Hafnia alvei impact on regulation of appetite and metabolic syndrome aspects

Abstract

The present invention relates to method of preventing, treating or attenuating obesity-related manifestations in a human or non-human mammal subject in need thereof, said method comprising orally administering to the subject a composition comprising an effective amount Hafnia alvei probiotics; wherein said obesity-related parameters are selected from the group of hyperphagia, increased fat mass on lean mass ratio, increased waist circumference, postprandial hyperglycemia, fasting hyperglycemia and hypercholesterolemia.

Claims

1. A method of treating or attenuating obesity-related manifestations in a human or non-human mammal subject in need thereof, said method comprising orally administering to the subject a composition comprising an effective amount of Hafnia alvei probiotics; wherein said obesity-related manifestations are selected from the group consisting of hyperphagia, increased fat mass on lean mass ratio, increased waist circumference, postprandial hyperglycemia, fasting hyperglycemia and hypercholesterolemia; and wherein the subject is obese or overweight.

2. The method according to claim 1, wherein the subject has at least 180 or at least 200 mg/dL of total cholesterol and/or at least 100 or at least 130 mg/dL of LDL cholesterol.

3. The method according to claim 1, wherein the subject has at least 80 or at least 100 mg/dL of glucose at a fasting state.

4. The method according to claim 1, wherein the subject has at least 80 or at least 90 waist-to-hip ratio.

5. The method according to claim 1, wherein Hafnia alvei probiotics are administered at a dose between 1000 million and 10000 million UFC.day-1.

6. The method according to claim 1, wherein the Hafnia alvei probiotic is administered to the subject in the form of a pharmaceutical composition.

7. The method according to claim 1, wherein the Hafnia alvei probiotic is administered to the subject in the form of companion composition to be administered simultaneously or sequentially with a pharmaceutical composition in a method of treatment of a metabolic syndrome condition selected from the group consisting of hyperglycemia, hypercholesterolemia, and hepatic disfunction.

8. The method according to claim 1, wherein the Hafnia alvei probiotic is administered to the subject in the form of a nutraceutical or food complement composition.

Description

FIGURES

(1) FIGS. 1A-E: Proteomic identification of molecular mimicry between E. coli K12 proteins and α-MSH.

(2) (a) 2D GE of E. coli cytoplasmic proteins. (b, c) Immunoblots of E. coli proteins detected with Rb anti-α-MSH IgG, preadsorbed (c) or not (b) with α-MSH. Circles in red surround the spots specifically recognized by α-MSH IgG which were used for protein identification. Circles in blue indicate nonspecific spots. Proteins identified in the spots 1-4 are isoforms of ClpB. (d) α-MSH and ClpB amino-acid sequence alignments using the Stretcher program. (e) Western blot of the recombinant ClpB, revealed with anti-α-MSH IgG. Lanes 1 and 2, 20 and 10 μg of ClpB, respectively.

(3) FIGS. 2A-K. ClpB immunization in mice.

(4) ClpB-immunized mice (ClpB+Adj) were compared with mice receiving adjuvant (Adj), PBS or controls (Ctr). (a) Body weight changes during 32 days of the study. Food intake and feeding pattern were studied during the last 2 weeks in the BioDAQ cages. Mean daily food intake (b), meal size (c) and meal number (d) during last 10 days of the study. (e) Food intake during 24 h after injection of α-MSH (100 μg kg-1 body weight, i.p.) or PBS. (f) Plasma levels of ClpB-reactive IgG before and after adsorption with 10.sup.−6M α-MSH. (g) Affinity of anti-ClpB IgG shown as the dissociation equilibrium constants (KD values). (h) Plasma levels of α-MSH-reactive total IgG. (i) Affinity (KD) of anti-α-MSH IgG. (j) cAMP assay in human embryonic kidney-293 cells overexpressing MC4R after stimulation by α-MSH alone or together with IgG (0.5 mg ml.sup.−1) pooled from ClpB-immunized or from Adj-injected mice. (k) The cAMP assay was performed with IgG depleted from anti-α-MSH IgG. (a) Two-way repeated measurement analysis of variance (ANOVA) before α-MSH injection (100 μg kg.sup.−1 body weight, i.p.), Po0.0001, Bonferroni post tests *a at least, Po0.05 ClpB group vs Ctr; *b at least, Po0.05 Adj group vs Ctr.; *c, Po0.05, Student's t-test ClpB group vs PBS; and *d, Po0.05, Student's t-test ClpB group vs Ctr. (b) ANOVA P=0.0002, Tukey's post tests ***Po0.001, **Po0.01, #Po0.05, Student's t-test. (c) ANOVA P=0.007, Tukey's post tests **Po0.01. (e) Student's t-test, *P00.05. (f, g) ANOVA Po0.0001, Tukey's posttests ***Po0.001 ClpB+Adj vs other groups, paired t-test ##Po0.01, ###Po0.001. (h) ANOVA P=0.0002, Tukey's post tests ***Po0.001, *P00.05; (i) Kruskal-Wallis test P=0.003, Dunn's post test **Po0.01, (mean±s.e.m., n=8). (j) ANOVA P=0.005, Tukey's post test *P00.05; ANOVA P=0.04, Student's t-test #Po0.05, aClpB vs α-MSH, bClpB vs Adj. (mean±s.e.m.; j, n=6, k, n=3).

(5) FIG. 3A-J: E. coli supplementation in mice.

(6) Effects of intragastric daily gavage (days 1-21) in mice with either E. coli K12 wild-type (WT), ClpBdeficient (ΔClpB) E. coli K12 or LB medium on body weight (a), food intake (b), meal size (c) and meal number (d). (e) PCR detection of a 180-base pair fragment of the bacterial ClpB DNA, first lane, molecular weight marker, second lane DNA from in vitro cultures of E. coli K12 WT, third lane DNA from in vitro cultures of E. coli K12 ΔClpB, and the remaining lanes DNA from mice feces collected at day 21. Plasma levels in optical density in enzyme-linked immunosorbent assay of anti-ClpB IgM (f) and IgG (g) before and after adsorption with 10.sup.−6M α-MSH.

(7) Plasma levels of anti-α-MSH IgM (h) and IgG (i). a) Affinity (equilibrium constant) of anti-α-MSH IgG. (a) Two-way repeated measurements analysis of variance (ANOVA), P=0.3, Bonferroni post test day 2, **Po0.01 control (Ctr) vs E. coli WT. (b) ANOVA days 1-2, P=0.0006, Tukey's post tests ***Po0.001, *P00.05, E. coli WT vs aCtr and bLB. (c) Kruskal-Wallis (K-W) test third week P=0.0001, Dunn's post tests, ***Po0.001, **Po0.01, E. coli WT vs aCtr, bLB and CΔClpB. (d) ANOVA days 1-2, P=0.006, Tukey's post tests **Po0.01, *P00.05, K-W test third week Po0.0001, Dunn's post tests, ***Po0.001, **Po0.01, E. coli WT vs aCtr, bLB and CΔClpB. (f) K-W test, before adsorption P=0.02, Dunn's post tests *P00.05, ANOVA after adsorption, Po0.0001, Tukey's post tests **Po0.01, E. coli WT vs other groups. (g) ANOVA before adsorption, P=0.01, Tukey's post tests *P00.05, E. coli WT vs other groups, paired t-test ##Po0.01. (h) Student's t-test, E. coli WT vs other groups *P00.05. (j) K-W test P=0.02, Dunn's post test *P00.05, Mann-Whitney test, #Po0.05. (mean±s.e.m., n=8).

(8) FIG. 4A-F. Anti-ClpB antibodies in ED patients.

(9) Plasma levels of anti-ClpB IgG (a) and IgM (b) in healthy women (control, Ctr) and in patients with AN, BN and BED. Plasma levels of ClpB IgG (c) and IgM (d) before and after adsorption with 10.sup.−6M α-MSH. Percentage of α-MSH crossreactive anti-ClpB IgG (e) and IgM (f). (b) Student's t-test *Po0.05. (c, d) Paired t-tests, ***Po0.001, **Po0.01. (e) Kruskal-Wallis test Po0.0001, Dunn's post test, **Po0.01, Mann-Whitney test #Po0.05. (f) Analysis of variance P=0.02, Tukey's post test *P00.05. (mean±s.e.m., Ctr, n=65, AN, n=27 BN, n=32 and BED, n=14).

(10) FIG. 5. Plasma concentrations of bacterial ClpB protein in patients with eating disorders and healthy controls (Ctr).

(11) AN, anorexia nervosa, BN, bulimia nervosa, BED, binge-eating disorder. *p<0.05 Student's t-test of mean ClpB concentrations vs. Ctr. Percentage (%) of patients having ClpB concentrations higher than mean+2 standard deviations (SD) of controls.

(12) FIG. 6: Body weight dynamics in obese ob/ob mice before and during (Days 0-21) intragastric gavage with E. coli K12, E. coli K12 ΔClpB, both in Mueller-Hilton (MH) medium, or with MH medium only, as a control (Ctr). 2-way ANOVA, Effect of treatment: p=0.01, Bonferroni post-tests Ctr. vs. E. coli K12, * p<0.05 and **p<0.01. Mean±SEM.

(13) FIG. 7: Percentage of mean body weight change (from the day of randomization=100%) in obese ob/ob mice after 3 weeks of intragastric gavage with E. coli K12 (n=8), E. coli K12 ΔClpB (n=8), both in Mueller-Hilton (MH) medium, or with the MH medium only, as a control (Ctr., n=7). ANOVA p=0.01, Tukey's post-tests * p<0.05. Mean±SEM.

(14) FIG. 8: Fat content in obese ob/ob mice measured by EchoMRI after 3 weeks of intragastric gavage with E. coli K12 (n=8), E. coli K12 ΔClpB (n=8), both in Mueller-Hilton (MH) medium, or with the MH medium only, as a control (Ctr., n=7). ANOVA p=0.005, Tukey's post-test **p<0.01. Mean±SEM.

