WO2015082633A1 - Bacterial influence on regulation of appetite via clpb protein mimicry of alpha-msh - Google Patents

Abstract

The present invention relates to bacterial Clp B protein and Clp B expressing bacteria and their impact on eating disorders. The invention further relates to compositions comprising antibiotic directed against at least one Clp B expressing bacterium as well as probiotics not expressing Clp B protein and their use in the treatment or prevention of eating disorders. The invention also relates to diagnostic tools for determining whether a subject is likely to respond to a method of treating eating disorders and to methods of immunization against eating disorders.

Description

Bacterial influence on regulation of appetite via ClpB protein mimicry of alpha-MSH

The present invention relates to bacterial ClpB protein and ClpB expressing bacteria and their impact on eating disorders. The invention further relates to compositions comprising antibiotic directed against at least one ClpB expressing bacterium as well as probiotics not expressing ClpB protein and their use in the treatment or prevention of eating disorders. The invention also relates to diagnostic tools for determining whether a subject is likely to respond to a method of treating eating disorders and to methods of immunization against eating disorders.

Eating disorders increased all over the world among both men and women over the last 50 years. There is evidence to suggest that in particular individuals in the western world are at the highest risk of developing them and the degree of westernization increases the risk. With recent advances, the scientists understand more and more the central processes of appetite. It is known that interactions between motivational, homeostatic and self-regulatory control processes are involved in eating behavior, which is a key component in eating disorders.

Recent science discovered a further regulator, the human microbiome. Advances of high-throughput DNA sequencing technologies allowed to explore the human microbiome and thus to make a major step towards the understanding of the molecular relationships between the host and its microbiota. This "second genome", the microbiome, has been described in a catalogue of more than 4-5 million, non-redundant microbial putative genes and 1 to 2,000 prevalent bacterial species. Each individual has at least 160 shared species and a number of well-balanced host-microbial molecular relationships that defines groups of individuals.

It is considered that understanding the essential features of symbiotic relationships between microbial communities and their human host may allow to predict host phenotypes, such as health status, from the particular features of indigenous communities.

The composition of gut microbiota for example has been associated with host metabolic phenotypes including obesity and diabetes as well as some neuropsychiatric disorders suggesting that gut bacteria may influence brain controlled functions and behavior.

In this context, determining the molecular mechanisms linking the microbiota to the host behavior appears, thus, as a necessary step for defining the role of specific microorganisms in host physiology. The molecular mechanisms at the origin of eating disorders, including anorexia nervosa (AN), bulimia (BN), and binge-eating disorder (BED), are currently unknown. Previous data indicated that immunoglobulins (Igs) or autoantibodies (auto-Abs) reactive with a-melanocyte-stimulating hormone (α-MSH) are involved in regulation of feeding and emotion; however, the origin of such auto-Abs is unknown.

The inventors discovered, using proteomics, that the ClpB chaperon protein of commensal gut bacteria E. Coli K12 is a conformational mimetic of a-melanocyte- stimulating hormone (a-MSH), a neuropeptide involved in the regulation of energy metabolism and emotion.

The inventors show that ClpB-immunized mice produce anti-ClpB IgG crossreactive with a-MSH, influencing food intake, body weight, anxiety and melanocortin receptor 4 signaling. Furthermore, they show that chronic intragastric delivery of E. coli in mice decreased food intake and stimulated formation of ClpB- and a-MSH-reactive antibodies, while CIpB-deficient E. coli did not affect food intake or antibody levels. Finally, they show that plasma levels of anti-ClpB IgG crossreactive with a-MSH are increased in patients with AN, BN and BED, and that the ED (eating disorders) lnventory-2 scores in eating disorders patients correlate with anti-ClpB IgG and IgM.

As a consequence, they shows that the bacterial ClpB protein, which is present in several commensal and pathogenic microorganisms, can be responsible for the production of auto-Abs crossreactive with a-MSH, associated with altered feeding and emotion in humans with eating disorders.

In addition, they show increased plasma concentrations of the ClpB protein in patients suffering from eating disorders.

Therefore, the presence of ClpB protein and/or anti-ClpB antibodies can be related to eating disorders and to a dysregulation of appetite and emotion.

The presence of the ClpB protein and/or anti-ClpB antibodies been correlated with eating disorders and dysregulation of appetite, in return reducing the level of ClpB protein and/or anti-ClpB antibodies can result in regulation of appetite and thus be used as a treatment of eating disorders.

Accordingly, the present invention relates to a composition comprising at least one antibiotic directed against at least one ClpB expressing bacterium for use in the treatment or prevention of eating disorders.

The present invention further relates to a non-therapeutic method of regulating appetite in a subject, comprising administering to said subject an effective amount of a composition comprising at least one antibiotic directed against at least one CIpB expressing bacterium.

Accordingly, the present invention also relates to a composition comprising probiotics not expressing CIpB for use in the treatment or prevention of an eating disorder.

The present invention further relates to a non-therapeutic method of regulating appetite in a subject, comprising administering to said subject an effective amount of a composition comprising probiotics not expressing CIpB.

The present invention also relates to a composition comprising a CIpB protein for use as a vaccine or as an immunogenic composition.

In particular, said composition for use, is used in the immunization against eating disorders.

In particular, said composition for use, is used for preventing eating disorders.

The invention also concerns an in vitro method for diagnosing an eating disorder in a subject, which method comprises:

a) measuring the level of CIpB protein and/or anti-ClpB antibodies in a biological sample from said subject;

b) comparing said measured level of CIpB protein and/or anti-ClpB antibodies to a reference value; and

c) deducing therefrom if the subject suffers from an eating disorder.

The present invention further relates to an in vitro method of selecting subjects suffering from an eating disorder as likely to respond to a treatment reducing the level of CIpB protein and/or anti-ClpB antibodies which comprises:

a) measuring the level of CIpB protein and/or anti-ClpB antibodies in a biological sample from said subject;

b) comparing said measured level of CIpB protein and/or anti-ClpB antibodies to a reference value; and

c) selecting the subject for a treatment reducing the level of CIpB protein and/or anti-ClpB antibodies, wherein said treatment reducing the level of CIpB protein and/or anti-ClpB antibodies comprises administering to said subject an effective amount of a composition comprising one antibiotic directed against at least one CIpB expressing bacterium and/or administering to said subject an effective amount of a composition comprising probiotics not expressing CIpB and/or a combination thereof.

The inventors having showed that intragastric delivery of E. coli in mice decreased food intake and body weight gain due to the presence of CIpB protein in bacteria, the invention further relates to a composition comprising probiotics surexpressing CIpB protein for use in the treatment or prevention of obesity. Detailed description of the invention

The inventors discovered, using proteomics, that the ClpB chaperon protein of commensal gut bacteria E.Coli K12 is a conformational mimetic of a-melanocyte- stimulating hormone (α-MSH), a neuropeptide involved in the regulation of energy metabolism and emotion and that the presence of ClpB expressing bacteria results in dysregulated appetite.

Accordingly, the present invention refers to a composition comprising at least one antibiotic directed against at least one ClpB expressing bacterium for use in the treatment or prevention of eating disorders.

As used herein, "ClpB expressing bacterium" refers to bacteria expressing the chaperone protein ClpB.

The "protein disaggregation chaperone", "chaperone protein ClpB", "ClpB protein" or "ClpB" also known as heat shock protein F84.1 is a member of the Hsp100/ClpB family of hexameric AAA+-ATPases. This family comprises bacterial, fungal, and plant Hsp100 ATPases, ClpB being the bacterial representative. As a part of a stress-induced multi- chaperone system, it is involved in the recovery of the cell from heat-induced damage, in cooperation with the Hsp70 system (DnaK, DnaJ and GrpE) in protein disaggregation, a crucial process for cell survival under stress conditions.

During the infection process, bacterial pathogens encounter stress conditions generated by the host defense to eliminate them and respond to this host defense by increasing the synthesis of heat shock and other stress proteins. In this context, ClpB has been described as an essential factor for acquired thermotolerance and for the virulence and infectivity of several Gram-negative and Gram-positive pathogenic bacteria, such as Staphylococcus aureus, Francisella turalensis, Listeria monocytogenes, Yersinia enterocolitica, and Salmonella thyphimurium.

In E.coli K12 the chaperone protein ClpB also known as heat shock protein F84.1 or htpM and is a protein of 857 amino acids.

Typically, the chaperone protein ClpB comprises or consists of the amino acid sequence of the chaperone protein ClpB from E. Coli K12 with SEQ ID NO: 1 (NCBI Reference Number : NP_417083.1 , as available on November 6, 2013 and/or UniProtKB/Swiss-Prot Number: P63284, as available on November 6, 2013). Preferably, the amino acid sequence of chaperone protein ClpB comprises or consists of an amino acid sequence 80 to 100% identical to the amino acid sequence of SEQ ID NO: 1 . Preferably, the amino acid sequence is 90 to 100% identical, more preferably 95 to 100%, most preferably 95, 96, 97, 98, 99 or 100% identical to the amino acid sequence of SEQ ID NO: 1 .

Thus, a CIpB expressing bacterium refers to a bacterium expressing or over- expressing the chaperone protein CIpB as defined above or a polypeptide comprising or consisting of an amino acid sequence 80 to 100% identical to the amino acid sequence of SEQ ID NO: 1 , more preferably 95 to 100%, most preferably 95, 96, 97, 98, 99 or 100% identical to the amino acid sequence of SEQ ID NO: 1 .

By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid.

In the context of the present application, the percentage of identity is calculated using a global alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. The « needle » program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used. The needle program is, for example, available on the ebi.ac.uk world wide web site. The percentage of identity in accordance with the invention is preferably calculated using the EMBOSS:: needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5, and a Blosum62 matrix.

Proteins consisting of an amino acid sequence "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may comprise mutations such as deletions, insertions and/or substitutions compared to the reference sequence. In case of substitutions, the protein consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence derived from another species than the reference sequence.

