typhimurium IgA in intestinal fluid by FACS plotted against total intestinal IgA concentration, from control mice (left bars), mice immunized with pHND10 harbouring attenuated S. typhimurium (middle bars) and mice immunized with pBAD28 harbouring attenuated S. typhimurium (right bars).

(9) FIG. 9 Recovery of virulent Salmonella from PPs, mesenteric lymph nodes (MLNs), spleen and liver in control non immunized mice (left bars), mice immunized with pHND10 harbouring attenuated S. typhimurium (middle bars) and mice immunized with pBAD28 harbouring attenuated S. typhimurium (right bars). CFU, colony forming unit; CTRL, non-immunized mice.

(10) FIGS. 10A-10B (FIG. 10A) Spleen size in mice immunized with attenuated Salmonella bearing pBAD28 or pHND10 and then challenged with virulent Salmonella, compared with control untreated mice. (FIG. 10B) Spleen weight in control, untreated mice (left bar), mice immunized with attenuated Salmonella bearing pHND10 and then challenged with virulent Salmonella (middle bar) and mice immunized with attenuated Salmonella bearing pBAD28 and then challenged with virulent Salmonella (right bar). CTRL, untreated mice.

(11) FIG. 11A-11B: (FIG. 11A) Images of liver tissue in mice immunized with attenuated Salmonella bearing pBAD28 or attenuated salmonella bearing pHND10 and then challenged with virulent Salmonella and of control, untreated mice. (FIG. 11B) Liver histological score in mice immunized with attenuated Salmonella bearing pBAD28 (right bar) or bearing pHND10 (middle bar) and then challenged with virulent Salmonella. The left bar shows liver histological score in control untreated mice.

(12) FIG. 12 Dextran amounts in mice immunized with pHND10 harbouring attenuated S. typhimurium (middle bar), non-immunized mice (CTRL; left bar) and mice immunized with pBAD28 harbouring attenuated S. typhimurium (right bar), after infection with virulent S. typhimurium.

(13) FIG. 13 Numbers of virulent Salmonella colony forming units (CFUs) recovered in mesenteric lymph nodes (MLNs), liver and spleen from recombinase-activating gene-1 (Rag-1) deficient mice at 48 hours after challenge with virulent Salmonella. Non-immunized mice (CTRL) are represented by the left bars, mice immunized with pBAD28 harbouring attenuated S. typhimurium by the right bars and mice immunized with pHND10 harbouring attenuated S. typhimurium by middle bars.

(14) FIG. 14 Fecal IgA concentrations in untreated mice (left bar—represented by triangles), mice treated with periplasmic proteins from pBAD28 bearing E. coli (middle bar—represented by diamonds) and mice treated with periplasmic proteins from pHND10 bearing E. coli.

MODES FOR CARRYING OUT THE INVENTION

(15) Mice and Administration of Antibiotics

(16) C57BL/6J and p2rx7.sup.−/− (B6.129P2-P2rx7tm1Gab/J, Jackson Lab) mice were bred in specific pathogen-free (spf) facility at Institute for Research in Biomedicine, Bellinzona, Switzerland. C57BL/6J germ free mice were maintained in flexible film isolators at the Clean Animal Facility, University of Bern, Switzerland. For antibiotic treatment, mice were given the following antibiotic associations in drinking water for 4 wk: ampicillin 1 g/l and chloramphenicol 0.5 g/l (bactericidal association active on endogenous flora but not pBAD28-transformed E. coli) or Stretpomycin 1 g/l, Penicillin 1 g/l and Vancomycin 0.5 g/l (bactericidal on both endogenous and pBAD28-transformed bacteria).

(17) Quantification of ATP

(18) For quantification of ileal ATP, intestinal content was collected by lavage with 10 ml of intestinal wash buffer (PBS, 0.5M EDTA, Soybean trypsin inhibitor, PMSF), spun at 14′000 rpm in a sterile tube, filtered (0.22 μm) to remove any bacteria-sized contaminants and immediately frozen in dry ice. ATP concentration in the intestinal washes was multiplied for the dilution factor to obtain the actual endoluminal ATP concentration. Bile and urine were collected from gallbladder and bladder through puncture with a 34G needle. For quantification of ATP secreted by commensal bacteria in culture, intestinal content was plated on BHI agar and cultured for 16 h at 37° C. Single colonies were picked and cultured in BHI broth. Medium from 16 h cultures of single colonies was centrifuged (15,000×g), the supernatant was collected and filtered (0.22 μm). For quantification of ATP in circulatory districts, inferior caval, jugular and portal veins, and heart were exposed and blood collected through puncture with a 34G needle. Blood was centrifuged at 1000×g and the serum collected and centrifuged a second time at 1000×g. Emolysed sera were discharged. The extracellular ATP concentration was evaluated by bioluminescence assay with recombinant firefly luciferase and its substrate D-luciferin according to the manufacturer's protocol.