(15) FIG. 9: Lean to fat mass ratios in obese ob/ob mice measured by EchoMRI after 3 weeks of intragastric gavage with E. coli K12 (n=8), E. coli K12 ΔClpB (n=8), both in Mueller-Hilton (MH) medium, or with the MH medium only, as a control (Ctr., n=7). ANOVA p=0.05, Student's t-test *p<0.05. Mean±SEM.

(16) FIG. 10: Mean daily food intake in obese ob/ob mice during 3 weeks of intragastric gavage with E. coli K12 (n=8), E. coli K12 ΔClpB (n=8), both in Mueller-Hilton (MH) medium, or with the MH medium only, as a control (Ctr., n=7). ANOVA p<0.0001, Tukey's post-test ***p<0.001. Mean±SEM.

(17) FIG. 11: Mean daily meal number in obese ob/ob mice during 3 weeks of intragastric gavage with E. coli K12 (n=8), E. coli K12 ΔClpB (n=8), both in Mueller-Hilton (MH) medium, or with the MH medium only, as a control (Ctr., n=7). ANOVA p<0.0001, Tukey's post-test ***p<0.001. Mean±SEM.

(18) FIG. 12: Daily body weight gain in obese ob/ob mice during intragastric gavage with E. coli K12, E. coli Niessle 1917, E. coli Niessle 1917 lyophilized (lyo), all in Mueller-Hilton (MH) medium, or with MH medium only, as a control (Ctr). 2-way ANOVA, p=0.02, Bonferroni post-tests a, Ctr. vs. E. coli Niessle 1917 lyo and b, Ctr. vs. E. coli Niessle 1917, * p<0.05 and **p<0.01. Mean±SEM.

(19) FIG. 13: Percentage of mean body weight change (from the day of randomization=100%) in obese ob/ob mice after 2 weeks of intragastric gavage with E. coli K12, E. coli Niessle 1917, E. coli Niessle 1917 lyophilized (lyo), all in Mueller-Hilton (MH) medium, or with MH medium only, as a control (Ctr). Kruskal-Wallis p<0.01, Dunn's post-test *p<0.05, Mann-Whitney test ##p<0.01. Mean±SEM.

(20) FIG. 14: Fat content in obese ob/ob mice measured by EchoMRI after 2 weeks of intragastric gavage with E. coli K12, E. coli Niessle 1917, E. coli Niessle 1917 lyophilized (lyo), all in Mueller-Hilton (MH) medium, or with MH medium only, as a control (Ctr). Student's t-test **p<0.01. Mean±SEM.

(21) FIG. 15: Body weight gain (in g) in obese ob/ob mice after 17 days of intragastric gavage with E. coli Niessle 1917 (Nissle) (n=15), E. coli K12 (K12 Δ-ClpB) (n=15) or Hafnia alvei AF036 (Hafnia) (n=15), all in Mueller-Hilton (MH) medium, or with MH medium only, as a control (Ctrl) (n=12).

(22) FIG. 16: Cumulative food intake (in g) in obese ob/ob mice after 17 days of intragastric gavage with E. coli Niessle 1917 (Nissle) (n=15), E. coli K12 (K12 Δ-ClpB) (n=15) or Hafnia alvei AF036 (Hafnia) (n=15), all in Mueller-Hilton (MH) medium, or with MH medium only, as a control (Ctrl) (n=12).

(23) FIGS. 17A-C: Fat and lean mass gain in obese ob/ob mice measured by EchoMRI after 17 days of intragastric gavage with E. coli Niessle 1917 (Nissle) (n=15), E. coli K12 (K12 ΔClpB) (n=15) or Hafnia alvei AF036 (H. alveri) (n=15), all in Mueller-Hilton (MH) medium, or with MH medium only, as a control (Ctrl) (n=12). A. Fat mass gain (in g). B. Lean mass gain (in g). C. Lean-to-fat mass ratios.

(24) FIGS. 18A-C: High fat diet validation. A. Body weight (in g) in mice fed with a high fat/high carbs diet (HFD) (n=48) and in mice fed with a control diet (Ctrl) (n=8). B. Fat mass (in g). C. Lean mass (in g).

(25) FIG. 19: Reduction in body weight gain from D0 in high fat diet (HFD)-induced obese mice after 14 days of intragastric gavage with E. coli K12 (K12 Δ-ClpB) or Hafnia alvei AF036 (H. alvei), all in Mueller-Hilton (MH) medium, or with MH medium only, as a control (Ctrl).

(26) FIGS. 20A-B: Fat and lean mass gain from D0 in high fat diet (HFD)-induced obese mice after 14 days of intragastric gavage with E. coli K12 (K12 Δ-ClpB) or Hafnia alvei AF036 (H. alvei), all in Mueller-Hilton (MH) medium, or with MH medium only, as a control (Ctrl). A. Fat mass gain (in g). B. Lean mass gain (in g).

(27) FIG. 21: Relative hormone-sensitive lipase protein (pHSL) expression rate (against actin expression rate as a standard) in obese ob/ob mice after 17 days of intragastric gavage with E. coli Niessle 1917 (E. coli Nissle) (n=15), E. coli K12 (E. coli K12) (n=15) or Hafnia alvei AF036 (Hafnia alvei) (n=15), all in Mueller-Hilton (MH) medium, or with MH medium only, as a control (Ctrl) (n=12). HSL and actine expression rates were measured by western blot.

(28) FIG. 22: Experimental protocol of the study of examples 7-10.

(29) FIGS. 23A-C: Effects of H. alvei and orlistat on body weight in high-fat-diet (HFD)-fed ob/ob mice as compared to standard diet (SDiet). (A) Body weight dynamics, vertical dashed line defines beginning of H. alvei or orlistat treatments (B) Body weight gain dynamics. (C) Body weight gain during treatment as an area under curve (AUC). (A,B) Two-way RM ANOVA, p<0.0001, Bonferroni's posttests, HFD vs. HFD+Orlistat, *** p<0.001, ** p<0.01, * p<0.05; C. ANOVA, p<0.0001, Tukey's posttests ays. HFD and bvs. HFD+H. alvei, both *** p<0.001, Student's Nests, * p<0.05, (mean±SEM; SDiet, n=16, all other groups, n=24).

(30) FIGS. 24A-C: Effects of H. alvei and orlistat on body composition in high-fat-diet (HFD)-fed ob/ob mice as compared to standard diet (SDiet). Percentage of fat (A) and lean (B) mass, as well as lean/fat mass ratio (C) at the end of the treatment. (A,C) ANOVA, p=0.0004, (B) ANOVA, p=0.0002, Bonferroni's posttests, *** p<0.001, * p<0.05. (A-C) Student's Nests, #p<0.05, (mean±SEM; SDiet, n=16, all other groups, n=24).

(31) FIGS. 25A-C: Effects of H. alvei and orlistat on food intake in high-fat-diet (HFD)-fed ob/ob mice as compared to standard diet (SDiet). (A) Dynamics of daily food intake. (B) Dynamics of cumulative food intake/mouse. (C) Mean cumulative food intake/mouse. (A) Two-way RM ANOVA, p<0.0001, Bonferroni's posttests, HFD vs. HFD+Orlistat. *** p<0.001, * p<0.05 days 8, 9, 14 and 19. HFD+H. alvei vs. HFD+Orlistat. *** p<0.001, * p<0.05 days 5 and 6. (B) Two-way RM ANOVA, p<0.0001, Bonferroni's posttests, HFD vs. HFD+Orlistat. *** p<0.001; ** p<0.01 days 24, 25; * p<0.05 days 22,23. HFD+H. alvei vs. HFD+Orlistat. *** p<0.001, ** p<0.01, days 17, 18; * p<0.05 days 15,16. (C). ANOVA p<0.0001, Tukey's posttests *** p<0.001, * p<0.05, (mean±SEM; SDiet, n=16, all other groups, n=24).

(32) FIGS. 26A-E: Effects of H. alvei HA4597™ and orlistat on glycemia and obesity-related metabolic parameters in high-fat-diet (HFD)-fed ob/ob mice as compared to standard diet (SDiet). (A) Plasma glucose levels in ad libitum feeding conditions. (B) OGTT after overnight fasting. Plasma levels of (C) triglycerides, (D) total cholesterol, and (E) alanine aminotransferase (ALAT) in ad libitum feeding conditions. (A). ANOVA p<0.05, Tukey's posttest vs. SDiet * p<0.05. Student's t-test #p<0.05. (B) Two-way RM ANOVA p<0.05, Bonferroni posttests, SDiet vs. HFD+orlistat * p<0.05 and a* p<0.05, ** p<0.01 for 90 and 120 min; HFD+H. alvei vs. HFD+Orlistat b* p<0.05, ** p<0.01 for 90 min. (C) ANOVA p<0.05, Tukey's posttest vs. SDiet * p<0.05. (D) ANOVA p<0.001, Tukey's posttests *** p<0.001, ** p<0.01, Student's t-test #p<0.05. (E) ANOVA p=0.0006, Tukey's posttests ** p<0.01, * p<0.05, (mean±SEM; SDiet, n=8, all other groups n=12).

EXAMPLES

(33) The following examples demonstrate that the ClpB chaperon protein of commensal gut bacteria E. coli K12 is a conformational mimetic of α-melanocyte-stimulating hormone (α-MSH), a neuropeptide involved in the regulation of energy metabolism and emotion. They also reveal a molecular link between ClpB expressing gut bacteria and the regulation of motivated behavior and emotion via production of ClpB protein and anti-ClpB antibodies crossreactive with α-MSH. They further support the involvement of ClpB-expressing microorganisms in increased ClpB protein and ClpB antibody production and establishment of abnormal feeding behavior and emotion.