Amino acid substitutions may be conservative or non-conservative. Preferably, substitutions are conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and/or chemical properties. The substitution preferably corresponds to a conservative substitution as indicated in the table below. Conservative substitutions Type of Amino Acid

Ala, Val, Leu, He, Met, Pro, Phe,

Amino acids with aliphatic hydrophobic side chains Trp

Ser, Tyr, Asn, Gin, Cys Amino acids with uncharged but polar side chains

Asp, Glu Amino acids with acidic side chains

Lys, Arg, His Amino acids with basic side chains

Gly Neutral side chain

CIpB expressing bacteria are well-known from the skilled person and may be identified by any conventional technique.

In one embodiment, the CIpB expressing bacterium is selected from the group constituted of the genus Staphylococcus aureus, Francisella turalensis, Listeria monocytogenes, Yersinia enterocolitica, Salmonella thyphimurium, Escherichia Coli, Enterobacteriaceae, Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Shigella boydii, Citrobacter youngae, Salmonella bongori and Salmonella enterica.

The CIpB protein comprises two nucleotide binding domains (ATP1 and ATP2) and two oligomerization domains (NBD1 and NBD2). The N-terminal domain might function as a substrate-discriminating domain, recruiting aggregated proteins to the CIpB hexamer and/or stabilizing bound proteins. The NBD2 domain is responsible for oligomerization, whereas the NBD1 domain stabilizes the hexamer probably in an ATP-dependent manner. The movement of the coiled-coil domain is essential for CIpB ability to rescue proteins from an aggregated state, probably by pulling apart large aggregated proteins, which are bound between the coiled-coils motifs of adjacent CIpB subunits in the functional hexamer.

The inventors identified that the CIpB chaperon protein of commensal gut bacteria E.coli K12 is a conformational mimetic of a-melanocyte-stimulating hormone (α-MSH), a neuropeptide involved in the regulation of energy metabolism and emotion.

"a-MSH", also known as "a-Melanocyte-stimulating hormone", "alpha-MSH", "a- MSH", alpha-melanotropin, alpha-melanocortin, or alpha-intermedin, is a naturally occurring endogenous peptide hormone of the melanocortin family, with a tridecapeptide structure. The amino acid sequence of a-MSH preferably comprises or consists of the amino acid sequence SYSMEHFRWGKPV (SEQ ID NO: 3) (Gen Pept Sequence ID, PRF: 223274, as available on December 2, 2013). However, there exist three types of alpha-melanocyte-stimulating hormone that differ in their acetyl status, the desacetyl alpha MSH, which lacks an acetyl group; mono-acetyl alpha MSH, in which the amino group of the Ser-1 of SEQ ID NO: 3 is acetylated; and di-acetyl alpha MSH, in which both amino and hydroxy groups of the Ser-1 of SEQ ID NO: 3 are acetylated. α-MSH as used herein refers in particular to the mono-acetylated a-MSH.

It is critically involved in the regulation of energy balance and increasing energy expenditure via activation of the melanocortin receptors type 4 (MC4R), acting both centrally and peripherally. In both humans and mice, plasma a-MSH levels are associated with body weight changes. Furthermore, a-MSH regulates mood and emotion by increasing anxiety.

"Mimetic" refers to a protein that imitates another protein. This imitation is possible since the protein shares certain characteristics with the protein it mimetics.

A "conformational mimetic" refers to a protein, that shares at least in part the same conformation as another protein.

The inventors demonstrated that the CIpB protein shares a discontinuous sequence homology between amino acids 542 to 548 from SEQ ID NO: 1 with a-MSH, of amino acid sequence 'RWTGIPV (referenced under SEQ ID NO: 2). Without being bound by theory, this conformation of CIpB allows stimulating the production of antibodies directed against both CIpB and a-MSH.

Thus "conformational mimetic" herein preferably refers to the capability to stimulate antibody production against CIpB as well as auto antibodies against a-MSH.

An "antibiotic directed against at least one CIpB expressing bacterium" refers herein to an antimicrobial compound that inhibits the growth of CIpB expressing bacteria and/or reduces the amount of CIpB expressing bacteria and/or destroys CIpB expressing bacteria.

The antibiotic directed against at least one CIpB expressing bacterium, preferably inhibits the growth, reduces the amount and/or destroys selectively or preferentially CIpB expressing bacteria. Said antibiotic preferably does not negatively affect eukaryotic cells.

Antibiotics targeting specifically CIpB expressing cells are well known from the skilled person and are further described in Martin, I et al. (Journal of Medicinal Chemistry, 2013, 56: 7177-7189).

The antibiotic directed against at least one CIpB expressing bacterium may bind to CIpB and modulate its activity.

In particular, the antibiotic directed against at least one CIpB expressing bacterium modulates

i) the ATPase activity of CIpB, and/or

ii) inhibit the free/and or substrate bond form of CIpB and/or

iii) inhibit the activation of CIpB. In one embodiment, the antibiotic directed against at least one ClpB expressing bacterium is a salicylaldehyde derivative over a benzylbenzene scaffold, in particular, 5- (2-chlorobenzyl)-2-hydroxy-3-nitrobenzaldehyde (CAS Number 292644-32-7, Sigma Aldrich) or (2-{[(3,4-dichlorophenyl)amino]thioxomethylthio}ethoxy)-N-benzamide (Supplier Number ST034398, TimTec).

A typical dose for an antibiotic directed against at least one ClpB expressing bacterium of the present invention can be, for example, in the range of from about 0.01 mg/kg to about 30 mg/kg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. The daily oral dosage regimen may be about 0.01 mg/kg to about 30 mg/kg of total body weight, in particular 0.01 , 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 mg/kg body weight. Patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of the disorder to which such term applies, or one or more symptoms of such disorder or condition.

"Preventing" refers to measures taken to prevent the disorder to which such term applies from occurrence or, in early stages of a disorder. Preventing further refers to the inhibition of further development of a disorder to which such term applies.

Treatment or prevention of eating disorders preferably refers herein to the reduction of the amount or concentration of ClpB expressing bacteria in the subject to be treated.

In the context of the present invention, a "subject" denotes a human or non-human mammal, such as a rodent (rat, mouse, rabbit), a primate (chimpanzee), a feline (cat), or a canine (dog). Preferably, the subject is human. The subject according to the invention may be in particular a male or a female.

Preferably, the subject according to the invention is older than 13.

Preferably, the subject is a female.

By "eating disorder" (ED) is meant psychiatric illnesses defined by criteria from the Diagnostic and Statistical Manual of Mental Disorders 5^th Edition (DSM-5) and earlier editions (DSM-4, etc). Abnormal eating habits may involve either insufficient or excessive food intake to the detriment of an individual's physical and mental health. Bulimia nervosa (BN), anorexia nervosa (AN) and binge eating disorder (BED) are the most common specific forms of eating disorders. According to the DSM-5 criteria, to be diagnosed as having anorexia nervosa a person must display: persistent restriction of energy intake leading to significantly low body weight (in context of what is minimally expected for age, sex, developmental trajectory, and physical health), either an intense fear of gaining weight or of becoming fat, or persistent behavior that interferes with weight gain (even though significantly low weight), disturbance in the way one's body weight or shape is experienced, undue influence of body shape and weight on self-evaluation, or persistent lack of recognition of the seriousness of the current low body weight.

According to the DSM-5 criteria, to be diagnosed as having bulimia nervosa a person must display recurrent episodes of binge eating. An episode of binge eating is characterized by both of the following: eating, in a discrete period of time (e.g. within any 2-hour period), an amount of food that is definitely larger than most people would eat during a similar period of time and under similar circumstances and a sense of lack of control over eating during the episode (e.g. a feeling that one cannot stop eating or control what or how much one is eating), recurrent inappropriate compensatory behavior in order to prevent weight gain, such as self-induced vomiting, misuse of laxatives, diuretics, or other medications, fasting, or excessive exercise. The binge eating and inappropriate compensatory behaviors both occur, on average, at least once a week for three months. Self-evaluation is unduly influenced by body shape and weight.

According to the DSM-5 criteria, to be diagnosed as having binge eating disorder a person must display recurrent episodes of binge eating. An episode of binge eating is characterized by both of the following: eating, in a discrete period of time (e.g. within any 2-hour period), an amount of food that is definitely larger than most people would eat during a similar period of time and under similar circumstances, a sense of lack of control over eating during the episode (e.g. a feeling that one cannot stop eating or control what or how much one is eating). The binge eating episodes are associated with three or more of the following:

- eating much more rapidly than normal;

- eating until feeling uncomfortably full;

- eating large amounts of food when not feeling physically hungry;

- eating alone because of feeling embarrassed by how much one is eating;

- feeling disgusted with oneself, depressed or very guilty afterward. Binge eating occurs, on average, at least once a week for three months.

Other types of eating disorders include Other Specified Feeding or Eating Disorder (OSFED).

According to the DSM-5 criteria, to be diagnosed as having OSFED a person must present with a feeding or eating behaviors that cause clinically significant distress and impairment in areas of functioning, but do not meet the full criteria for any of the other feeding and eating disorders. A diagnosis might then be allocated that specifies a specific reason why the presentation does not meet the specifics of another disorder (e.g. bulimia nervosa- low frequency). The following are further examples for OSFED:

- Atypical anorexia nervosa: all criteria are met, except despite significant weight loss, the individual's weight is within or above the normal range;

- Binge eating disorder (of low frequency and/or limited duration): all of the criteria for BED are met, except at a lower frequency and/or for less than three months; Bulimia nervosa (of low frequency and/or limited duration): all of the criteria for bulimia nervosa are met, except that the binge eating and inappropriate compensatory behavior occurs at a lower frequency and/or for less than three months;

Purging disorder: recurrent purging behavior to influence weight or shape in the absence of binge eating;

Night eating syndrome: recurrent episodes of night eating, eating after awakening from sleep, or by excessive food consumption after the evening meal. The behavior is not better explained by environmental influences or social norms. The behavior causes significant distress/impairment. The behavior is not better explained by another mental health disorder (e.g. BED);Unspecified Feeding or Eating Disorder (UFED). According to the DSM-5 criteria this category applies to where behaviors cause clinically significant distress/impairment of functioning, but do not meet the full criteria of any of the Feeding or Eating Disorder criteria. This category may be used by clinicians where a clinician chooses not to specify why criteria are not met, including presentations where there may be insufficient information to make a more specific diagnosis (e.g. in emergency room settings). In the context of the present invention, an eating disorder can thus refer to the above mentioned list of disorders.