(19) Treatment of Bacterial Culture with Antibiotics

(20) Ampicillin (2.5 μg/ml), vancomycin (1 μg/ml), metronidazole (1 μg/ml) were added to intestinal bacterial culture at 0.5 OD. Supernatants from bacterial cultures were collected 3, 4 and 5 h after addition of antibiotics, spun at 14′000 rpm in a sterile tube and filtered (0.22 μm). ATP concentration was evaluated by bioluminescence assay (see above).

(21) Antibodies and Flow Cytometry

(22) The following mAbs were purchased from BD Biosciences: biotin conjugated anti-CXCR5 (clone: 2G8, Cat.#: 551960) and phycoerythrin (PE) conjugated anti-ICOS (clone: 7E.17G9, Cat.#: 552146). PE-Cy7 conjugated anti-CD4 (Clone:GK1.5, Cat.# 100422) and APC conjugated streptavidin (Cat.#: 405207) were from Biolegend. Percp-eFluor710 conjugated anti-CD3 (Clone: 17A2, Cat.#: 46-0032-80) was obtained from eBioscience. Annexin V staining was performed in Biolegend Annexin V binding buffer (Cat.#422201) (1×10.sup.6 cells/ml) following the manufacturer's protocol. Data were analysed using FlowJo software (TreeStar, Ashland, Oreg.) or FACS Diva software (BD Biosciences).

(23) Plasmids

(24) Full length phoN2::HA fusion of S. flexneri was cloned into the polylinker site of plasmid pBAD28 (ATCC 8739387402), under the control of the P.sub.BAD L-arabinose inducible promoter, generating plasmid pHND10 (ref. 8).

(25) Oral Immunization with E. coli and Flow Cytometry for Detection of Anti-E. coli IgA

(26) E. coli transformed with pBAD28 or pHND10 were aseptically inoculated into LB medium containing arabinose (0.3%) and ampicillin (100 μg/ml), and incubated at 37° C. for 18 h. Bacteria were harvested by centrifugation, washed in sterile PBS and concentrated to a density of 2×10.sup.10 CFUs/ml in PBS. Bacterial suspensions (10.sup.10 CFUs in 300 μl) were gavaged into the stomach. The procedure was repeated every 3 day for 3 weeks and mice were sacrificed at day 28. Intestinal contents were collected by lavages with 5 ml of intestinal wash buffer (PBS, 0.5M EDTA, Soybean trypsin inhibitor, PMSF), spun at 14′000 rpm in a sterile tube and filtered (0.22 μm) to remove any bacteria-sized contaminants.sup.4. For flow cytometry analysis of anti-E. coli IgA, 3 ml of LB broth were inoculated with single colonies and cultured overnight at 37° C. Cultures were subsequently centrifuged (3 min at 7000 rpm), washed 3 times with sterile-filtered PBS, 2% BSA, 0.005% NaN.sub.3 and resuspended at a density of approximately 10.sup.7 bacteria per ml. Intestinal contents and bacteria were then mixed and incubated at 4° C. for 1 h. Bacteria were washed twice, before being resuspended in monoclonal FITC-anti-mouse IgA (Southern Biotech, Cat.#: 1040-02, working dilution 1:200). After 1 h incubation bacteria were washed twice and resuspended in 2% paraformaldehyde in PBS for acquisition on a FACSCanto using FSC and SSC parameters in logarithmic mode. For each animal analyzed, ELISA was used to determine the total IgA concentration in an undiluted aliquot of the same intestinal wash sample used for surface staining of E. coli. This value was used to calculate the total IgA concentration at each dilution of intestinal wash used for flow cytometry of E. coli and was plotted against the geometric mean fluorescence obtained in flow cytometry.