Example 1

Materials and Methods

(34) E. coli K12 Culture and Protein Extraction

(35) The bacterial strain used in this study was E. coli K12, provided by UMR 6270 CNRS Laboratory in Rouen University, France. E. coli K12 was grown in 250 ml Luria Bertani (LB) broth (MP Biomedicals, Illkirch, France) at 37° C. for 24 h. Protein extraction was performed as described by Marti et al. (PLoS ONE 2011, e26030). In brief, bacteria were harvested by centrifugation at 4000 g for 30 min at 4° C. and the resulting pellet was resuspended in extraction buffer (300 mM NaCl and 20 mM Tris-HCl, pH 8). The suspension was disrupted by sonication (3×3 min, pulse ON 1 s, OFF 1 s at 21% of amplitude) and centrifuged at 10 000 g for 10 min at 4° C. The supernatant was recovered and ultracentrifuged at 4° C. for 45 min at 60 000 g to further separate proteins into cytoplasmic (supernatant) and envelope (pellet) fractions. Protein concentrations were measured using 2-D Quant Kit (GE Healthcare, Piscataway, N.J., USA).

(36) Two-Dimensional Polyacrylamide Gel Electrophoresis

(37) For two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE), 400 μg of E. coli K12 protein extract were added to iso-electro focusing buffer (7M urea, 2M thiourea and 0.5% ampholytes, pH 4-7, 20 mM DTT, 2 mM TBP, 2% CHAPS and 0.005% bromophenol blue) and solubilized for 60 min at room temperature with slight shaking. The first-dimensional gel separation was carried out using ReadyStrip IPG Strip (18 cm, pH 4-7 NL, Bio-Rad, Marnes-la-Coquette, France). After 24 h of passive rehydration of the strip with iso-electro focusing buffer, the protein sample was added to the strips through a loading cup placed at 1.5 cm from the cathode. Isoelectro focusing was performed with the Ettan IPGphor 3 System (GE Healthcare, Orsay, France) in four steps (31 500 Vh): 500 V for 1 h, 1000 V gradient, 10 000 V gradient and 10 000 V for 2 h. After two equilibration steps with 2% DTT and 2.5% iodoacetamide, respectively, the second dimension, that is, a SDS-PAGE, (10% polyacrylamide gel, 20 cm×18-cm×1 mm) was performed on an Ettan Daltsix vertical electrophoresis system (GE Healthcare) with 12 mA per gel. After SDS-PAGE, the 2D gel was fixed for 2 h in 2% (vol:vol) orthophosphoric acid and in 50% (vol:vol) methanol at room temperature. Gels were then rinsed with water, and the protein spots were visualized by CBB G-250 (Bio-Rad) staining (34% (vol:vol) methanol, 17% (wt:vol) ammonium sulfate, 2% (vol:vol) orthophosphoric acid and 0.66 g CBB G-250 per liter).

(38) Immunoblotting

(39) Following 2D-PAGE, E. coli cytoplasmic proteins were transferred onto Hybond-ECL PVDF membrane (GE Healthcare) via a dry transfer method (Trans Blot Cell, Bio-Rad, USA) and a constant current of 0.8 mA.cm.sup.−2 of the membrane size for 2 h. After transfer, membranes were blocked with 5% (wt:vol) milk (Regilait, France) in phosphate-buffered saline (PBS; 10 mmol.Math.l.sup.−1 Tris, pH 8, and 150 mM.Math.l.sup.−1 NaCl) plus 0.05% (vol:vol) Tween 20. After washes, membranes were incubated overnight at 4° C. with polyclonal rabbit anti-α-MSH IgG (1:1000, Peninsula Laboratories, San Carlos, Calif., USA), followed by washes and incubation with polyclonal swine anti-rabbit horseradish peroxidase-conjugated Igs (1:3000; Dako, Trappes, France). Immunoblots were revealed by the ECL detection system (GE Healthcare) and were scanned with ImageScanner 11 (GE Healthcare) previously calibrated by using a greyscale marker (Kodak) and digitalized with Labscan 6.00 software (GE Healthcare). The same procedure was performed after adsorption of rabbit anti-α-MSH IgG with 10.sup.−6M of α-MSH peptide (Bachem AG, Bubendorf, Switzerland) overnight at 4° C.

(40) Protein Identification

(41) The protein spots of interest were excised from CBB G-250-stained 2D gels using the Ettan Spot Picker (GE Healthcare), and automated in-gel digestion of proteins was performed on the Ettan Digester (GE Healthcare). Protein extracts were then resuspended in 10 μl of 5% (vol:vol) acetonitrile/0.1% (vol:vol) formic acid and then analyzed with a nano-LC1200 system coupled to a 6340 Ion Trap mass spectrometer equipped with a nanospray source and an HPLC-chip cube interface (Agilent Technologies, Courtaboeuf, France). In brief, peptides were enriched and desalted on a 40-nl RPC18 trap column and separated on a Zorbax (30-nm pore size, 5-μm particle size) C18 column (43 mm long×75 μm inner diameter; Agilent Technologies). A 9-min linear gradient (3-80% acetonitrile in 0.1% formic acid) at a flow rate of 400 nl.Math.min.sup.−1 was used, and the eluent was analyzed with an Ion Trap mass spectrometer. For protein identification, MS/MS peak lists were extracted and compared with the protein databases by using the MASCOT Daemon version 2.2.2 (Matrix Science) search engine. The searches were performed with the following specific parameters: enzyme specificity, trypsin; one missed cleavage permitted; no fixed modifications; variable modifications, methionine oxidation, cysteine carbamidomethylation, serine, tyrosine and threonine phosphorylation; monoisotopic; peptide charge, 2+ and 3+; mass tolerance for precursor ions, 1.5 Da; mass tolerance for fragmentations, 0.6 Da; ESI-TRAP as instrument; taxonomy, E. coli; National Center for Biotechnology Information (NCBI) database (NCBInr 20120531 (18280215 sequences, 6265275233 residues); Bethesda, Md., USA). Protein hits were automatically validated if they satisfied one of the following criteria: identification with at least two top-ranking peptides (bold and red) each with a MASCOT score of 454 (Po0.01), or at least two top-ranking peptides each with a MASCOT score of 447 (Po0.05). To evaluate false-positive rates, all the initial database searches were performed using the ‘decoy’ option of MASCOT. Results were considered relevant if the false-positive rate never exceeded 1%.

(42) Protein Identification from OFFGEL

(43) High-resolution E. coli K12 protein separation into 24 fractions was done onto the 3100 OFFGEL fractionator using the OFFGEL pH3-10 kit (Agilent Technologies). Protein samples (400 μg) preparation and assembly of all parts of the OFFGEL systems were done according to the procedures described in the Agilent Quick start Guide. OFFGEL fractionation was performed using the standard program OG24PRO with maximum limited current parameters (8000 V, 50 μA and 200 mW) until 64 KVh was reached after 30 h. At the end of the experiment, all fractions were transferred into a 0.8-ml deep well (Thermo Fisher Scientific, Illkirch, France) and stored at −20° C. Nine protein-containing fractions recovered from the central part of the OFFGEL were studied by western blot using rabbit anti-α-MSH IgG (Peninsula Laboratories) followed by protein identification as described above.

(44) Immunization and Behavior in Mice

(45) All experimental protocols were conducted according to US National Institutes of Health guidelines and EU directives, and animal experiments were approved by the Institutional Ethical Committees. Two-month-old male C57Bl6 mice (Janvier Laboratories, L'Arbresle, France) were acclimated to the animal facility for 1 week with 12 h light-dark cycle, lights on at 0700 hours and were kept in standard mouse-holding cages (n=8) each. Mice were fed ad libitum with standard pelleted rodent chow (RM1 diet, SDS, UK) with drinking water always available and were manipulated daily by gentle holding and measuring body weight. During acclimatization, mice were distributed between four cages to obtain the similar mean body weight per mouse per cage. After 1 week of acclimatization, mice from each cage were assigned to one of four study group and received following treatments: (i) Group 1, ClpB immunization (n=8): ClpB protein (Delphi Genetics, Gosselies, Belgium) 50 μg per mouse in 200 μl of 1:1 (vol:vol) of PBS with Complete Freund's Adjuvant (Sigma, St Louis, Mo., USA), intraperitoneally (i.p.); (ii) Group 2, adjuvant injection controls (n=8): 200 μl of Complete Freund's Adjuvant in PBS (1:1 (vol:vol), i.p.); (iii) Group 3, PBS injection controls (n=8): 200 μl of PBS (i.p.); and (iv) Group 4, intact controls (n=8): received no injections, and then all mice were returned to their holding cages. Fifteen days later, mice were given a boost immunization and the following treatments: (i) Group 1 (n=8), ClpB protein (Delphi Genetics) 50 μg per mouse in 200 μl of 1:1 (vol:vol) of PBS with Incomplete Freund's Adjuvant (Sigma) i.p.; (ii) Group 2 (n=8): 200 μl of Incomplete Freund's Adjuvant in PBS (1:1 (vol:vol), i.p.); (iii) Group 3, (n=8): 200 μl of PBS (i.p.); and iv) Group 4, (n=8): received no injections. Next day after the boost, mice were placed individually into the BioDAQ mouse cages (Research Diets, Inc., New Brunswick, N.J., USA) each equipped with an automatic feeding monitor. Food (Serlab, Montataire France) and drinking water were available ad libitum and body weight was measured daily. After 3 days of acclimatization to the BioDAQ cages, mice received the following treatments: Groups 1, 2 and 3 (each n=8), that is, mice that have been immunized with ClpB, injected with adjuvants and with PBS, respectively, all received an acute injection of α-MSH peptide (Bachem AG), 100 μg.Math.kg.sup.−1 body weight in 100 μl of PBS (i.p.) at 1000 hours. The control mice (n=8) received PBS only (i.p.). Feeding data was continuously monitored and analyzed using the BioDAQ data viewer 2.3.07 (Research Diets). For the meal pattern analysis, the inter-meal interval was set at 300 s. After the feeding study, mice were placed in individual mouse-holding cages with food and water available ad libitum, and were analyzed for locomotor activity and anxiety in 0-maze (Med Associate, Inc., St Albans, Vt., USA) tests performed during 2 consecutive days. Two hours after the 0-maze test, mice were killed by decapitation in a guillotine and trunk blood was collected into EDTA-containing tubes. Plasma was separated by centrifugation at 3500 r.p.m. (1.4 g) for 10 min at 4° C. and stored at −80° C. before assay.