In one embodiment, the eating disorder is selected from the group consisting of anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED), overeating, hyperphagia, wasting diseases such as cachexia

By "appetite" is meant the desire to eat food, felt as hunger. Appetite exists in all higher life-forms, and serves to regulate adequate energy intake to maintain metabolic needs. It is regulated by a close interplay between the digestive tract, energy storage such as in adipose tissue and liver and the brain. Appetite is assessed in a subject by measuring the amount of food ingested and by assessing the subject's desire to eat.

"Dysregulation of appetite" refers to an abnormal appetite which includes increased appetite as well as decreased appetite which is permanently present or reoccurs several times a week and thus contributes to anorexia nervosa, bulimia nervosa, cachexia, wasting disease, overeating, binge eating disorder and hyperphagia.

In one embodiment, the subject suffers from an eating disorder selected from the group consisting of anorexia nervosa, bulimia nervosa, binge eating disorder, cachexia and wasting diseases and more particularly, anorexia nervosa, bulimia nervosa and binge eating disorder.

"Anorexia" relates to the decreased sensation of appetite thus resulting in a reduced appetite. While the term in non-scientific publications is often used interchangeably with anorexia nervosa, many possible causes exist for a decreased appetite, some of which may be harmless, while others indicate a serious clinical condition or pose a significant risk.

"Anorexia nervosa" (AN) refers in the context of the invention to an eating disorder characterized by immoderate food restriction that is characterized by failure to maintain body weight of at least 85% of the normal body weight. Furthermore the subject suffering from anorexia nervosa has an irrational fear of gaining weight, as well as a distorted body self-perception, where the subject sees him/herself as overweight despite overwhelming evidence to the contrary.

Accordingly, a person suffering from anorexia nervosa preferably disposes at least one of the psychological traits selected from the group consisting of body dissatisfaction, drive for thinness, perfectionism, ineffectiveness, interpersonal distrust, social insecurity and anhedonia. Preferably, a person suffering from anorexia nervosa disposes at least one of the psychological traits selected from the group consisting of body dissatisfaction, drive for thinness and perfectionism. Still preferably, a person suffering from anorexia nervosa disposes at least one of the psychological traits selected from the group consisting of ineffectiveness, interpersonal distrust, social insecurity and anhedonia.

In a further embodiment, a subject suffering from anorexia nervosa has a BMI inferior to 17.

The "BMI" or "body mass index" is defined as the subject's body mass divided by the square of his height. The formulae universally used in medicine produce a unit of measure of kg/m².

"Bulimia" or "Bulimia nervosa" is an eating disorder characterized by binge eating and purging, or consuming a large amount of food in a short amount of time followed by an attempt to rid oneself of the food consumed (purging), typically by vomiting, taking a laxative or diuretic, and/or excessive exercise. Some subjects may tend to alternate between bulimia nervosa and anorexia nervosa. Subject suffering from bulimia may be characterized by a normal BMI range, usually inferior to 25. "Binge eating disorder" or "BED" refers to an eating disorder characterized by binge eating consisting of eating, in a discrete period of time (e.g. within any 2-hour period), an amount of food that is larger than most people would eat in a similar period of time under similar circumstances, and is accompanied by a feeling of loss of control. The binge eating occurs, on average, at least twice a week for 6 months. Contrary to bulimia the binge eating is not associated with the recurrent use of inappropriate compensatory behavior. Subjects suffering from BED are seriously worried about the binge eating. Furthermore, subjects suffering from BED eat until being physically uncomfortable and nauseated due to the amount of food consumed and/or eat when bored or depressed and/or eat large amounts of food even when not really hungry and/or eat alone during periods of normal eating, owing to feelings of embarrassment about food and/or feel disgusted, depressed, or guilty after binge eating. Subjects with binge eating disorder act impulsively and feel a lack of control over their eating. Furthermore, subject suffering from binge eating disorder have problems coping with stress, anxiety, anger, sadness, boredom and worry.

In the context of this invention, subjects suffering of BED are preferably obese and have a BMI superior to 30.

"Overeating" is the consumption of excess food in relation to the energy that an organism expends (or expels via excretion), leading to weight gaining. Overeating can sometimes be a symptom of binge eating disorder or bulimia. Compulsive over eaters depend on food to comfort themselves when they are stressed, suffering bouts of depression, and have feelings of helplessness.

"Hyperphagia", also known as "polyphagia" refers to excessive hunger (compulsive) or increased appetite. In one embodiment hyperphagia may be caused by disorders such as Diabetes, Kleine-Levin Syndrome, the genetic disorders Prader-Willi Syndrome and Bardet Biedl Syndrome.

"Wasting disease" refers to the process by which a debilitating disease causes muscle and fat tissue to "waste" away. Wasting can be caused by an extremely low energy intake (e.g., caused by famine), nutrient losses due to infection, or a combination of low intake and high loss.

"Cachexia" is a wasting syndrome associated with loss of weight and/or muscle atrophy and/or fatigue, weakness, and significant loss of appetite which may be caused by cancer, AIDS, chronic obstructive lung disease, multiple sclerosis, congestive heart failure, tuberculosis, familial amyloid polyneuropathy, mercury poisoning (acrodynia) and hormonal deficiency. Cachexia, or wasting, as it may also be called, is seen with several diseases, such as AIDS, cancer, post hip fracture, chronic heart failure, chronic lung disease such as chronic obstructive lung disease (COLD) and/or chronic obstructive pulmonary disease (COPD), liver cirrhosis, renal failure, and autoimmune diseases such as rheumatoid arthritis and systemic lupus, sepsis and severe infection. Furthermore, wasting is also seen in aging.

In some embodiments, cachexia is caused by cancer.

The foremost sign of cachexia is weight loss, not only of fatty tissue but also of muscle tissue and even bone. This non-fatty tissue is also known as "lean body mass."

In addition, there is loss of appetite, weakness (asthenia), and a drop in hemoglobin level (anemia).

Cachexia is found as the terminal state of many different clinical conditions or in chronic diseases such as cancer, infections, AIDS, congestive heart failure, rheumatoid arthritis, tuberculosis, post-hip fracture, cystic fibrosis and Crohn's disease. It can also occur in elderly people who do not have any obvious symptoms of disease.

"Increased appetite" refers to an appetite wherein a subject has a higher food intake than the body requires. This may or may not result in weight gain.

"Normal appetite" thus refers to a subject having a food intake that corresponds to the amount of food the body requires.

"Decreased appetite" and/or "reduced appetite" refers an appetite wherein a subject has a lower food intake than the body requires. This may or may not result in weight loss.

"Food intake" can be measured using a multitude of techniques including self- reporting using e.g. diaries or questionnaires, measurements of calorie-intake from a buffet meal, using weighing of food prior to ingestion, or weighing and analysis of paired quantities of food. The food intake may be measured on a meal basis, a daily basis, a weekly basis or a monthly basis.

In one embodiment the composition is for decreasing weight gain in the subject.

The composition may be used, for example, to decrease appetite, wherein the increased appetite is due to binge eating disorder or overeating.

In an embodiment of the invention, the treatment results in at least 1 % decrease in food intake, such as a decrease of 2%, more preferably 3% or 5% or 7%, and even more preferred 10% below average food intake prior to initiation of treatment.

In another embodiment, the treatment leads to decrease in calorie intake irrespective of changes in food intake, since amount of food ingested may not be directly related to the ingested calorie intake, as the various food items such as fat, carbohydrates and proteins contain different amounts of calories per amount food. In an embodiment of the invention, the treatment results in a at least 1 % decrease in calorie intake, such as a decrease of 2%, more preferably 3%, or 5% or 7%, and even more preferred a decrease of 10% in calorie intake below average calorie intake prior to initiation of treatment.

In one embodiment the composition is for increasing weight gain in the subject.

The composition may be used, for example, to stimulate appetite, wherein the reduced appetite is due to anorexia, anorexia nervosa, bulimia nervosa, cachexia or wasting diseases, in particular anorexia, anorexia nervosa or bulimia nervosa.

In one embodiment of the invention, the treatment results in a 1 % increase in food intake, such as an increase of 2%, more preferably 3% or 5% or 7%, and even more preferred 10% above average food intake compared to prior to initiation of treatment.

In another embodiment, the treatment leads to increase in calorie intake irrespective of changes in food intake, since amount of food ingested may not be directly related to the ingested calorie intake, as the various food items such as fat, carbohydrates and proteins, contain different amounts of calories per amount food.

In a preferred embodiment of the invention, the treatment results in a at least 1 % increase in calorie intake, such as an increase of 2%, more preferably 3%, or 5% or 7%, and even more preferred 10% in calorie intake compared to prior to initiation of treatment.

In some embodiments, the composition is for use in the treatment or prevention of a disorder selected from the group consisting of anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED), overeating, hyperphagia, wasting diseases such as cachexia, and more particularly from anorexia nervosa (AN), bulimia nervosa (BN) and binge eating disorder (BED).

Also provided is a method of treating or reducing the chances of occurrence of eating disorders in a subject, said method comprising administering to a subject in need thereof a composition comprising at least one antibiotic directed against at least one CIpB expressing bacterium and/or administering to said subject an effective amount of a composition comprising probiotics not expressing CIpB and/or a combination thereof as described below.

A subject in need thereof is a subject suffering from an eating disorder, as herein defined, in particular selected from the group consisting of anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED), hyperphagia, wasting diseases such as cachexia, and more particularly from anorexia nervosa (AN), bulimia nervosa (BN) and binge eating disorder (BED).