(27) Oral Immunization with Attenuated Salmonella typhimurium and Flow Cytometry for Detection of Anti-Salmonella typhimurium IgA

(28) Avirulent gyrA1816 Δcya1 Δcrp1 S. typhimurium (which have mutations in cya and crp genes and are incapable of producing functional adenylate cyclase as well as cyclic AMP receptor protein) (ATCC® 53648™) transformed with pBAD28 or pHND10 were aseptically inoculated into LB medium containing arabinose (0.05%) and chloramphenicol (30 μg/ml), and incubated at 37° C. for 18 hours. Bacteria were harvested by centrifugation, washed in sterile PBS and concentrated to a density of 5×10.sup.10 CFU/ml in PBS. Bacterial suspensions (5×10.sup.9 CFUs in 100 μl) were gavaged into the stomach of normally colonized C57BL/6 mice every three days for three times. Arabinose 0.05% was added in the drinking water to ensure maximal expression of apyrase by pHND10 transformants. One month after the last immunization, mice were tested for anti-Salmonella secretory IgA as described above for E. coli colonized mice.

Example 1

E. coli in Normally Colonized Mice

(29) Detection of Extracellular ATP Levels Produced by Commensals

(30) Extracellular ATP had previously been detected in the supernatant of in vitro cultured intestinal commensals derived from murine faeces.sup.5,6. In order to address whether the metabolic activity of commensals contributed to the level of intestinal ATP, the levels of ATP in the small intestine was observed in mice from a specific pathogen free facility and in entirely germ free mice. Micromolar concentrations of ATP were detected in the specific pathogen free mice, whereas ATP was barely detectable in the entirely bacteria free mice. It was found that fluids such as urine, bile and serum which originate from sterile (or almost sterile) epithelial or endothelial organs did not demonstrate a substantial amount of endoluminal ATP (see FIG. 1(a)). This finding indicates that mucosal colonization by commensals plays a role in increasing extracellular ATP levels.

(31) To test whether bacteria present in the small intestine release ATP, cultures of aerobic and anaerobic colonies isolated from murine ilea were tested for ATP in the medium in comparison to cell growth. FIG. 1(b) shows that ATP in the medium was found to increase proportionally with bacterial growth. These data therefore show that bacteria present in the small intestine contribute to the generation of luminal ATP.

(32) The inventors also investigated whether bacterial cell death results in an additional increase in extracellular ATP due to release of ATP during cell lysis. Ileal bacterial cell cultures were treated with vancomycin, ampicillin and metronidazole (VAM). Cell death was monitored using DAPI (4′,6-diamidino-2-phenylindole) staining and membrane damage was monitored using DIBAC (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) staining in flow cytometry as described in reference 7. FIG. 1(d)-(f) shows that bacterial cell death and membrane permeability were associated with prominent ATP release. In vivo oral administration with VAM also resulted in an acute and significant increase in endoluminal ATP as shown in FIG. 1(g) in association with increased phosphatidyl serine exposure (which is a signal of cell death) in Tfh cells from Peyer's patches of wild type mice but not P2rx7.sup.−/− mice. This indicates that release of ATP by bactericidal death affects the abundance of Tfh cells via P2X7.

(33) Epithelial Permeability to ATP

(34) In order to assess epithelial permeability to ATP in the small intestine, mice were gavaged daily with ATPγS (a non-hydrolysable analogue of ATP). As Tfh cells are sensitive to extracellular ATP via P2X7 the inventors analysed Tfh cell recovery in Peyer's patches two weeks after treatment. Administration of ATPγS resulted in significant reduction of Tfh cells in wild type mice but not in p2rx7.sup.−/− mice. This finding suggests that luminal ATP can permeate Peyer's patches and can affect the abundance of Tfh cells via P2X7.

(35) Analysis of ATP concentrations in blood collected from portal or jugular veins, vena cava and heart revealed a 30-50 fold increase in ATP concentration in the blood from the portal vein compared to the other samples (see FIG. 1(c)), indicating that ATP is readily absorbed in the small intestine.

(36) Reducing Endoluminal ATP Levels Using Apyrase

(37) The inventors used a recombinant E. coli K-12 strain expressing pHND10 (a pBAD28-based recombinant plasmid carrying the phoN2::HA fusion.sup.8 which encodes a periplasmic apyrase (ATP-diphosphohydrolase) from Shigella flexneri.sup.9.