(46) Locomotor Activity and Anxiety Tests

(47) After feeding study in the BioDAQ cages, mice were analyzed for locomotor activity using a Versamax Animal Activity Monitor (AccuScan Instruments, Inc., Columbus, Ohio, USA). Next day after the locomotor activity test, all mice were tested for their anxiety in an elevated O-maze. The elevated-O-maze is a variation of more commonly used elevated plusmaze pharmacologically validated for anxiety testing in rodents. The advantage of the O-maze is that it lacks the ambiguous central square of the traditional plus-maze. The O-maze consisted of a circular infrared platform (outer diameter 120 cm) elevated 80 cm above the floor, featuring two open and two closed segments made of gray plastic. The closed segments were enclosed by walls extending 20 cm above the surface of the maze and covered with a black infrared plexiglas lid. Each test started by placing the mouse into one of the two closed segments. The test lasted 5 min and was recorded using a video camera placed above the O-maze and the EthoVision video tracking software (Noldus IT, Wageningen, The Netherlands). Measurements of distance and time spent in the open and closed segments were analyzed. Between each mouse tests, the O-maze was cleaned with 30% ethanol.

(48) ClpB and α-MSH Autoantibody Assay

(49) Plasma levels of auto-Abs reacting with ClpB, α-MSH and adrenocorticotropic hormone were measured using enzyme-linked immunosorbent assay according to a published protocol (Fetissov S O., Methods Biol Mol 2011). In brief, ClpB protein (Delphi Genetics), α-MSH or adrenocorticotropic hormone peptides (Bachem AG) were coated onto 96-well Maxisorp plates (Nunc, Rochester, N.Y., USA) using 100 μl and a concentration of 2 μg.Math.ml.sup.−1 in 100 mM NaHCO3 buffer, pH 9.6, for 72 h at 4° C. Plates were washed (5 min for three times) in PBS with 0.05% Tween 200, pH 7.4, and then incubated overnight at 4° C. with 100 μl of mouse plasma diluted 1:200 in PBS to determine free auto-Ab levels or diluted 1:200 in dissociative 3M NaCl and 1.5M glycine buffer, pH 8.9, to determine total auto-Ab levels. The plates were washed (three times) and incubated with 100 μl of alkaline phosphatase (AP)-conjugated goat antimouse IgG (1:2000) or anti-mouse IgM (1:1000), all obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa., USA). Following washing (three times), 100 μl of p-nitrophenyl phosphate solution (Sigma) was added as AP substrate. After 40 min of incubation at room temperature, the reaction was stopped by adding 3 N NaOH. The optical density was determined at 405 nm using a microplate reader Metertech 960 (Metertech Inc., Taipei, Taiwan). Blank optical density values resulting from the reading of plates without addition of plasma samples were subtracted from the sample optical density values. Each determination was done in duplicate. The variation between duplicate values was inferior to 5%. Similar protocol was used to measure anti-ClpB IgG and IgM in human plasma (1:400) using corresponding anti-human IgG or IgM AP-conjugated antibodies (1:2000, Jackson ImmunoResearch Laboratories, Inc.).

(50) Absorptions of ClpB Antibodies with α-MSH

(51) Plasma samples of mice, diluted 1:200 in PBS, or humans, diluted 1:400 in PBS, were preincubated with 10.sup.−6M α-MSH peptide (Bachem AG) overnight at 4° C. before adding the samples to 96-well Maxisorp plates (Nunc) coated with ClpB protein (Delphi Genetics). IgG and IgM antibodies reactive with ClpB were detected by enzyme-linked immunosorbent assay using corresponding anti-mouse or anti-human AP-conjugated antibodies (Jackson ImmunoResearch Laboratories, Inc.) as described above. Percentage of ClpB antibodies crossreactive with α-MSH were calculated relative to levels of anti-ClpB antibodies detected without absorption in each individual plasma sample equal 100%.

(52) IgG Purification from Plasma

(53) IgG purification and affinity assay were performed according to a published protocol (Legrand et al., Protoc Exch 2014, doi:10.1038/protex2014.004). Extraction of plasma globulins was done by plasma acidification and separation on C18 SEP column (Phoenix Pharmaceuticals, Burlingame, Calif., USA), then 500 μl of mouse plasma was mixed with 500 μl of buffer A (1% trifluoroacetic acid in water). The column was activated in 1 ml of buffer B (60% acetonitrile in 1% trifluoroacetic acid) by 3 min centrifugation with 700 r.p.m. and rinsed three times with 3 ml of buffer A. Diluted plasma (1:1 in buffer A) was added to the column and the effluent (1 ml) was saved (frozen at −20° C.) for further purification of IgG. Total IgG were purified from the effluents of mouse plasma samples using the Melon Gel Kit (Thermo Fisher Scientific, Rockford, Ill., USA). Plasma effluents diluted 1:4 in kit's purification buffer was added on washed melon gel deposited in a column. Column was spun 1 min at 6000 r.p.m., and the IgG containing effluent was saved and frozen at −20° C. before lyophilization. Lyophilized IgG were reconstituted in the HBS-EP buffer (GE Healthcare, Piscataway, N.J., USA). For the cyclic adenosine monophosphate (cAMP) experiment, IgG purified from eight mice of the ClpB and of the adjuvant control group were combined, respectively, into two pools that were divided in two parts. One part was used directly in cAMP assay and the other was further purified using affinity chromatography for α-MSH (Bachem AG) coated on activated UltraLink beads (Pierce, Rockford, Ill., USA). The α-MSH IgGdepleted IgG effluents were saved, lyophilized and diluted in PBS.

(54) Affinity Kinetics Assay

(55) Affinity kinetics of mouse IgG for ClpB and α-MSH was determined by a biospecific interaction analysis based on the surface plasmon resonance phenomenon on a BIAcore 1000 instrument (GE Healthcare). α-MSH (Bachem AG) or ClpB protein (Delphi Genetics) were diluted to 0.5 mg.Math.ml.sup.−1 in 10 mM sodium acetate buffer, pH 5.0 (GE Healthcare), and were covalently coupled on the sensor chips CM5 (GE Healthcare) by using the amine coupling kit (GE Healthcare). All measures were performed on the same α-MSH or ClpB-coated chips. For the affinity kinetic analysis, a multicycle method was run with five serial dilutions of each IgG sample: 3360, 1680, 840, 420 and 210 (nmol), including a duplicate of 840 nmol and a blank sample (HBS-EP buffer only). Each cycle included 2 min of analyte injection and 5 min of dissociation with flow speed 30 μl.min.sup.−1 at 25° C. Between injections of each sample, the binding surface was regenerated with 10 mM NaOH, resulting in the same baseline level of the sensorgram. The affinity kinetic data were analyzed using BiaEvaluation 4.1.1 program (GE Healthcare). For fitting kinetic data, the Langmuir's 1:1 model was used, and the sample values were corrected by subtracting the blank values.

(56) In Vitro cAMP Assay

(57) Stable cell line of human embryonic kidney-293 cells expressing human MC4R was generated using a lentiviral transduction technology and purchased from Amsbio (Oxon, UK). High expression of MC4R mRNA in transfected cells was validated by reverse transcription PCR in Amsbio and in our laboratory. The presence of the transgene in cells before each experiment was verified by the visualization at a fluorescence microscope of the green fluorescent protein, which gene was inserted in the same with MC4R lentivector but under a different promoter. The α-MSH peptide (Bachem AG) was diluted in the induction buffer: PBS, 500 μM IBMX, 100 μM RO 20-1724 (Sigma), 20 mM MgCl2 to the final concentrations of 2, 1, 750, 500, 250, 100, 75, 50 and 10 nM corresponding to the α-MSH doses of 0.6, 3, 4.5, 6, 15, 30, 45, 60 and 120 μmol, respectively, and also included one blank sample. After unfreezing, the cells were cultured in 250 ml tissue culture flasks (BD-Falcon, Beckton-Dickinson, Bedford, Mass., USA) in Dulbecco's modified Eagle medium 4.5 g.Math.l.sup.−1 glucose (Eurobio, Courtaboeuf, France) supplemented with (2 mM L-glutamine, 10% fetal calf serum, 0.1 mM nonessential amino acids and 1% penicillin-streptavidin) in humidified cell culture incubator at 37° C., 5% CO2 for 8-10 days. At the day of experiment, cultured MC4R human embryonic kidney-293 cells were treated with 0.25% trypsin-EDTA (Sigma-Aldrich) and cell pellets were resuspended in PBS to obtain 5000 cells per well (10 μl) in a nontreated bioluminescence white 96-microwell plate (Nunc, Roskilde, Denmark). The cAMP production was measured using the bioluminescent assay cAMP-Glo Max Assay kit (Promega, Madison, Wis., USA) according to the manufacturer's instructions. In brief, the cells were incubated for 15 min at room temperature with different concentrations of α-MSH peptide alone or α-MSH together with mouse IgG pools from ClpB-immunized or adjuvant control groups, and which were added to the cells just before α-MSH. Serial dilutions of cAMP standard (provided by the kit) were assayed on the same microplate. cAMP detection solution was added to each well, then the cells were homogenized by agitation and centrifuged 2 min at 1000 r.p.m. and then incubated for 20 min at 23° C. Kinase-Glo reagent substrate was added in each well and after 10 min of incubation at 23° C., the luminescence was read with a bioluminescence instrument (Safas Spectrometer, Monaco). Three tests for each dilution were performed in separate wells and were repeated at two separate days resulting in n=6 for each point of the cAMP activation curve when native IgG were used. After depletion of native IgG from anti-α-MSH IgG fraction, the same experiment was performed with each α-MSH concentration and IgG as described above.