Also provided is the use of a composition comprising at least one antibiotic directed against at least one CIpB expressing bacterium for the manufacture of a medicament intended for the treatment or prevention of eating disorders, as herein defined.

The inventors discovered that disregulated appetite is associated with the presence of CIpB protein and/or anti-ClpB antibodies and increased plasma levels of anti- a- MS H- reactive antibodies, preferably anti-ClpB IgG and/or IgM and anti-a-MSH-reactive IgG and/or IgM.

In one embodiment, reducing the amount or concentration of CIpB expressing bacteria in a subject results in the normalization of appetite in said subject.

Without being bound by theory, the composition comprising at least one antibiotic directed against at least one CIpB expressing bacterium reduces the amount of CIpB expressing bacteria, thus reducing the level or concentration of CIpB protein and/or anti- ClpB antibodies and/or reducing the level of anti-a-MSH-reactive antibodies, thus resulting in a normalized appetite.

In one embodiment, the composition used in the context of the invention is a pharmaceutical composition.

Pharmaceutical compositions used in the context of the invention are preferably designed to be appropriate for the selected mode or route of administration, and pharmaceutically acceptable diluents, carriers, and/or excipients such as dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents, and the like are used as appropriate. Such compositions can be designed in accordance with conventional techniques as disclosed, for example, in Remington, The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA 1995.

Suitable carriers for pharmaceutical compositions include any material which, when combined with the antibiotic of the invention retains the molecule's activity and is non-reactive with the subject's immune system.

The pharmaceutical compositions according to the invention may be administered orally in the form of a suitable pharmaceutical unit dosage form. The pharmaceutical compositions of the invention may be prepared in many forms that include tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow- release formulations, such as shaped polymeric gels.

The mode of administration and dosage forms are closely related to the properties of the therapeutic agents or compositions which are desirable and efficacious for the given treatment application. Suitable dosage forms include, but are not limited to, oral, intravenous, rectal, sublingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular, transdermal, spinal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial, and lymphatic administration, and other dosage forms for systemic delivery of active ingredients.

Pharmaceutical compositions of the invention may be administered by any method known in the art, including, without limitation, transdermal (passive via patch, gel, cream, ointment or iontophoretic); intravenous (bolus, infusion); subcutaneous (infusion, depot); transmucosal (buccal and sublingual, e.g., orodispersible tablets, wafers, film, and effervescent formulations; conjunctival (eyedrops); rectal (suppository, enema)); or intradermal (bolus, infusion, depot).

Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.

Pharmaceutical compositions of the invention may also be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion containers or multi-dose containers with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the pharmaceutical compositions of the invention may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

Pharmaceutical compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art, and the suppositories may be conveniently formed by admixture of the pharmaceutical composition with the softened or melted carrier(s) followed by chilling and shaping in molds.

For administration by inhalation, the pharmaceutical compositions according to the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Alternatively, for administration by inhalation or insufflation, the pharmaceutical compositions of the invention may take the form of a dry powder composition, for example, a powder mix of the pharmaceutical composition and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.

For intra-nasal administration, the pharmaceutical compositions of the invention may be administered via a liquid spray, such as via a plastic bottle atomizer. Typical of these are the Mistometerg (isoproterenol inhaler-Wintrop) and the Medihaler® (isoproterenol inhaler-Riker).

Pharmaceutical compositions of the invention may also contain excipient such as flavorings, colorings, anti-microbial agents, or preservatives.

The administration regimen may be for instance for a period of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 days.

The dose range depends on the composition to be administered and is defined above.

As is well known in the medical arts, dosages for any one subject depend on many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

In one aspect of the invention, the method of treatment or prevention of the invention enables maintaining a stable body-weight by administering the composition comprising at least one antibiotic directed against at least one ClpB expressing bacterium.

Overweight or too low body weight might also be considered in certain cultures as a physical appearance that is considered to be less attractive.

Therefore in one embodiment, the invention refers to a non-therapeutic method of regulating appetite in a subject, comprising administering to said subject an effective amount of a composition comprising at least one antibiotic directed against at least one ClpB expressing bacterium.

All the definitions previously mentioned also apply to the said non therapeutical method.

In one embodiment, an effective amount or a therapeutically effective amount is at least the minimal dose, but less than a toxic dose, of an active agent which is necessary to impart therapeutic benefit or benefit to a subject. Stated another way, such an amount for treating eating disorders, for example, is an amount that induces, ameliorates, or otherwise causes an improvement in the state of the subject, as the regulation of food intake. In one embodiment said non-therapeutic method is a cosmetic method.

As mentioned above, the invention also relates to a composition comprising probiotics not expressing ClpB protein for use in the treatment or prevention of an eating disorder.

All the definitions previously mentioned also apply to the said composition.

In one embodiment, the eating disorder is selected from the group consisting of anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED), hyperphagia, wasting diseases such as cachexia, and more particularly from anorexia nervosa (AN), bulimia nervosa (BN) and binge eating disorder (BED).

It further relates to a non-therapeutic method of regulating appetite in a subject, comprising administering to said subject an effective amount of a composition comprising probiotics not expressing ClpB protein.

All the definitions previously mentioned also apply to the said non therapeutical method.

By "probiotic" is meant a food additive comprising an effective amount of a microorganism, intended to be introduced in the diet. According to WHO, probiotics are live microorganisms which, when administered in adequate amounts confer a benefit to the host health (WHO, 2001 ).

In the context of the invention, the bacterium introduced in the diet is a bacterium not expressing ClpB protein. It is, in particular, a bacterium normally expressing the ClpB protein in which the expression of the protein would have been deleted.

ClpB expressing bacteria are well-known from the skilled person and may be identified by any conventional technique.

In one embodiment, the ClpB expressing bacterium in which the expression of the protein is deleted is selected from the group constituted of bacteria known as probiotic or commensal non-pathogenic bacteria in humans, e.g. non-pathogenic Escherichia coli.

A bacteria not expressing ClpB protein can be prepared by methods known to the skilled person. This can for example be done by DNA recombination.

By "effective amount" is meant an amount of bacteria that allows the manifestation of the desired effect, in the context of the invention, the treatment or prevention of eating disorders. In particular, it is meant an amount of between 1000 million and 10000 million UFC.day -1 .

As mentioned above, the invention also relates to a composition comprising a ClpB protein for use as a vaccine or as an immunogenic composition.

By "immunogenic composition" is meant in the context of the present invention a composition comprising a ClpB protein which provokes or immunomodulates {i.e. immunosuppress or immunostimulate) an immune response when administered to an individual or which induces the production of antibodies when administered to an individual.

By "vaccine" is meant a composition, such as the immunogenic composition described herein which is administered to immunomodulate an immune response, that will protect or treat an individual from illness, in particular due to that agent. The vaccine of the present invention is in particular a preventive (prophylactic) vaccine, for administration to the individual prior to the development of the eating disorder.

In particular, said composition is for use in the immunization against eating disorders.

More particularly, said composition is for use for preventing eating disorders.

Also provided is a method of immunization or vaccination in a subject, said method comprising administering to a subject in need thereof a composition comprising a CIpB protein.

In particular, said method is for reducing the chances of occurrence of eating disorders in a subject.

A subject in need thereof is a subject susceptible to suffer from an eating disorder. Also provided is the use of a composition comprising a CIpB protein for the manufacture of a vaccine or immunogenic composition for the immunization against eating disorders.

In particular, said composition is for the prevention of eating disorders.

In particular, said eating disorder is selected from anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED), overeating, hyperphagia, wasting diseases such as cachexia, in particular anorexia nervosa, bulimia nervosa and binge eating disorder.

In one embodiment, vaccine or immunogenic compositions can also comprise combinations of CIpB proteins disclosed herein.

Methods of obtaining a vaccine composition or immunogenic composition are well known in the art. Generally such methods involve extracting proteins from bacterial preparations using techniques such as sonication, proteolytic digestion, heat treatment, freeze-thaw treatment, osmotic shock treatment etc. ... Examples of artificial bacterial preparations include protein preparations either in part or entirely obtained by synthetic or recombinant methods.

A typical dose for a vaccine or immunogenic composition comprising CIpB can be, for example, in the range of from about 0.01 nmol/kg to about 1000 nmol/kg; however, doses below or above this exemplary range are envisioned. In particular, said dose is from about 0.1 nmol/kg to about 100 nmol/kg of total body weight, preferably from about 0.1 nmol/kg to about 20 nmol/kg, more particularly from 0.1 nmol/kg to about 10 nmol/kg, in particular 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 nmol/kg body weight.

As regards to said vaccine and immunogenic compositions, forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intraarterial, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, (see for example, Remington: The Science and Practice of Pharmacy, 21 ^st Edition, Lippincot and Williams, 2005). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent by filtered sterilization. As used herein, 'carrier' includes, without limitation, solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, liposomes and virosomes such as those described in Bomsel et al (201 1 ) Immunity 34: 269-280. The use of such media and agents for pharmaceutical active substances is well known in the art.

The phrase 'pharmaceutically-acceptable' or 'pharmacologically-acceptable' refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.

In a particular embodiment of the invention, the immunogenic or vaccine composition further comprises one or several adjuvant(s).

The term "adjuvant" as used herein denotes a product which, added to the content of an immunogenic composition, in particular to a vaccine, increases the intensity of the immune reaction induced in the host to which said composition is administered. An adjuvant may in particular increase the quantity of specific antibodies which said mammal is capable of producing after administration of said composition and thus increases the efficiency of immunization.

Preferably, the adjuvant(s) is devoid of side effect in a human host.