(38) Extracellular ATP released concomitantly with E. coli growth was undetectable in bacteria carrying pHND10 as shown in FIGS. 2(a) and (b), indicating that apyrase efficiently abrogated ATP secretion. Colonization of mice with pHND10 expressing E. coli resulted in significantly reduced ATP concentrations in the small intestine following VAM administration indicating that release of PhoN2 (apyrase) following bacterial cell death efficiently hydrolysed endoluminal ATP.

(39) Enhancement of Antigen Specific Immune Responses

(40) The enhancement of the anti-E. coli IgA response using PhoN2 was demonstrated by the inventors. C57BI/6 mice were gavaged with E. coli harbouring the pHND10 plasmid and the pBAD28 plasmid (which does not encode the apyrase PhoN2). The level of E. coli specific IgA was significantly increased in mice gavaged with bacteria transfected with pHND10 (which encodes apyrase). Therefore ATP released by bacteria limits the development of a high affinity IgA response and reducing ATP levels by expressing apyrase as demonstrated allows a high affinity IgA response to occur as shown in FIGS. 2(c) and 3. Anti-E. coli IgA elicited by apyrase expressing bacteria was equally reactive on both pBAD28 and pHND10 containing bacteria, indicating that apyrase did not promote an “apyrase specific” IgA response (see FIGS. 2(d) and (e)).

(41) The inventors addressed the role of apyrase as an adjuvant for high-affinity IgA response by measuring endoluminal ATP and anti-E. coli IgA after oral administration of chloramphenicol and ampicillin (CA; which are active on endogenous flora but not pBAD28-transformed E. coli) or penicillin/streptomycin/vancomycin (PSV; which are bactericidal on both endogenous and pBAD28 transformed bacteria). Anti-E. coli IgA in mice gavaged with pBAD28 transformed bacteria was reduced by PSV but not CA administration concomitantly with an increase in endoluminal ATP. Neither endoluminal ATP nor anti-E. coli IgA response were influenced by CA or PSV in mice colonized with pHND10 harboring bacteria (see FIGS. 2(f)-(h)).

(42) The finding explained above was found to be specific to the bacterium in which the apyrase was delivered, i.e. E. coli. IgA antibodies specific for other bacterial species were tested for, and no increase was recorded when pHND10 harbouring E. coli were administered to the mice compared to when E. coli harbouring pBAD28 were administered as shown in FIG. 2(i).

(43) Therefore, the data provided herein show that compositions comprising apyrase or another agent which is capable of reducing the level of binding of ATP to the P2X7 receptor can increase the specific IgA response to an immunogen included in the composition. Therefore, the compositions of the invention may be useful as vaccines.

Example 2

E. coli in Germ-Free, Monocolonized Mice

(44) The inventors monocolonized germ-free mice in order to demonstrate the effect of apyrase on endoluminal ATP levels, Tfh and germinal centre cell number and anti-E. coli IgA levels in a controlled experimental setting, in which the same amount of bacterial stimuli were present in the gut apart from extracellular ATP.

(45) Reducing Endoluminal ATP in the Presence of Apyrase in Monocolonized Mice

(46) Germ-free mice were monoclolonized by E. coli transformed with pHND10 or pBAD28. FIG. 4 shows that mice monocolonized with pHND10 harbouring E. coli showed significantly reduced concentration of endoluminal ATP in the intestine compared to mice monocolonized with pBAD28 harbouring E. coli and control mice. Germ-free mice which were not colonized with E. coli were used as a control.

(47) Number of Tfh Cells and Germinal Centre B Cells are Increased in the Presence of Apyrase in Monocolonized Mice

(48) Germ-free mice monocolonized with E. coli transformed with either pHND10 or pBAD28 were tested for number of Tfh cells (CD3.sup.+CD4.sup.+CXCR5.sup.+ICOS.sup.+) and number of germinal centre B cells (CD19.sup.+Fas.sup.+PNA.sup.+) by enumerating total cells in the Peyer's patches and extrapolating relative abundances of Tfh and GC B cells by their frequencies at FACS. FIG. 5 shows that the number of Tfh cells and germinal centre B cells were increased in mice monocolonized with pHND10 harbouring E. coli compared with mice monocolonized with pBAD28 harbouring E. coli or control mice.