(58) E. coli Gavage in Mice

(59) One-month-old male C57Bl6 mice (Janvier Laboratories) were acclimated to the animal facility for 1 week and maintained as described above. Mice were distributed into four groups (n=8 in each) as follows: (i) gavaged with 108 E. coli K12 bacteria; (ii) gavaged with 108 E. coli K12 bacteria deficient for ClpB; (iii) gavaged with LB medium only; and (iv) controls that did not receive any treatments. The ClpB mutant strain was generated in the Bernd Bukau's Laboratory (ZMBH, Heidelberg University, Heidelberg, Germany) and was kindly provided together with the corresponding wildtype (WT) E. coli bacteria by Dr Axel Mogk. Mice were placed individually into the BioDAQ cages (Research Diets) and intragastrically gavaged daily before the onset of dark phase for 21 days with 0.5 ml of LB medium with or without bacteria. During the last day of gavage, mice feces were collected and frozen. After gavage, mice were killed by decapitation and trunk blood was collected into EDTA-containing tubes. Plasma was separated by centrifugation at 3500 r.p.m. (1.4 g) for 10 min at 4° C. and stored at −80° C. before assay. Plasma levels of anti-ClpB and anti-α-MSH IgG and IgM were assayed as described above.

(60) ClpB DNA Assay

(61) DNA was extracted from the cultures of the WT and ClpB mutant strains, and was also purified from mice feces using the QIAampR DNA Stool Mini Kit (Qiagen, France). Bacteria were dissolved in water and boiled at 100° C. during 5 min, after 1 min of centrifugation at 11 000 r.p.m., the supernatant containing the DNA was stored at −20° C. Using the NCBI primer design tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), we designed the following nucleotide primers that amplify 180-base pair DNA region coding for the ClpB protein fragment containing one identified α-MSH-like epitope (FIG. 1e), forward: 5′-GCAGCTCGAAGGCAAAACTA-3′ (SEQ ID NO: 4) and reverse: 5′-ACCGCTTCGTTCTGACCAAT-3′ (SED ID NO: 5) (Invitrogen Custom Primers, Cergy Pontoise, France). PCR was performed in a thermocycler with MicroAmp tubes (Eppendorf, Hambourg, Germany). The reaction was carried out in a 50-μl volume containing 25 μl of Go Taq Green Master Mix 2× (Promega), 1 μl (20 μmol) of each primer, 21 μl of bi-distilled water and 1 μl of bacterial DNA. PCR conditions were as follows: 3 min at 94° C. followed by 35 cycles at 94° C. for 30 s, 60° C. for 30 s and 72° C. for 1.5 min. PCR products were visualized on a 1% agarose gel (Sigma), with the expected size of 180 base pair and the specificity validated using ClpB mutant strain.

(62) Plasma Concentrations of Bacterial ClpB Protein

(63) Plasma concentrations of bacterial ClpB were measured using enzyme-linked immunosorbent assay (ELISA) according to the following protocol. Rabbit polyclonal anti-ClpB IgG, customly generated by Delphi Genetics (Gosselies, Belgium), were coated on to 96-well Maxisorp plates (Nunc, Rochester, N.Y.) using 100 μl and a concentration of 2 μg/ml in 100 mM NaHCO3 buffer, pH 9.6 for 24 h at 4° C. Plates were washed (5 min×3) in phosphate-buffered saline (PBS) with 0.05% Tween 200, pH 7.4. The recombinant ClpB protein, customly generated by Delphi Genetics, as a standard, was serially diluted to 5, 10, 25, 50, 70, 100 and 150 μM in the sample buffer (PBS, sodium azide 0.02%, pH 7.4) and added to the wells in duplicates. The plasma samples from patients with eating disorders and healthy controls (1:25 in sample buffer) were added to the remaining wells in duplicates and the ClpB standards and plasma samples were incubated 2 h at room temperature (RT). Plates were washed (5 min×3) in PBS with 0.05% Tween 200, pH 7.4. Mouse monoclonal anti-ClpB IgG (1:500 in sample buffer), customly generated by Delphi Genetics and pre-screened for having no cross-reactivity with α-MSH, were added to the wells and incubated 90 min at room temperature. Plates were washed (5 min×3) in PBS with 0.05% Tween 200, pH 7.4. Goat anti-mouse IgG conjugated with alkaline phosphatase (1:2000 in sample buffer) from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa.) were added to the wells and incubated for 90 min at RT. Plates were washed (5 min×3) in PBS with 0.05% Tween 200, pH 7.4 and then 100 μl of p-nitrophenyl phosphate solution (Sigma, St. Louis, Mo.) was added as alkaline phosphatase substrate. After 40 min of incubation at room temperature, the reaction was stopped by adding 3N NaOH. The optical density (OD) was determined at 405 nm using a microplate reader Metertech 960 (Metertech Inc., Taipei, Taiwan). Blank OD values resulting from the reading of plates without addition of plasma samples or ClpB protein standard dilutions were subtracted from the sample OD values. Plasma concentrations of ClpB was calculated based on the OD of the ClpB standard curve and was adjusted for the plasma dilution.

(64) Statistical Analysis

(65) Data were analyzed and the graphs were plotted using the GraphPad Prism 5.02 (GraphPad Software Inc., San Diego, Calif., USA). Normality was evaluated by the Kolmogorov-Smirnov test. Group differences were analyzed by the analysis of variance or the nonparametric Kruskal-Wallis test with the Tukey's or Dunn's post tests, respectively, according to the normality results. Body weight changes were analyzed with two-way repeated measurements analysis of variance and the Bonferroni post tests. Individual groups were compared using the Student's t-test or the Mann-Whitney test according to the normality results. Effects of absorptions of ClpB antibodies with α-MSH were analyzed using paired t-test. Pearson's or Spearman's correlation coefficients were calculated according to the normality of the variable. The cAMP production was analyzed using a nonlinear regression fit (log(α-MSH) vs normalized cAMP response), which equation was Y=100/(1+10(Log EC50-X)×HillSlope). Data are shown as mean±s.e.m., and for all test, Po0.05 was considered statistically significant.

(66) Results

(67) Proteomic Identification of Bacterial α-MSH Mimetics

(68) To identify bacterial proteins with molecular mimicry to α-MSH, a research strategy based on proteomic technology was developed. Total protein was extracted from E. coli K12 cultures, the cytoplasmic fraction was resolved by 2D gel electrophoresis (FIG. 1a) and transferred to a polyvinylidene difluoride membrane. To increase the probability of detection of multiple α-MSH-like epitopes in bacterial proteins, the membrane was revealed with polyclonal anti-α-MSH IgG. 13 immunopositive protein spots were found (FIG. 1b), among which the spots 1-8 disappeared after preadsorption of antibodies with 10.sup.−6M α-MSH (FIG. 1c), confirming specific α-MSH-mimetic epitopes. Using mass spectrometry, protein spots 1, 2, 3 and 4, displaying the strongest α-MSH-like staining, were identified as isoforms of the heat-shock protein named ClpB, a 857-a.a. protein, 857 amino acid protein disaggregation chaperone or ClpB, MW 95526 (molecular weight: 95526 Da, accession number: NP_417083.1, SEQ ID NO: 1). Less intensely stained α-MSH-like spots 5-8 (with the highest MASCOT scores of 880, 877, 874 and 800, respectively) were isoforms of the 548-a.a. protein chaperonin GroEL, (molecular weight: 57293 Da; accession number: YP_001732912.1). An alternative strategy of E. coli protein separation was also used, using an OFFGEL fractionator followed by one-dimensional gel electrophoresis and western blot with anti-α-MSH IgG preadsorbed or not with α-MSH (data not shown). One band was specifically recognized by anti-α-MSH IgG and was found to contain the ClpB protein (with the highest MASCOT score of 1065). Based on these results, ClpB was selected as a target protein for further validation of its molecular mimicry with α-MSH. To analyze the amino-acid sequence homology between α-MSH and bacterial ClpB, both sequences were aligned in the Emboss Stretcher program that uses the Needleman-Wunsch algorithm (http://www.ebi.ac.uk/Tools/emboss/). The alignments revealed a site of the ClpB protein displaying discontinuous 5 a.a. sequence homology with α-MSH (FIG. 1d). This putative α-MSH-like epitope was located in an inter-helical loop of the ClpB protein structure indicating that it is exposed on the protein surface, that is, accessible to auto-Abs binding. Western blot of the recombinant ClpB protein revealed with anti-α-MSH IgG showed a 96-kDa band (FIG. 1e), confirming that the ClpB protein contains α-MSH-like epitope(s). These results show that the presence of at least five consecutive amino-acid sequence homology, according to the molecular mimicry concept is not an obligatory condition for bacterial proteins to be recognized by IgG crossreacting with a neuropeptide.