Adjuvants include any compound or compounds that act to increase a protective immune response. Adjuvants can include for example, emulsifiers; muramyl dipeptides; avridine; aqueous adjuvants such as aluminum hydroxide; oxygen-containing metal salts; chitosan-based adjuvants, and any of the various saponins, oils, and other substances known in the art, such as Ampfaigen, LPS, bacterial cell wall extracts, bacterial DNA, CpG sequences, synthetic oligonucleotides and combinations thereof (Schijns et al (2000) Curr. Opin. Immunol, 12:456), Mycohacterialplilei ( phlei) cell wall extract ( CWE) (U.S. Patent No. 4,744,984), M. phlei DNA (M-D A), and M-DNA-M phlei ceil wall complex (MCC), heat-labile enterotoxin (LT), cholera toxin (CT), cholera toxin B subunit (CTB), polymerised liposomes, mutant toxins, e.g. LTK63 and LTR72, microcapsules, interleukins (e.g. I L- 1 β , IL-2, IL-7, IL-12, INFy), GM-CSF, MDF derivatives, CpG oligonucleotides, LPS, MPL, phosphophazenes, Adju-Phos®, glucan, antigen formulation, liposomes, DDE, DHEA, DMPC, DMPG, DOC/Alum Complex, Freund's incomplete adjuvant, ISCOMs®, LT Oral Adjuvant, muramyl dipeptide, monophosphoryl lipid A, muramyl tripeptide, and phospatidylethanolamine. Compounds which can serve as emulsifiers include natural and synthetic emulsifying agents, as well as anionic, cationic and nonionic compounds. Oxygen-containing metal salts include salts of Al, K, Ca, Mg, Zn, Ba, Na, Li, B, Be, Fe, Si, Co, Cu, Ni, Ag, Au, and Cr which are sulphates, hydroxides, phosphates, nitrates, iodates, bromates, carbonates, hydrates, acetates, citrates, oxalates, and tartrates, and mixed forms thereof, including aluminium hydroxide, aluminium phosphate, aluminium sulphate, potassium aluminium sulphate, calcium phosphate, Maalox (mixture of aluminium hydroxide and magnesium hydroxide), beryllium hydroxide, zinc hydroxide, zinc carbonate, zinc chloride, and barium sulphateAmong the synthetic compounds, anionic emulsifying agents include, for example, the potassium, sodium and ammonium sails of lauric and oleic acid, the calcium., magnesium and aluminum salts of fatty acids, and organic sulfonates such as sodium lauryl sulfate. Synthetic cationic agents include, for example, cetyltrhethylarnmonlum bromide, while synthetic nonionic agents are exemplified by glycerylesters (e.g., glyceryl monostearate), polyoxyethylene glycol esters and ethers, and the sorbitan fatty acid esters (e.g., sorbitan monopalmitate) and their polyoxyethylene derivatives (e.g., polyoxyethylene sorbitan. monopalmitate). Natural emulsifying agents include acacia, gelatin, lecithin and cholesterol.

Other suitable adjuvants can be formed with an oil component, such as a single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion. The oil can be a mineral oil, a vegetable oil, or an animal oil. Mineral oils are liquid hydrocarbons obtained from petrolatum via a distillation technique, and are also referred to in the art as liquid paraffin, liquid petrolatum, or white mineral oil. Suitable animal oils include, for example, cod liver oil, halibut oil, menhaden oil, orange roughy oil and shark liver oil, all of which are available commercially. Suitable vegetable oils, include, for example, canola oil, almond oil, cottonseed oil, com oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and the like. Freund's Complete Adjuvant (FCA) and Freund's incomplete Adjuvant (FIA) are two common adjuvants that are commonly used in vaccme preparations, and are also suitable for use in the present invention. Both FCA and FIA are water-in-mineral oil emulsions; however, FCA also contains a killed Mycobacterium sp. Particularly preferred adjuvants for mucosal vaccines include galactosyl ceramide (GalCer), as described in Lee et al (201 1 ) Vaccine 29: 417-425.

Immunomodulatory cytokines can also be used in the vaccine compositions to enhance vaccine efficacy, for example, a an adjuvant, Non-limiting examples of such cytokines include interferon alpha (IFN-a), interleukin-2 (IL-2), and granulocyte rnacrophage-colony stimulating factor (GM--CSF), or combinations thereof. The vaccine and immunogenic compositions, may be administered by intravenous administration, intrarterially, endoscopically, intralesionally, percutaneously, subcutaneously, intramuscular, intrathecal^, intraorbital^, intradermally, intraperitoneally, transtracheal^, subcuticularly, by intrasternal injection, intravenous, intrarterial, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, by inhalation or intranasal spraying, by endotracheal route and the like. Administration of the compositions can be by infusion or injection (e.g., intravenously, intramuscularly, intracutaneousiy, subcutaneously, intrathecal, intraduodenally, intraperitoneally, and the like). Additionally, said compositions can be administered by "needle-free" delivery systems.

The injections can be split into multiple injections, with such split inoculations administered preferably substantially concurrently. When administered as a split inoculation, the dose of the immunogen is preferably, but not necessarily, proportioned equally in each separate injection. If an adjuvant is present in the vaccine composition, the dose of the adjuvant is preferably, but not necessarily, proportioned equally in each separate injection. The separate injections for the split inoculation are preferably administered substantially proximal to each other on the patient's body. In some preferred aspects, the injections are administered at least about 1 cm apart from each other on the body. In some preferred aspects, the injections are administered at least about 2.5 cm apart from each other on the body. In highly preferred aspects, the injections are administered at least about 5 cm apart from each other on the body, in some aspects, the injections are administered at least about 10 cm apart from each other on the body, in some aspects, the injections are administered more than 10 cm apart from each other on the body, for example, at least about 12,5. 15, 17,5, 20, or more cm apart from each other on the body.

Various alternative pharmaceutical delivery systems may be employed. Non- limiting examples of such systems include liposomes and emulsions. Certain organic solvents such as dimethylsulfoxide also may he employed. Additionally, the vaccine compositions may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the protein. The various sustained- release materials available are well known by those skilled in the art. Sustained -release capsules may, depending on their chemical nature, release the vaccine compositions over a range of several days to several weeks to several months.

The compositions are ideally administered to a subject in advance of evidence of eating disorder, or in advance of any symptom of an eating disorder. Administration may be via a parenteral or non-parenteral route. Routes of administration include, e.g. intravenous, intrarterial, intradermal, transdermal, intramuscular, mucosal subcutaneous, percutaneous, intratracheal, intraperitoneal, perfusion and lavage. For example, administration is via a mucosal route, for example via a nasal, oral (via the mucosa of the digestive system), vaginal, buccal, rectal, sublingual, ocular, urinal, pulmonal or otolar (vie the ear) route.

The immunisation may include various 'unit doses.'

A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. A unit dose may contain from about 0.01 nmol/kg to about 1000 nmol/kg. In particular, said dose is from about 0.1 nmol/kg to about 100 nmol/kg of total body weight, preferably from about 0.1 nmol/kg to about 20 nmol/kg, more particularly from 0.1 nmol/kg to about 10 nmol/kg, in particular 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 nmol/kg body weight.

In one embodiment, the vaccine composition may be administered in a single daily dose, or the total daily dosage may be administered in divided doses, for example, two, three or four times daily. Furthermore, the vaccine composition may be administered in intranasal form via topical use of suitable intranasal vehicles, via transdermal routes, using those forms of transdermal skin patches well known to persons skilled in the art, by implantable pumps; or by any other suitable means of administration. To be administered in the form of a transdermal delivery system, for example, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

Vaccine administration may further comprise a prime-boost regimen. In these methods, one or more priming immunisations are followed by one or more boosting immunisations. The actual immunogenic composition can be the same or different for each immunisation and the type of immunogenic composition, the route, and formulation of the immunogens can also be varied. One useful prime-boost regimen provides for two priming immunisations, four weeks apart, followed by two boosting immunisations at 4 and 8 weeks after the last priming immunisation.

Immunisation schedules (or regimens) are well known for animals (including humans) and can be readily determined for the particular subject and composition. Hence, the compositions can be administered one or more times to the subject. Preferably, there is a set time interval between separate administrations of the immunogenic composition. While this interval varies for every subject, typically it ranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks. For humans, the interval is typically from 2 to 6 weeks. In a particularly advantageous embodiment of the present invention, the interval is longer, advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks or 70 weeks.

The immunisation regimes typically have from 1 to 6 administrations of the composition, but may have as few as 1 , 2, 3, 4 or 5. The methods of inducing an immune response can also include administration of an adjuvant with the composition. In some instances, annual, biannual or other long interval (5-10 years) booster immunisation can supplement the initial immunisation protocol.

A specific embodiment of the invention provides methods of inducing an immune response against an eating disorder in a subject by administering a vaccine or immunogenic composition of the invention, one or more times to a subject wherein the ClpB protein is at a level sufficient to induce a specific immune response in the subject. Such immunisations can be repeated multiple times at time intervals of at least 2, 4 or 6 weeks (or more) in accordance with a desired immunisation regime.

The inventors discovered a correlation between ClpB protein and anti-ClpB antibodies, in particular anti-ClpB IgG and eating disorders, in particular anorexia nervosa and bulimia nervosa.

As a consequence, the present invention also concerns an in vitro method for diagnosing an eating disorder in a subject, which method comprises:

a) measuring the level of ClpB protein and/or anti-ClpB antibodies in a biological sample from said subject;

b) comparing said measured level of ClpB protein and/or anti-ClpB antibodies to a reference value; and

c) deducing if the subject suffers from an eating disorder.

In the context of the invention, the subject produces antibodies directed against the bacterial protein ClpB, called anti-ClpB antibodies.

The antibodies produced by the subject in the context of the invention may be IgM, IgD, IgG, IgA and/or IgE antibodies, in particular IgG and/or IgM antibodies.

In the context of the invention, the presence of said ClpB protein in form of a ClpB expressing bacterial strain increases the production of antibodies directed against ClpB and/or antibodies against a-MSH.

The antibodies produced by the subject that are directed against a-MSH are also called auto-antibodies. The term "auto-antibody" refers to an antibody produced by the immune system that is directed against a subject's own protein.