(49) This finding is consistent with endoluminal ATP having a role in regulating Tfh cell and germinal centre B cells in the Peyer's patches of the small intestine. The presence of apyrase expressed in the pHND10 harbouring E. coli reduces levels of endoluminal ATP, which prevents ATP from reducing the number of Tfh cells and germinal centre cells.

(50) Anti-E. coli IgA is Increased in the Presence of Apyrase in Monocolonized Mice

(51) Germ-free mice monocolonized with E. coli transformed with either pHND10 or pBAD28 were tested for anti-E. coli IgA as described above. FIG. 6 shows that the level of E. coli specific IgA was significantly increased in mice monocolonized with pHND10 compared with mice monocolonized with pBAD28 or control mice.

(52) This finding confirms that the IgA produced as a result of the presence of apyrase is specific to an immunogen administered simultaneously with the apyrase.

Example 3

Attenuated Salmonella typhimurium in Normally Colonized Mice

(53) Generating Salmonella typhimurium which Expresses Apyrase

(54) To address whether apyrase expression in live attenuated Salmonella typhimurium could increase the specific IgA response and confer enhanced protection from infection by a virulent strain, the inventors used avirulent gyrA1816 Δcya1 Δcrp1 Salmonella typhimurium (ATCC® 53648™) (which includes mutations in cya and crp genes and is incapable of producing functional adenylate cyclase as well as cyclic AMP receptor protein) as a model vaccine. The inventors transformed S. typhimurium , with either pBAD28 or pHND10, as described above. As observed in E. coli, FIG. 7 shows that ATP was undetectable in culture medium of S. typhimurium carrying pHND10 (and therefore expressing apyrase) (see FIG. 7b) but was detected in increasing amounts that correlated with bacterial cell density in S. typhimurium carrying pBAD28 (and therefore not expressing apyrase) (see FIG. 7a).

(55) Anti-S. typhimurium IgA is Increased in the Presence of Apyrase

(56) Normally colonized mice were immunized by gavage every three days for three times with 5×10.sup.9 avirulent S. typhimurium transformed with either pHND10 or pBAD28. Arabinose 0.05% was added to the animals' drinking water to ensure maximal expression of apyrase by pHND10 transformants during immunization. After one month from the last immunization mice were tested for anti-Salmonella secretory IgA as described above for E. coli colonized mice. FIG. 8 shows that the Salmonella specific IgA response was significantly increased in mice immunized per os with pHND10 carrying attenuated S. typhimurium as compared to attenuated S. typhimurium carrying pBAD28 or control mice.

(57) This finding confirms that the IgA produced as a result of the presence of apyrase is specific to the immunogen administered simultaneously with the apyrase.

(58) Immunization with Attenuated S. typhimurium Carrying Apyrase Protects against Virulent S. typhimurium

(59) Colonization resistance by commensal flora limits infection with virulent S. typhimurium . In contrast, pretreatment of mice with streptomycin allows efficient development of enterocolitis and typhoid. Immunization of mice with attenuated S. typhimurium transformed with either pHND10 or pBAD28 was tested for its ability to protect against infection by 5×10.sup.7 virulent Salmonella (s. Tm.sup.wt: SB300 S. enterica serovar Thyphimurium SL1344 (wildtype) resistant to streptomycin, as disclosed in reference 10) upon streptomycin administration at one month from the last immunization. Infection of mice that have been previously immunized with avirulent Salmonella bearing pHND10 resulted in significantly reduced recovery of virulent Salmonella from Peyer's patches and barely detectable levels of virulent Salmonella in mesenteric lymph nodes (MLNs), spleen and liver when compared with mice immunized with pBAD28 transformants or control non-immunized mice at 48 h post-infection (see FIG. 9).

(60) This is consistent with the decrease in pathophysiological changes following infection observed in mice immunized with pHND10 containing attenuated S. typhimurium compared with mice immunized with pBAD28 containing S. typhimurium or non-immunized mice. FIG. 10 shows that spleen size and weight is greatly increased in infected mice immunized with pBAD28 harbouring S. typhimurium but not in infected mice immunized with pHND10 harbouring attenuated S. typhimurium as compared to a control spleen of untreated mice. FIG. 11 shows that liver histology is worsened in infected mice immunized with pBAD28 harbouring S. typhimurium but not in infected mice immunized with pHND10 harbouring attenuated S. typhimurium as compared to a control liver of untreated mice.