(69) Immunization of Mice with ClpB

(70) To investigate whether E. coli ClpB may induce auto-Abs crossreactive with α-MSH, influencing feeding and anxiety, mice were immunized with the recombinant bacterial ClpB protein. Mice that received ClpB together with adjuvant or adjuvant alone displayed lower body weight for a few days after injections (FIG. 2a). However, 4 weeks later, ClpB-immunized mice had higher body weight (+5%) vs controls (FIG. 2a). The mean daily food intake, as measured during the last 10 days of the experiment, was also higher (+13%) in ClpB-immunized mice as compared with other groups (FIG. 2b). The increase in food intake was owing to increased meal size (FIG. 2c), as meal number did not change (FIG. 2d), indicating that the ClpB immunization interfered with satiation rather than with hunger mechanisms. This is in agreement with the known role of α-MSH to induce satiation. To further validate the relevance of ClpB immunization to α-MSH anorexigenic effect, mice received i.p. injection of α-MSH. The following 24 h food intake and body weight were not affected in ClpB-immunized mice (FIG. 2e), indicating that they were not sensitive to the anorexigenic effect of administered α-MSH that was present in nonimmunized mice. After the feeding experiments, locomotor activity and anxiety related behavior in mice were studied in the open field and O-maze tests. The total locomotor activity and the time spent in the open vs border areas did not significantly differ between the study groups (data not shown). However, in the closed arms of the O-maze, the ClpB-immunized mice moved a shorter distance as compared with controls (data not shown) and spent less time as compared with all other groups (data not shown), indicating decreased anxiety. To confirm the efficiency of immunization, plasma levels of anti-ClpB IgG were assayed and their affinity measured. In ClpB immunized mice, a strong increase in anti-ClpB IgG levels (FIG. 2f) with lower affinities (FIG. 2g) were found, in agreement with recent IgG induction. Increased plasma levels of α-MSH-reactive IgG were also found in ClpB-immunized mice (FIG. 2h); these IgG were similarly characterized by lower affinities for α-MSH, as compared with controls (FIG. 2i). Adsorption of mouse plasma with α-MSH, significantly reduced plasma levels of anti-ClpB IgG, confirming that a fraction, but not all of the anti-ClpB IgG, were crossreactive with α-MSH (FIG. 2f). Plasma levels of α-MSH IgM auto-Abs did not significantly differ between the groups (data not shown). Whether ClpB immunization may induce auto-Abs crossreacting with the adrenocorticotropic hormone, a 39-a.a. peptide containing the α-MSH sequence was also analyzed. No significant differences in plasma adrenocorticotropic hormone-reactive IgG were found (data not shown), showing the selectivity of the conformational mimicry between ClpB and α-MSH.

(71) Mouse IgG Effects on MC4R Signaling

(72) To determine the impact of ClpB immunization-induced α-MSH crossreactive IgG on MC4R signaling, their effects on α-MSH-induced cAMP production in MC4R-expressing cells were studied. cAMP concentrations were found to be lower when α-MSH was preincubated with IgG from ClpB-immunized mice, as compared with α-MSH alone or α-MSH preincubated with IgG from adjuvant injected mice, with a reduction of 8-10% at the two highest α-MSH concentrations (FIG. 2j). After depletion of α-MSH-reactive IgG from the pooled IgG, the remaining IgG from the ClpB immunized mice did not show any effect on α-MSH-induced cAMP release (FIG. 2k), indicating that anti-α-MSH crossreactive IgG in ClpB-immunized mice were responsible for lowering cAMP production in response to α-MSH. The reduction in MC4R activation and signaling may, hence, account for the increased food intake and decreased anxiety observed in ClpB-immunized mice.

(73) Intragastric Delivery of E. coli in Mice

(74) To test whether E. coli may induce immunogenic response against the ClpB protein, resulting in production of anti-ClpB auto-Abs crossreactive with α-MSH, WT and ΔClpB strains of E. coli K12 were given daily to mice by intragastric gavage during 3 weeks. Another group of mice was gavaged with the bacterial culture medium only, and the control group did not receive any treatment. The first days of gavage were accompanied by a decrease in body weight and food intake in mice receiving WT E. coli, which then gradually returned to control levels (FIGS. 3a and b). Again, during the last week of gavage, feeding pattern was affected in mice receiving E. coli WT showing a decrease in meal size but increase in meal number (FIGS. 3c and d). Remarkably, mice receiving ΔClpB E. coli did not significantly differ from controls in either body weight gain, food intake or feeding pattern at any time point. These data support specific involvement of bacterial ClpB in the host acute decrease of food intake as well as in the chronic regulation of feeding pattern following E. coli infection. Expectedly, ClpB DNA was more abundant in feces of mice receiving E. coli WT, although its low level was detected in some control mice (FIG. 3e). After 3 weeks of gavage, plasma levels of both anti-ClpB IgM and IgG were elevated in mice that received E. coli WT as compared with controls and ΔClpB E. coli groups (FIGS. 3f and g). Adsorption of plasma with α-MSH reduced anti-ClpB IgG levels in E. coli WT-gavaged mice (FIG. 3g), indicating the presence of anti-ClpB IgG crossreactive with α-MSH. Interestingly, plasma levels of the IgM class of anti-ClpB auto-Abs were increased after adsorption with α-MSH, suggesting that α-MSH caused dissociation of α-MSH IgM immune complexes crossreactive with ClpB that were increased in E. coli WT-gavaged mice (FIG. 3f). Plasma levels of anti-α-MSH IgM were also increased by E. coli WT delivery as compared with all other groups (FIG. 3h), while anti-α-MSHreactive IgG were only slightly increased without reaching significance (FIG. 3i). Nevertheless, affinity kinetic analysis of α-MSH IgG revealed lower values of the dissociation equilibrium constants in E. coli-gavaged mice (FIG. 3j), without significant changes of the association or dissociation rates (data not shown). These changes, including increased levels of the IgM class of α-MSH reactive auto-Abs, might reflect an immune response towards ClpB as to a novel antigen. In fact, low or undetectable levels of ClpB DNA in feces of mice that did not receive E. coli WT indicates that ClpB-expressing microorganisms were not major gut commensals in the studied mice. Thus, in contrast to ClpB-immunized mice, which showed increased levels of low-affinity anti-α-MSH IgG associated with increased meal size and body weight gain, E. coli-gavaged mice showed increased production of both anti-α-MSH-reactive IgM and IgG with increased affinities associated with decreased meal size and body weight.

(75) Anti-ClpB Antibodies in ED Patients

(76) As the ability of the E. coli ClpB protein to stimulate production of α-MSH crossreactive auto-Abs was validated, the relevance of bacterial ClpB to ED was next determined by studying anti-ClpB antibodies in patients with AN, BN or BED. It was found that both anti-ClpB IgG and IgM were readily detectable in plasma of ED patients as well as healthy subjects with no significant differences of their mean levels (FIGS. 4a and b). However, there was high variability in all study groups, indicating a different individual history in encountering ClpB-like antigens. To verify whether human anti-ClpB antibodies similarly were crossreactive with α-MSH, plasma samples were adsorbed with 10.sup.−6M α-MSH, leading to significant reduction of anti-ClpB IgG and IgM detectable levels in all study groups (FIGS. 4c and d). Further, the relative levels of α-MSH crossreactive anti-ClpB IgG were increased in all three groups of ED patients, in particular BN and BED vs healthy controls (FIG. 4e). Elevated levels of α-MSH crossreactive anti-ClpB IgM were found in AN as compared with BN (FIG. 4f). To further determine the relevance of anti-ClpB IgG and IgM to ED, it was studied whether their plasma levels may correlate with behavioral traits in ED patients and controls measured by the EDI-2. It was found that in controls, ClpB IgG correlated inversely with the normal range of a few psychological traits, but in AN patients,

(77) ClpB IgG levels correlated positively with the core psychopathological traits such as body dissatisfaction and drive for thinness (Table 1). Moreover, in AN and BED patients, EDI-2 subscale scores correlated with ClpB IgM in the opposite way, being negative in AN but positive in BED (Table 1). However, in BED patients, ClpB IgM correlated negatively with age, suggesting that the highest anti-ClpB IgM levels were associated with the acute form of the disease. Remarkably, the correlations found in AN patients between ClpB IgG or IgM and drive for thinness or interpersonal distrust, respectively, resembled closely the correlations between the same psychological traits and α-MSH-reactive IgG or IgM found in a different group of AN patients in a previous study.

(78) TABLE-US-00002 TABLE 1 Significant correlations between plasma levels of anti-ClpB IgG and IgM and psychological traits in eating disorder patients and controls (Contr.) assayed by the Eating Disorder Inventory-2. ClpB Maturity fears Impulse regulation Social insecurity IgG r = −0.31 * r = −0.26 * r = −0.26 * (Contr.) ClpB Body dissatisfaction Drive for thinness Perfectionism IgG (AN) r = 0.4 * r = 0.35 * r = 0.38 * ClpB Ineffectiveness Interpersonal distrust Social insecurity Anhedonia IqM (AN) r = −0.42 * r = −0.58 ** r = −0.52 ** r = −0.35 * ClpB Bulimia Perfectionism Age IgM r = 0.53 * r = 0.6 * r = −0.74 ** (BED) All Spearman's r * p < 0.05, ** p < 0.01, except Pearson's r * p < 0.05 for perfectionism. (n = 65 Contr., n = 27 AN, and n = 14 BED).
Plasma Concentrations of Bacterial ClpB Protein

(79) ClpB protein was detected in plasma samples of all study subjects ranging from 10 μM to 180 μM with a mean level of about 30 μM in healthy controls. Mean plasma levels of ClpB were significantly elevated in all groups of patients with eating disorders, including AN, BN and BED (see FIG. 5). By applying the common criteria of a diagnostically-relevant changes of concentrations equal or exceeding 2 standard deviations, 21.7% of AN, 32% of BN and 25% of BED patients showed increased levels of plasma ClpB.