Preferably, the anti-ClpB antibody cross-reacts with α-MSH as defined above. Thus, in one embodiment, the anti-ClpB antibody is also an anti-a-MSH antibody. An "antibody" or "immunoglobulin" (Ig) may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (I) and kappa (k). There are five main heavy chain classes (or isotopes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1 -L, CDR2-L, CDR3-L and CDR1 -H, CDR2-H, CDR3-H, respectively. A conventional antibody antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

"Framework Regions" (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1 -L, FR2-L, FR3-L, FR4-L, and FR1 -H, FR2-H, FR3-H, FR4-H, respectively. As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

As used herein, a "method for diagnosing" or "diagnosing method" or simply "diagnosis" refers to a method for determining or identifying a possible disease or disorder in a subject. In the context of the invention the method of diagnosis relates to determining or identifying an eating disorder, in particular an eating disorder selected from anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED), overeating, hyperphagia, wasting diseases such as cachexia, in particular anorexia nervosa, bulimia nervosa and binge eating disorder.

As used herein, the term "biological sample" means a substance of biological origin. Examples of biological samples include, but are not limited to blood, plasma, serum or saliva. Preferably, a biological sample according to the present invention is a blood, serum and/or plasma sample, in particular plasma sample. The biological sample according to the invention may be obtained from the subject by any appropriate means of sampling known from the skilled person.

The method of diagnosing an eating disorder according to the invention comprises a step b) of comparing the measured level of CIpB protein and/or anti-ClpB antibodies to a reference value.

Preferably the reference value is measured in a sample from the same tissular origin as the biological sample of the subject of step a) of the methods of the invention.

Preferably, the "reference value" corresponds to the normal level of CIpB protein and/or anti-ClpB antibodies.

As intended herein a "normal level" of CIpB protein and/or anti-ClpB antibodies means that the level of CIpB protein and/or anti-ClpB antibodies in the biological sample is within the norm cut-off values for those CIpB protein and/or anti-ClpB antibodies. The norm is dependant on the biological sample type and on the method used for measuring the level of CIpB protein and/or anti-ClpB antibodies in the biological sample. In particular, the normal level is the mean level of CIpB protein and/or anti-ClpB antibodies in a healthy population.

In particular, the reference value corresponds to the level of CIpB protein and/or anti-ClpB antibodies in a healthy subject. Accordingly, the reference value may correspond to the level of CIpB protein and/or anti-ClpB antibodies in a reference sample derived from a healthy subject. In particular, the reference value corresponds to the level of ClpB protein and/or anti-ClpB antibodies in a healthy subject, in particular when the level of ClpB protein and/or anti-ClpB antibodies is measured in step a).

As used herein, a "healthy population" means a population constituted of subjects who have not previously been diagnosed with an eating disorder. Subjects of a healthy population preferably also do not otherwise exhibit any symptoms of an eating disorder. In other words, such subjects, if examined by a medical professional, would be characterized as healthy and free of symptoms of disease.

Accordingly, a "healthy subject" means a subject who has not previously been diagnosed with an eating disorder. A healthy subject also does not otherwise exhibit symptoms of an eating disorder. In other words, such healthy subject, if examined by a medical professional, would be characterized as healthy and free of symptoms of disease.

Preferably, a healthy subject does not display any of the eating disorders previously mentioned.

In the context of the invention the level of ClpB protein anti-ClpB antibodies may be measured by any method known to the skilled person, for example by enzyme-linked immunosorbent assay.

In particular, levels of ClpB protein is measured by immunoassay or immunoblots or by analytical methods, like for example mass spectrometry (MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography coupled to mass spectrometry (LC-MS, LC-MS/MS), for example.

The term "immunoassay" as used according to the present invention includes competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, agglutination test, enzyme-labelled and mediated immunoassays, such as ELISA, biotin/avidin type assay, radioimmunoassay, Immunoelectrophoresis, and immunoprecipitation, and is more directly related to ELISA.

Mass spectrometry (MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography coupled to mass spectrometry (LC-MS/MS), are all analytical methods very well known by the man skilled in the art which are suitable to carry out the method according to the invention.

In a particular embodiment, levels of ClpB DNA is measured by quantifying the ClpB DNA in the stool of the subject, for example by following the method disclosed in the experimental part below under ClpB DNA assay, or as follows: DNA is extracted from the cultures of bacteria strains and purified from feces using the QIAamp^R DNA Stool Mini Kit (QIAGEN, France). Bacteria are dissolved in water and boiled at 100 °C during 5 min, after 1 min of centrifugation at 1 1 ,000 r.p.m., the supernatant containing the DNA is stored at -20°C. Using the NCBI primer design tool (http://www.ncbi.nlm.nih.gov/tools/primer- bjast/), the following nucleotide primers are designed that amplify 180 bp DNA region coding for the ClpB protein fragment containing one identified a-MSH-like epitope Forward: 5'- GCAGCTCGAAGGCAAAACTA -3' (SEQ ID NO: 4) and Reverse: 5'- ACCGCTTCGTTCTGACCAAT -3' (SEQ ID NO: 5) (Invitrogen Custom Primers, Cergy Pontoise, France). PCR is performed in a thermocycler with MicroAmp tubes (Eppendorf, Hambourg, Germany). The reactionis carried out in a 50 μΙ volume containing 25 μΙ_ of Go Taq ^R Green Master Mix 2X (Promega, Madison, Wl), 1 μΙ (20 pmol) of each primer, 21 μΙ of bi-distilled water and 1 μΙ of bacterial DNA. PCR conditions are 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 are visualized on a 1 % agarose gel (Sigma), with the expected size of 180 bp and the specificity validated using ClpB mutant strain.

In particular, levels of antibodies reacting with ClpB are measured using enzyme- linked immunosorbent assay (ELISA).

Typically, the ClpB protein (Delphi Genetics) is coated on a typically Maxisorp plates (Nunc, Rochester, NY) using for exemple 100 μΙ and a concentration of 2 μg/ml in typically 100 mM NaHC0₃ buffer, pH 9.6 for typically 72 h at 4°C. Plates are washed for example (5 min x 3) in phosphate-buffered saline (PBS) with typically 0.05% Tween 200, pH 7.4, and then incubated for example overnight at 4°C with 100 μΙ of mouse or rat plasma diluted 1 :200 in PBS to determine free antibody levels or diluted 1 :200 in typically dissociative 3 M NaCI, 1 .5 M glycine buffer, pH 8.9 to determine total Antibody levels. The plates are washed (3x) and incubated with typically 100 μΙ of alkaline phosphatase (AP)- conjugated goat anti-rat IgG (1 :2000), anti-rat IgM (1 :1000), anti-mouse IgG (1 :2000), or anti-mouse IgM (1 :1000) all from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Following washing (3x), typically 100 μΙ of p-nitrophenyl phosphate solution (Sigma) is added as alkaline phosphatase substrate. After typically 40 min of incubation at room temperature, the reaction is stopped by adding 3N NaOH. The OD (optiocal density) is determined typically at 405 nm using typically a microplate reader Metertech 960 (Metertech Inc., Taipei, Taiwan). Blank OD values resulting from the reading of plates without addition of plasma samples are subtracted from the sample OD values. Each determination is typically done in duplicate. The variation between duplicate values is typically less than 5%.

Typically a similar protocol is used to measure anti-ClpB IgG and IgM antibodies in human plasma typically diluted 1 :400 using typically corresponding anti-human IgG or IgM AP-conjugated antibodies. Preferably, in particular when the level of anti-ClpB antibodies is measured using enzyme-linked immunosorbent assay, the reference value of anti-ClpB antibodies is in particular measured in OD for anti-ClpB IgG in plasma and the OD value is between 0 and 3, more preferably between 2.0 and 3.0 and/or between 0.0 and 1 .0.

Preferably, in the methods of the invention, it is determined whether the measured level of CIpB protein and/or anti-ClpB antibodies is higher (increased) or lower (decreased) than the reference value according to the invention.

The inventors further discovered, that while, in controls, anti-ClpB IgG correlated negatively with the normal range of several psychological traits, in AN patients, anti-ClpB IgG correlated positively with the core psychopathological traits such as body dissatisfaction and drive for thinness.

The method may thus further comprise a step b2) wherein the psychological traits characterizing an eating disorder are evaluated.

In one embodiment, an increased value of anti-ClpB IgG antibodies than the reference value is indicative of the presence of an eating disorder in particular, AN.

In one embodiment psychopathological traits indicating the presence of an eating disorder are body dissatisfaction and drive for thinness.

In a particular embodiment, an increased value of anti-ClpB IgG than the reference value and/or the detection of physical traits associated with an eating disorder in step b2) is indicative for the presence of an eating disorder, in particular, AN.

Methods to evaluate "psychological traits" connected with an eating disorder are known to the skilled in the art.

In one example psychological traits connected with an eating disorder can be assayed using the eating disorder inventory-2.

"Eating Disorder Inventory (EDI)" is a self-report questionnaire used to assess the presence of eating disorders, (a) Anorexia Nervosa both restricting and binge- eating/purging type; (b) Bulimia Nervosa; and (c) Eating disorder not otherwise specified including Binge Eating Disorder (BED). The original questionnaire consisted of 64 questions, divided into eight subscales. EDI-2 refers to a reviewed version of 1991 .

In one embodiment, step c) of the method of diagnosis of the invention refers to deducing if the subject suffers from an eating disorder wherein an increased value of anti- ClpB antibodies than the reference value is preferably indicative for the presence of an eating disorder.

In another embodiment, the detection of physical traits with an eating disorder as detected in step b2) is indicative for the presence of an eating disorder. The method may thus comprise a further step pre-c) of determining the ratio of the level of anti-ClpB IgG that cross-react with α-MSH in said sample compared to the total level of anti-ClpB IgG determined in step a).

The ratio of the level of anti-ClpB IgG that cross-react with α-MSH may be determined for example by preincubating plasma samples of humans diluted 1 :400 in PBS with typically 10^~6 M a-MSH peptide (Bachem) for example overnight at 4°C before adding the samples to typically 96-well Maxisorp plates (Nunc) coated with CIpB protein (Delphi Genetics). IgG antibodies reactive with CIpB are detected typically by ELISA using corresponding anti-human AP-conjugated antibodies (Jackson). The percentage of CIpB IgG that are cross-reactive with a -MSH may be calculated relative to levels of anti-ClpB IgG detected without absorption in each individual plasma sample equal 100%.