(61) A further way in which the inventors demonstrated that immunization with pHND10 containing attenuated S. typhimurium resulted in protection from virulent Salmonella was by monitoring the extent to which infection with virulent Salmonella causes leaking of the gut endothelial barrier. This leakage was monitored by analyzing the permeability of the gut to dextran administered to the mice by gavage. To this end, C57BL/6J mice immunized with avirulent Salmonella were orally infected with 5×10.sup.7 virulent Salmonella and gavaged with 5 mg of 70 KDa FITC-dextran. After 4 hours peripheral blood was collected and tested for the presence of the fluorophore in the serum. From each value, background fluorescence value of serum collected from untreated mice was subtracted.

(62) Mice that have been effectively protected from the effects of virulent Salmonella infection do not display gut permeability to dextran therefore sera from such mice contain less dextran that mice suffering from the effects of virulent Salmonella infection (i.e. which have been less efficiently protected).

(63) FIG. 12 shows that sera from mice immunized with pHND10 harbouring attenuated S. typhimurium contained significantly reduced amounts of dextran after infection with virulent S. typhimurium compared with control non-immunized mice and mice immunized with pBAD28 harbouring attenuated S. typhimurium . These results indicate that the IgA response provided by immunization with apyrase-expressing bacteria confers protection from systemic spreading of Salmonella.

(64) Adaptive IgAs are Responsible for the Protection Observed in the Presence of Apyrase

(65) The inventors immunized recombinase-activating gene-1 (Rag-1) deficient mice with attenuated S. typhimurium carrying either pBAD28 or pHND10, as described above for C57BL/6 mice, in order to observe the effect that such immunization had on mice unable to produce mature B or T lymphocytes. The presence of apyrase (in mice immunized with attenuated S. typhimurium carrying pHND10) did not result in enhanced protection from infection with virulent Salmonella as shown in FIG. 13 by comparable numbers of virulent Salmonella colony forming units (CFUs) recovered in mesenteric lymph nodes (MLNs), liver and spleen from control non-immunized mice or mice immunized with pBAD28 or pHND10 harbouring S. typhimurium at 48 h after challenge with virulent Salmonella. These results indicate that lymphocytes (e.g. adaptive IgAs) are responsible for the observed protection by immunization in wild-type mice.

(66) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

Example 4

Treatment of Mice with an Apyrase Composition in the Absence of an Immunogen in the Form of a Bacterial Carrier

(67) Extraction of Periplasmic Proteins

(68) E. coli transformed with pBAD28 or pHND10 were aseptically inoculated into LB medium containing arabinose (0.3%) and ampicillin (100 μg/ml), and incubated at 37° C. for 18 hours. Bacteria (10.sup.11) were spun at 6000 rpm for 20 min at 4° C., washed twice in PBS, resuspended in 1 ml of 30 mM Tris-HCl (pH 8.0), 4 mM EDTA, 1 mM PMSF, 20% sucrose and 0.5 mg/ml lysozime and incubated for 3 min at 30° C.; then, MgCl.sub.2 was added at a final concentration of 10 mM and bacteria incubated for 1 h a 30° C. Bacteria suspension was centrifuged at 10′000 rpm for 10 min at 4° C. and 100 μl supernatant (i.e. periplasmic proteins) administered to C57BL/6 mice by gavage.

(69) Fecal IgA Increased in Mice Treated with Periplasmic Proteins from Apyrase Bearing E. coli

(70) Fecal IgA concentrations were measured in mice that were either untreated or gavaged daily for 15 days with periplasmic proteins from arabinose induced pBAD28 (empty vector) or pHND10 (apyrase bearing vector) E. coli transformants.

(71) FIG. 14 shows that mice treated with an apyrase containing composition have increased fecal IgA compared with untreated mice or mice treated with a non-apyrase containing composition. These data indicate that apyrase is capable of providing an IgA immune response within an immunologically inert composition in the gut (as shown by lack of modification of IgA concentration by periplasmic proteins from pBAD28 transformants) and in the absence of an immunogen in the form of a bacterial carrier.

REFERENCES

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