(80) Conclusion

(81) The results reveal a molecular link between ClpB expressing gut bacteria and the regulation of motivated behavior and emotion via production of ClpB protein and anti-ClpB antibodies crossreactive with α-MSH. It shows that specific alterations of gut microbiota may lead to behavioral and emotional abnormalities as observed in ED patients. The findings of increased levels of ClpB protein and anti-ClpB IgG crossreactive with α-MSH in ED patients and correlations of anti-ClpB antibodies with patient's psychopathological traits support the involvement of ClpB-expressing microorganisms in increased ClpB antibody production and establishment of abnormal feeding behavior and emotion. In conclusion, the results identify ClpB as a protein responsible for the origin of auto-Abs crossreactive with α-MSH, associated with psychopathological traits in ED patients and, hence, that ClpB-expressing microorganisms as a novel specific target for diagnostics and treatment of ED.

Example 2

(82) This example demonstrates the effect of ClpB-expressing bacteria on obese ob/ob mice.

(83) Genetically obese ob/ob mice were acclimated to the animal facility for 1 week and maintained as described above. Mice were intragastrically gavaged with (i) 10.sup.8 E. coli K12 bacteria (expressing ClpB); (ii) 10.sup.8 E. coli K12 bacteria deficient for ClpB; both in Mueller-Hilton (MH) medium or with (iii) MH medium only, as a control. The ClpB mutant strain was generated in the Bernd Bukau's Laboratory (ZMBH, Heidelberg University, Heidelberg, Germany) and was kindly provided together with the corresponding wildtype (WT) E. coli bacteria by Dr. Axel Mogk. Mice were placed individually into the BioDAQ cages (Research Diets) and intragastrically gavaged daily for 21 days as indicated.

(84) The inventors showed that gavage with E. coli K12 WT bacteria induced a 56% reduction in weight gain (FIGS. 6 and 7), a reduced fat mass/lean mass ratio (FIGS. 8 and 9) and a reduction of 20% of the total food intake (FIGS. 10 and 11), which was not observed with E. coli K12 bacteria deficient for ClpB.

Example 3

(85) This example demonstrates the effect of other strains of bacteria expressing ClpB on obese ob/ob mice.

(86) Genetically obese ob/ob mice were acclimated to the animal facility for 1 week and maintained as described above. Mice were intragastrically gavaged with (i) 10.sup.8 E. coli K12 bacteria (expressing ClpB); (ii) 10.sup.8 E. coli Niessle 1917 bacteria (expressing ClpB) (iii) 10.sup.8 E. coli Niessle 1917 bacteria (expressing ClpB) in lyophilized form; all in Mueller-Hilton (MH) medium or with (iv) MH medium only, as a control. Mice were intragastrically gavaged daily for 14 days as indicated.

(87) The inventors showed that gavage with any strain of E. coli ClpB-expressing bacteria induced a reduction in weight gain (FIGS. 12 and 13) and a reduction in fat content (FIG. 14).

Example 4

(88) This example demonstrates the effect of ClpB-expressing Hafnia alvei on obese ob/ob mice.

(89) Genetically obese ob/ob mice were acclimated to the animal facility for 1 week and maintained as described above. Mice were intragastrically gavaged with (i) 10.sup.8 Hafnia alvei AF036 bacteria (expressing ClpB); (ii) 10.sup.8 E. coli K12 bacteria deficient for ClpB; (iii) 10.sup.8 E. coli Niessle 1917 bacteria (expressing ClpB); all in Mueller-Hilton (MH) medium or with (iv) MH medium only, as a control.

(90) The inventors show that treatment with Hafnia alvei induced a significant decrease in body weight gain as compared to obese controls (FIG. 15). Moreover, the decrease in body weight gain was associated with a decrease of cumulative food intake (FIG. 16), and with a decrease of both fat and lean masses, without alteration of the lean/fat mass ratio (FIG. 17).

Example 5

(91) This example demonstrates the effect of ClpB-expressing Hafnia alvei on high fat diet-induced obese mice.

(92) One-month-old male C57Bl6 mice (Janvier Laboratories) were induced with high fat/high carbs diet for 2 weeks. Mice were then intragastrically gavaged with (i) 10.sup.8 Hafnia alvei AF036 bacteria (expressing ClpB); (ii) 10.sup.8 E. coli K12 bacteria deficient for ClpB; both in Mueller-Hilton (MH) medium or with (iii) MH medium only, as a control. Mice were placed individually into the BioDAQ cages (Research Diets) and intragastrically gavaged daily for 21 days as indicated.

(93) Induction of obesity by high fat diet was validated by measurement of mean body weight (FIG. 18A), fat mass (FIG. 18B) and lean mass (FIG. 18C) in a group induced and a group non-induced for obesity.

(94) The inventors showed that gavage with any strain of Hafnia alvei ClpB-expressing bacteria induced a reduction in weight gain (FIG. 19) and a reduction in fat content (FIG. 20).

Example 6

(95) Hormone-sensitive lipase (HSL) is an intracellular neutral lipase that is capable of hydrolyzing triacylglycerols, diacylglycerols, monoacylglycerols, and cholesteryl esters, as well as other lipid and water-soluble substrates. In particular, activation of HSL induces lipolysis. Hormonal activation of HSL occurs via cyclic AMP dependent protein kinase (PKA), which phosphorylates HSL. An increase of the expression of the phosphorylated form of HSL protein (pHSL) means thus activation of lipolysis. Expression of pHSL was measured by western blot.

(96) To evaluate the possible effects of H. alvei HA4597 supplementation on fat tissue lipolysis, the levels of pHSL protein, a lipolytic marker, were studied by WB in the epididymal fat tissue of both models using anti-pHSL antibodies (Cell Signaling Technology, Mass., USA).

(97) The inventors showed that gavage with E. coli Niessle 1917 and Hafnia alvei bacteria increased pHSL expression (FIG. 21) as compared to control and gavage with E. coli K12 bacteria. Therefore, E. coli Niessle 1917 and Hafnia alvei bacteria activate the mechanism of lipolysis.

Example 7

(98) Previous data on leptin-deficient ob/ob obese mice show the effects of H. alvei on the food intake reduction with no impact on the fat mass/lean mass ratio. However, in the following High Fat Diet (HFD) model, the anti-obesity effects as well as the reduction of fat mass/lean mass ratio were confirmed. Therefore, the administration of H. alvei not only decreased food intake/body weight gain, but also improved body composition.

(99) Experimental Conditions

(100) Animal care and experimentation were done in accordance with guidelines established by the National Institutes of Health, French and European Community regulations (Official Journal of the European Community L 358, 18 Dec. 1986) and were approved by the Local Ethical Committee of Normandy (n° 5986). Six-to-seven-week-old male ob/ob mice (B6.V-Lep ob/ob JRj−n=88) were purchased from Janvier Labs (Le Genest-Saint-Isle, France), and, upon arrival, they were kept in a specialized air-conditioned animal facility (22±2° C., relative humidity 40±20%, reverse 10:00 p.m. to 10:00 a.m. light/dark cycle). During acclimation to the animal facility (from day (D)−19 to D−12), mice were housed in standard holding cages (n=3 per cage) with pelleted standard rodent chow (3430 standard diet, KlibaNafag, Kaiseraugst, Switzerland) and drinking water available ad libitum. After acclimation, the mice were weighed and placed in a restraint cylinder, and their body composition, including lean and fat mass, as well as body fluids, was measured, using the MiniSpec LF50 (Bruker, Rheinstetten, Germany). Then, according to body weight, the animals were randomly divided into two groups: (i) fed with a HFD (n=72, 45 kcal % fat, 35 kcal % carbohydrates, 20 kcal % proteins, energy density 4.7 kcal/g, D12451, Research Diets, New Brunswick, N.J., USA) and (ii) fed with a standard diet (SD; n=16, 5 kcal % fat, 75 kcal % carbohydrates, 20 kcal % proteins, energy density 3.8 kcal/g, 3430 KlibaNafag). The animals underwent a 5-day “induction” to both diets, followed by 1 week of daily handling protocol, where they were sham-treated (oral gavage of 200 μL of NaCl 0.9%, using rounded-end cannulas, Socorex, Ecublens, Switzerland) twice a day (approximatively 10:00 a.m. and 17:00 p.m.) from D−7 to D0 (FIG. 1). This procedure has been found to reduce stress-related effects on the body weight in chronic studies. During the baseline period and the later treatment sessions, body weight, as well as food intake (by cage), were measured daily, before the morning gavage (at approximatively 9:30 a.m.).

(101) Toward the end of the baseline (D0), body composition was measured for a second time, using the MiniSpec LF50 (Bruker), and the HFD-fed animals were randomly divided into 3 subgroups (n=24 in each). All animals from the same cage were affiliated to the same subgroup. From D0 onward, animals from each of the 3 HFD-fed groups were treated daily for 38 days, at approximatively 7:00 p.m., by intragastric gavage, with a volume of 5 mL/kg containing either: (i) H. alvei HA4597™ provided by Biodis (Noyant, France) for TargEDys SA (1.4×1010 CFU/day in NaCl 0.9%); (ii) orlistat 80 mg/kg/day in NaCl 0.9% (Tocris BioScience, Bristol, UK); or (iii) NaCl 0.9% as a vehicle. The standard diet (SDiet)-fed controls received the same 5 mUkg volume of NaCl 0.9% by intragastric gavage (FIG. 22).

(102) After five days of the DIO induction phase (Diet induced obesity), HFD-fed mice presented a significant increase of body weight as compared to SDiet-fed mice (44.7±0.4 g vs. 39.5±0.9 g at D−7, p<0.001), which persisted during the sham-treatment period of the DIO induction. The total daily food intake and the cumulative caloric intake were increased in HFD-fed vs. SDiet-fed mice during the sham-treatment period. At the end of the sham period, an increase of fat mass and percentage of body fat was found in HFD-fed mice. In spite of a small increase of the lean mass, its body percentage was lower in HFD-fed vs. SDiet-fed mice. The changes of body composition led to an overall decrease of lean/fat mass ratio in the HFD group. No significant changes of the body composition were observed in the SDiet group during sham treatment.