In a particular embodiment, an increased ratio of the level of anti-ClpB IgG that cross-react with a-MSH compared to the total level of anti-ClpB antibodies than the reference value is indicative for the presence of an eating disorder.

The invention further relates to a method of treating or reducing the chances of occurrence of an eating disorder in a subject in need thereof, comprising administering to said subject a composition comprising one antibiotic directed against at least one CIpB expressing bacterium and/or administering to said subject a composition comprising probiotics not expressing CIpB and/or a combination thereof, wherein said subject was diagnosed as suffering from an eating disorder by:

a) measuring the level of CIpB protein and/or anti-ClpB antibodies in a biological sample from said subject;

b) comparing said measured level of CIpB protein and/or anti-ClpB antibodies to a reference value; and

c) deducing if the subject suffers from an eating disorder.

All the definitions previously mentioned also apply to the said method.

The invention further relates to an in vitro method of selecting subjects suffering from an eating disorder as likely to respond to a treatment reducing the level of CIpB protein and/or anti-ClpB antibodies which comprises:

a) measuring the level of CIpB protein and/or anti-ClpB antibodies in a biological sample from said subject;

b) comparing said measured level of CIpB protein and/or anti-ClpB antibodies to a reference value; and

c) selecting the subject for a treatment reducing the level of CIpB protein and/or anti-ClpB antibodies, wherein said treatment reducing the level of CIpB protein and/or anti-ClpB antibodies comprises administering to said subject an effective amount of a composition comprising one antibiotic directed against at least one CIpB expressing bacterium and/or administering to said subject an effective amount of a composition comprising probiotics not expressing CIpB and/or a combination thereof.

In one embodiment, a subject having an increased value of CIpB protein and/or CIpB antibodies than the reference value is selected as being likely to respond to a treatment reducing the level of CIpB protein and/or anti-ClpB antibodies.

In one embodiment, the method thus may further comprise a step b2) wherein the psychological traits characterizing an eating disorder are evaluated as defined above.

Step c) of the method of diagnosis of the invention refers to selecting the subject for a treatment decreasing the level of protein CIpB and/or anti-ClpB antibodies, wherein a subject having an increased value of CIpB protein and/or CIpB antibodies than the reference value is selected as being likely to respond to a treatment reducing the level of CIpB protein and/or anti-ClpB antibodies.

In a particular embodiment, a subject having an increased value of CIpB protein and/or CIpB antibodies than the reference value and/or having physical traits associated with an eating disorder as detected in step b2) is selected as being likely to respond to a treatment decreasing the level of CIpB protein and/or anti-ClpB antibodies.

In one embodiment psychopathological traits indicating the presence of an eating disorder are body dissatisfaction and drive for thinness.

Furthermore, the inventors having showed that chronic intragastric delivery of E. coli in mice decreased food intake, the invention further relates to a composition comprising probiotics overexpressing CIpB protein for use in the treatment or prevention of obesity.

Obesity" refers herein to a medical condition wherein the subject preferably has a

BMI superior to 30.

By "overexpressing" is meant the artificial expression of a protein due to expression of a gene in increased quantity, here the increased expression of the gene encoding for CIpB protein.

A bacteria overexpressing CIpB protein can be prepared by methods known to the skilled person. This can for example be done by bacterial transformation with vectors expressing CIpB DNA.

In one embodiment, the CIpB overexpressing bacterium is selecting from bacteria known as probiotic or commensal non-pathogenic bacteria in humans, e.g. non- pathogenic Escherichia coli. By "effective amount" is meant an amount of bacteria that allows the manifestation of the desired effect. In particular, it is meant an amount of between 1000 million and 10000 million UFC.day-1 . Throughout the instant application, the term "comprising" is to be interpreted as encompassing all specifically mentioned features as well optional, additional, unspecified ones. As used herein, the use of the term "comprising" also discloses the embodiment wherein no features other than the specifically mentioned features are present (i.e. "consisting of").

The invention will now be described in more detail with reference to the following figures and examples.

Sequences

SEQ ID NO: 1 shows the amino acid sequence of the chaperon protein CIpB from E. Coli K12 referenced under NCBI Reference Number NP 417083.1 and referenced under Uni-Prot entry P63284.

SEQ ID NO: 2 shows amino acids 542 to 548 of the amino acid sequence of the chaperon protein CIpB from E. Coli K12 of SEQ ID NO: 1 .

SEQ ID NO: 3 shows the amino acid sequence of α-MSH from Homo sapiens referenced under Gen Pept Sequence ID, PRF: 223274.

SEQ ID NO: 4 shows the nucleic acid sequence of the nucleotide primers CIpB.

SEQ ID NO: 5 shows the nucleic acid sequence of the nucleotide primers CIpB.

Figures

Figure 1 : Proteomic identification of molecular mimicry between E. coli K12 proteins and a-MSH.

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

Figure 2. CIpB immunization in mice.

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 a-MSH (100 μg kg- 1 body weight, i.p.) or PBS. (f) Plasma levels of ClpB-reactive IgG before and after adsorption with 10^"6M α-MSH. (g) Affinity of anti-ClpB IgG shown as the dissociation equilibrium constants (KD values), (h) Plasma levels of a-MSH-reactive total IgG. (i) Affinity (KD) of anti-a-MSH IgG. (j) cAMP assay in human embryonic kidney-293 cells overexpressing MC4R after stimulation by a- MSH alone or together with IgG (0.5 mg ml^"1) pooled from ClpB-immunized or from Adj- injected mice, (k) The cAMP assay was performed with IgG depleted from anti-a-MSH IgG. (a) Two-way repeated measurement analysis of variance (ANOVA) before a-MSH injection (100 μg kg^"1 body weight, i.p.), PoO.0001 , Bonferroni post tests ^*a at least, PoO.05 CIpB group vs Ctr; ^*b at least, PoO.05 Adj group vs Ctr.; ^*c, PoO.05, Student's t- test CIpB group vs PBS; and ^*d, PoO.05, Student's t-test CIpB 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, ^*Po0.05. (f, g) ANOVA PoO.0001 , Tukey's posttests ^***Po0.001 CIpB +Adj vs other groups, paired t-test ##Po0.01 , ###Po0.001 . (h) ANOVA P =0.0002, Tukey's post tests ^***Po0.001 , ^*Po0.05; (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 ^*Po0.05; ANOVA P =0.04, Student's t-test #Po0.05, aClpB vs a-MSH, bClpB vs Adj. (mean ± s.e.m.; j, n =6, k, n=3).

Figure 3: E. coli supplementation in mice.

Effects of intragastric daily gavage (days 1-21 ) in mice with either E. coli K12 wild-type (WT), ClpBdeficient (AClpB) 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 CIpB 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 AClpB, 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^"6M a-MSH.

Plasma levels of anti-a-MSH IgM (h) and IgG (i). (j) Affinity (equilibrium constant) of anti- a-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 , ^*Po0.05, E. coli WT vs aCtr and bl_B. (c) Kruskal-Wallis (K-W) test third week P=0.0001 , Dunn's post tests, ^***Po0.001 , ^**Po0.01 , E. coli WT vs aCtr, bl_B and CACIpB. (d) ANOVA days 1 -2, P =0.006, Tukey's post tests ^**Po0.01 , ^*Po0.05, K-W test third week PoO.0001 , Dunn's post tests, ^***Po0.001 , ^**Po0.01 , E. coli WT vs aCtr, bl_B and CACIpB. (f) K-W test, before adsorption P =0.02, Dunn's post tests ^*Po0.05, ANOVA after adsorption, PoO.0001 , Tukey's post tests ^**Po0.01 , E. coli WT vs other groups, (g) ANOVA before adsorption, P=0.01 , Tukey's post tests ^*Po0.05, E. coli WT vs other groups, paired t-test ##Po0.01 . (h) Student's t-test, E. coli WT vs other groups ^*Po0.05. (j) K-W test P =0.02, Dunn's post test ^*Po0.05, Mann- Whitney test, #Po0.05. (mean ± s.e.m., n=8).

Figure 4. Anti-ClpB antibodies in ED patients.

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 CIpB IgG (c) and IgM (d) before and after adsorption with 10^"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 PoO.0001 , Dunn's post test, ^**Po0.01 , Mann-Whitney test #Po0.05. (f) Analysis of variance P =0.02, Tukey's post test ^*Po0.05. (mean ±s.e.m., Ctr, n =65, AN, n=27 BN, n =32 and BED, n=14).

Figure 5. Plasma concentrations of bacterial CIpB protein in patients with eating disorders and healthy controls (Ctr).

AN, anorexia nervosa, BN, bulimia nervosa, BED, binge-eating disorder. ^*p<0,05 Student's t-test of mean CIpB concentrations vs. Ctr. Percentage (%) of patients having CIpB concentrations higher than mean+2 standard deviations (SD) of controls. Examples

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

Example 1

Materials and Methods

E.coli K12 culture and protein extraction

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, lllkirch, France) at 37 °C for 24 h. Protein extraction was performed as described by Marti et al. (PLoS ONE 201 1 , 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 (300mM NaCI and 20mM Tris-HCI, pH 8). The suspension was disrupted by sonication (3 x 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, NJ, USA).

Two-dimensional polyacrylamide gel electrophoresis

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, 20mM DTT, 2mM 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 χ 18- cm χ 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% (vokvol) orthophosphoric acid and in 50% (vohvol) 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% (vohvol) orthophosphoric acid and 0.66 g CBB G-250 per liter).

Immunoblotting

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^{" 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.l^"1 Tris, pH 8, and 150mM.I^"1 NaCI) plus 0.05% (vohvol) Tween 20. After washes, membranes were incubated overnight at 4 °C with polyclonal rabbit anti-a- MSH IgG (1 :1000, Peninsula Laboratories, San Carlos, CA, 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 II (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-a-MSH IgG with 10^"6M of a-MSH peptide (Bachem AG, Bubendorf, Switzerland) overnight at 4 °C.