(103) At D38, the body composition of all animals was measured, using the MiniSpec LF50, and treatment groups were divided into two sub-groups, for the oral glucose tolerance test (OGTT) after overnight fasting and for the baseline plasma glucose assay. An overnight fasting, after the removal of food during D22 light phase, (approximatively at 17:00 p.m.) was performed on half of each group, while food remained available ad libitum for the rest of the animals. The next morning, the mice received 30% glucose solution (2 g/kg) via intragastric gavage. Blood samples (one drop from tail tip) were taken every 15 min during 2 h, for glucose assay, using a glucometer (FreeStylePapillon Vision, Abbott Diabetes Care, Oxon, UK). Mice which didn't undergo the fasting procedure were anesthetized right after body composition measurements on D38 by intraperitoneal injection of ketamine/xylazine solution (80:10 mg/kg). Terminal blood samples (approximately 0.5 mL) were taken by a puncture of abdominal aorta in tubes coated with lithium heparin, which were stored at 4° C., until biochemical measurements of obesity-related metabolic markers using IDEXX Catalyst® One technology (Catalyst® Chem 17 CLIP+Catalyst® TRIG, IDEXX, Laboratories, Inc., Westbrook, Me., USA). Results, expressed as mean±standard error of means (SEM), were analyzed, using GraphPad Prism 5.02 (GraphPad Software Inc., San Diego, Calif., USA), with a p-value <0.05 considered as statistically significant. Longitudinal group differences were compared by the two-way repeated measurement (RM) analysis of variance (ANOVA) with Bonferroni's posttests. Individual differences were analyzed, using Student's t-test or Mann-Whitney's test according to normality evaluated by the D'Agostino-Pearson's test.

(104) Confirmation of Effects on Body Weight

(105) HFD-fed control mice displayed increased body weight gain as compared to SDiet-fed mice (FIG. 23A-C). Supplementation of H. alvei in HFD mice resulted in a decreased body weight gain vs. untreated HFD group, as compared by the area under curve (FIG. 3C), reaching a difference of 15.3% at the end of the treatment (FIG. 23B). Accordingly, the body weight gain of the H. alvei-treated group was 58.1% lower as compared to the difference between HFD and SDiet groups, although such decrease did not reach significance by ANOVA (FIG. 23B). The orlistat-treated group showed a significant decrease in body weight gain as compared to both the HFD control and H. alvei groups, reaching to the level of the SDiet-fed group (FIG. 23A-C).

(106) Body Composition Effect

(107) HFD increased percentage of fat mass and decreased percentage of lean mass, resulting in lower lean/fat mass ratio vs. SDiet-fed mice (FIG. 24A-C). H. alvei treatment alleviated the increase of adiposity and loss of lean mass associated with HFD (FIG. 24A,B). These changes led to a significant improvement of the lean/fat mass ratio in mice treated with H. alvei (FIG. 24C). Orlistat-treated HFD mice displayed similar improvements of body composition to the mice treated with H. alvei (FIG. 24A-C).

(108) The reduction of the fat mass on lean mass ratio is further confirmed by the clinical data of example 9.

(109) Food Intake and Weight-Loss without Induction of Hyperphagia

(110) Daily food intake was higher in HFD controls vs. SDiet-fed mice during the first three weeks, and then it declined to the level of the SDiet group (FIG. 25A). H. alvei HA4597™-treated HFD-fed mice displayed similar daily food intake to SDiet-fed mice (FIG. 25A). Cumulative food intake was also reduced in H. alvei HA4597™-treated mice as compared to HFD controls (FIG. 25B,C). In the end of H. alvei HA4597™ treatment, at D38, the cumulative food intake was lower by 26.7 g vs. the HFD controls (FIG. 25C). Orlistat-treated mice displayed hyperphagia throughout the treatment (FIG. 25A), resulting in an increased cumulative food intake (FIG. 25B,C).

Example 8

(111) In the experimental conditions of example 7 the following obesity-related parameters were assessed.

(112) At D38, the body composition of all animals was measured, using the MiniSpec LF50, and treatment groups were divided into two sub-groups, for the oral glucose tolerance test (OGTT) after overnight fasting and for the baseline plasma glucose assay. An overnight fasting, after the removal of food during D22 light phase, (approximatively at 17:00 p.m.) was performed on half of each group, while food remained available ad libitum for the rest of the animals. The next morning, the mice received 30% glucose solution (2 g/kg) via intragastric gavage. Blood samples (one drop from tail tip) were taken every 15 min during 2 h, for glucose assay, using a glucometer (FreeStylePapillon Vision, Abbott Diabetes Care, Oxon, UK). Mice which didn't undergo the fasting procedure were anesthetized right after body composition measurements on D38 by intraperitoneal injection of ketamine/xylazine solution (80:10 mg/kg). Terminal blood samples (approximately 0.5 mL) were taken by a puncture of abdominal aorta in tubes coated with lithium heparin, which were stored at 4° C., until biochemical measurements of obesity-related metabolic markers using IDEXX Catalyst® One technology (Catalyst® Chem 17 CLIP+Catalyst® TRIG, IDEXX, Laboratories, Inc., Westbrook, Me., USA).

(113) Effects on Glycaemia

(114) Ad libitum HFD-fed H. alvei-treated mice displayed a significant decrease, by about 1.5-fold, of the basal glucose plasma levels as compared to both the SDiet and HFD-fed controls (FIG. 26A). The OGTT test after an overnight fasting showed elevated levels of fasting glycemia in HFD-fed control mice (Student's t-test p<0.05) and in orlistat-treated mice as compared to SDiet-fed controls (FIG. 26B). After the oral glucose load, a 15 min peak of glucose was observed in all groups, without significant difference. A return to the baseline was delayed in the orlistat-treated mice as compared to both SDiet-fed and H. alvei HA4597™-treated mice (FIG. 26B).

(115) Effects on Lipidemic Parameters: Blood Lipids Cholesterol and Alanine Aminotransferase

(116) Triglycerides plasma level increased, although not significantly, in HDF-fed mice as compared to SDiet, and no significant effect of H. alvei on plasma triglycerides was observed (FIG. 26C). In HFD+Orlistat mice, triglycerides plasma level increased significantly (FIG. 26C). Total cholesterol plasma level increased significantly in HFD-mice as compared to control HFD-mice, and this increase was prevented in H. alvei-treated HFD-mice (FIG. 26D). Orlistat reduced total cholesterol even more than H. alvei (FIG. 26D). Plasma levels of alanine aminotransferase (ALAT), an indicator of obesity-induced steatohepatitis, were increased in HFD-mice as compared to SDiet-mice; treatment with either H. alvei HA4597™ or orlistat prevented this ALAT increase (FIG. 6E).

(117) In contrast to the anorexigenic effect of H. alvei, orlistat, although efficient to reduce body weight in HFD-fed mice, induced a strong hyperphagic effect, which was most likely due to the decreased fat absorption in the gut. At the end of treatment period, food intake in the orlistat-treated HFD-mice was markedly increased, and, consequently, the difference between orlistat and H. alvei effects on the reduction of body weight gain tended to decrease. Moreover, such compensatory hyperphagia was accompanied at the end of the treatment period by increased basal and OGTT-induced plasma glucose levels. Although these orlistat-induced unwanted effects might be specific to this animal model, they suggest that H. alvei-induced moderate but sustained reduction of body mass gain through decreased food intake, along with beneficial metabolic effects, may represent a safer and more-efficient strategy for long-term body weight management. The present example shows that a daily provision of the H. alvei strain in genetically obese and hyperphagic ob/ob mice with HFD-exacerbated obesity decreased body weight gain, improved body composition, decreased food intake, and ameliorated several metabolic parameters, including plasma glucose and total cholesterol levels. These data further preclinically validate the anti-obesity efficacy of H. alvei as a potential new-generation nutraceutical or Life biotherapeutic for appetite and body weight control in obesity as well as preventing or treating diet-induced alterations of glucose and lipid metabolism.

Example 9

(118) 10.sup.11 cells of Hafnia alvei HA4597™ per diem were orally administered in Double-blind, randomised, placebo-controlled Clinical Trial that lasted 12 weeks. The subjects (20-65 years old, mean age 46.4; 127 female subjects and 103 male subjects; mean BMI 27.84; mean waist circumference 101.3 cm; mean hip circumference 106.2 cm) were divided in a 2-arm trial

(119) There were statistically significant differences in the proportion of subjects who lost at least 3% of baseline body weight at 12 weeks between the study groups (V: 57.7% vs. P: 41.7%; pexF=0.028). The results were confirmed by the absolute body weight and BMI. Furthermore, there were statistically significant differences in the reduction in hip circumference from baseline to visit v4 (2 months) between the study groups at 8 weeks (p.sub.U=0.002). The reduction in hip circumference was also significant from baseline to visit v5 (3 months) between the study groups at 12 weeks (p.sub.U<0.001).

(120) There were statistically significant differences in the feeling of fullness between the study groups at 12 weeks (p.sub.U=0.009).

(121) There clinical trial showed statistically significant differences in Glucose between the study groups at 12 weeks (p.sub.U=0.027).

(122) Lastly, in further endpoints of the study: statistically significant changes in Cholesterol from visit v1 to visit v5 at week 12 (p.sub.Wil=0.011) that were present in the H. alvei treated group and not in the placebo group, statistically significant changes in LDL-cholesterol from visit v1 to visit v5 at week 12 (p.sub.Wil=0.039) that were present in the H. alvei treated group and not in the placebo group.

(123) The study further showed no statistically significant adverse effects as well as an overall good tolerability.

(124) The above clinical data confirm the previous in vivo evidence, the oral administration of H. alvei having a significant effect on body weight, BMI, hip circumference reduction, fullness sensation induction. Such treatment further shows a positive impact on the metabolic parameters of glycemia, total cholesterol and LDL-cholesterol.