Protein identification

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 μΙ of 5% (vohvol) acetonitrile/0.1 % (vohvol) 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-μηι particle size) C18 column (43mm long χ 75 μηι inner diameter; Agilent Technologies). A 9-min linear gradient (3-80% acetonitrile in 0.1 % formic acid) at a flow rate of 400 nl.min^"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 (PoO.01 ), or at least two top-ranking peptides each with a MASCOT score of 447 (PoO.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 %.

Protein identification from OFFGEL

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 μΑ 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, lllkirch, 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-a-MSH IgG (Peninsula Laboratories) followed by protein identification as described above.

Immunization and behavior in mice

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 C57BI6 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 μΙ of 1 :1 (vohvol) of PBS with Complete Freund's Adjuvant (Sigma, St Louis, MO, USA), intraperitoneal^ (i.p.); (ii) Group 2, adjuvant injection controls (n = 8): 200 μΙ of Complete Freund's Adjuvant in PBS (1 :1 (vohvol), i.p.); (iii) Group 3, PBS injection controls (n = 8): 200 μΙ 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 μΙ of 1 :1 (vohvol) of PBS with Incomplete Freund's Adjuvant (Sigma) i.p.; (ii) Group 2 (n = 8): 200 μΙ of Incomplete Freund's Adjuvant in PBS (1 :1 (vohvol), i.p.); (iii) Group 3, (n = 8): 200 μΙ 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, NJ, 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 a-MSH peptide (Bachem AG), 100 g.kg^"1 body weight in 100 μΙ 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 O-maze (Med Associate, Inc., St Albans, VT, USA) tests performed during 2 consecutive days. Two hours after the O-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.

Locomotor activity and anxiety tests

After feeding study in the BioDAQ cages, mice were analyzed for locomotor activity using a Versamax Animal Activity Monitor (AccuScan Instruments, Inc., Columbus, OH, 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.

ClpB and a-MSH autoantibody assay

Plasma levels of auto-Abs reacting with ClpB, a-MSH and adrenocorticotropic hormone were measured using enzyme-linked immunosorbent assay according to a published protocol (Fetissov SO., Methods Biol Mol 201 1 ). In brief, ClpB protein (Delphi Genetics), a-MSH or adrenocorticotropic hormone peptides (Bachem AG) were coated onto 96-well Maxisorp plates (Nunc, Rochester, NY, USA) using 100 μΙ and a concentration of 2 μg.ml^"1 in 100mM NaHC03 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 μΙ of mouse plasma diluted 1 :200 in PBS to determine free auto-Ab levels or diluted 1 :200 in dissociative 3M NaCI and 1 .5M glycine buffer, pH 8.9, to determine total auto-Ab levels. The plates were washed (three times) and incubated with 100 μΙ 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 μΙ 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.).

Absorptions of ClpB antibodies with a-MSH

Plasma samples of mice, diluted 1 :200 in PBS, or humans, diluted 1 :400 in PBS, were preincubated with 10^"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 a-MSH were calculated relative to levels of anti-ClpB antibodies detected without absorption in each individual plasma sample equal 100%.

IgG purification from plasma

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, CA, USA), then 500 μΙ of mouse plasma was mixed with 500 μΙ 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, IL, 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, NJ, USA). For the cyclic adenosine monophosphate (cAMP) experiment, IgG purified from eight mice of the CIpB 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 a-MSH (Bachem AG) coated on activated UltraLink beads (Pierce, Rockford, IL, USA). The a-MSH IgGdepleted IgG effluents were saved, lyophilized and diluted in PBS.

Affinity kinetics assay

Affinity kinetics of mouse IgG for CIpB and a-MSH was determined by a biospecific interaction analysis based on the surface plasmon resonance phenomenon on a BIAcore 1000 instrument (GE Healthcare). a-MSH (Bachem AG) or CIpB protein (Delphi Genetics) were diluted to 0.5 mg.ml^"1 in 10mM 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 a-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 μΙ. Γπ ί η ¹ at 25 °C.

Between injections of each sample, the binding surface was regenerated with 10mM 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.

In vitro cAMP assay

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 a-MSH peptide (Bachem AG) was diluted in the induction buffer: PBS, 500 μΜ IBMX, 100 μΜ RO 20-1724 (Sigma), 20mM MgCI2 to the final concentrations of 2, 1 , 750, 500, 250, 100, 75, 50 and 10 nM corresponding to the a- MSH doses of 0.6, 3, 4.5, 6, 15, 30, 45, 60 and 120 pmol, respectively, and also included one blank sample. After unfreezing, the cells were cultured in 250 ml tissue culture flasks (BD-Falcon, Beckton-Dickinson, Bedford, MA, USA) in Dulbecco's modified Eagle medium 4.5 g. ¹ glucose (Eurobio, Courtaboeuf, France) supplemented with (2mM 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% C02 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 μΙ) 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, Wl, 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 CIpB-immunized or adjuvant control groups, and which were added to the cells just before a-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-a-MSH IgG fraction, the same experiment was performed with each a-MSH concentration and IgG as described above.

E. coli^' gay age in mice

One-month-old male C57BI6 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 CIpB; (iii) gavaged with LB medium only; and (iv) controls that did not receive any treatments. The CIpB 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-a-MSH IgG and IgM were assayed as described above.

ClpB DNA assay

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 1 1 000 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 a-MSH-like epitope (Figure 1 e), 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-μΙ volume containing 25 μΙ of Go Taq Green Master Mix 2 ^χ (Promega), 1 μΙ (20 pmol) of each primer, 21 μΙ of bi-distilled water and 1 μΙ 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. Plasma concentrations of bacterial ClpB protein

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, NY) using 100 μΙ and a concentration of 2 μg/ml in 100 mM NaHC03 buffer, pH 9.6 for 24 h at 4°C. Plates were washed (5 min x 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 pM 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 2h at room temperature (RT). Plates were washed (5 min x 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 x 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 x 3) in PBS with 0.05% Tween 200, pH 7.4 and then 1 00 μΙ 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 CIpB protein standard dilutions were subtracted from the sample OD values. Plasma concentrations of CIpB was calculated based on the OD of the CIpB standard curve and was adjusted for the plasma dilution.

Statistical analysis

Data were analyzed and the graphs were plotted using the GraphPad Prism 5.02 (GraphPad Software Inc., San Diego, CA, 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 CIpB 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(a-MSH) vs normalized cAMP response), which equation was Y = 100/(1 +10(LogEC50-X) ^χ HillSlope). Data are shown as mean ± s.e.m., and for all test, Po0.05 was considered statistically significant.

Results

Proteomic identification of bacterial a-MSH mimetics

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 K1 2 cultures, the cytoplasmic fraction was resolved by 2D gel electrophoresis (Figure 1 a) and transferred to a polyvinylidene difluoride membrane. To increase the probability of detection of multiple a-MSH-like epitopes in bacterial proteins, the membrane was revealed with polyclonal anti-a-MSH IgG. 13 immunopositive protein spots were found (Figure 1 b), among which the spots 1 -8 disappeared after preadsorption of antibodies with 1 0^"6M a-MSH (Figure 1 c), confirming specific a-MSH-mimetic epitopes. Using mass spectrometry, protein spots 1 ,2,3 and 4, displaying the strongest a-MSH-like staining, were identified as isoforms of the heat-shock protein named CIpB, a 857-a.a. protein, 857 amino acid protein disaggregation chaperone or CIpB, MW 95526 (molecular weight: 95526 Da, accession number: NP_417083.1 , SEQ ID NO: 1 ). Less intensely stained a- 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-a-MSH IgG preadsorbed or not with a-MSH (data not shown). One band was specifically recognized by anti-a-MSH IgG and was found to contain the CIpB protein (with the highest MASCOT score of 1065). Based on these results, CIpB was selected as a target protein for further validation of its molecular mimicry with α-MSH. To analyze the amino-acid sequence homology between a-MSH and bacterial CIpB, 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 CIpB protein displaying discontinuous 5 a.a. sequence homology with a-MSH (Figure 1 d). This putative a-MSH-like epitope was located in an inter-helical loop of the CIpB protein structure indicating that it is exposed on the protein surface, that is, accessible to auto-Abs binding. Western blot of the recombinant CIpB protein revealed with anti-a-MSH IgG showed a 96-kDa band (Figure 1 e), confirming that the CIpB protein contains a-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.

Immunization of mice with CIpB

To investigate whether E. coli CIpB may induce auto-Abs crossreactive with a-MSH, influencing feeding and anxiety, mice were immunized with the recombinant bacterial CIpB protein. Mice that received CIpB together with adjuvant or adjuvant alone displayed lower body weight for a few days after injections (Figure 2a). However, 4 weeks later, ClpB-immunized mice had higher body weight (+5%) vs controls (Figure 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 (Figure 2b). The increase in food intake was owing to increased meal size (Figure 2c), as meal number did not change (Figure 2d), indicating that the CIpB immunization interfered with satiation rather than with hunger mechanisms. This is in agreement with the known role of a-MSH to induce satiation. To further validate the relevance of CIpB immunization to a-MSH anorexigenic effect, mice received i.p. injection of a-MSH. The following 24 h food intake and body weight were not affected in ClpB-immunized mice (Figure 2e), indicating that they were not sensitive to the anorexigenic effect of administered a-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 CIpB immunized mice, a strong increase in anti-ClpB IgG levels (Figure 2f) with lower affinities (Figure 2g) were found, in agreement with recent IgG induction. Increased plasma levels of a-MSH-reactive IgG were also found in ClpB-immunized mice (Figure 2h); these IgG were similarly characterized by lower affinities for a-MSH, as compared with controls (Figure 2i). Adsorption of mouse plasma with a-MSH, significantly reduced plasma levels of anti-ClpB IgG, confirming that a fraction, but not all of the anti-ClpB IgG, were crossreactive with a- MSH (Figure 2f). Plasma levels of a-MSH IgM auto-Abs did not significantly differ between the groups (data not shown). Whether CIpB immunization may induce auto-Abs crossreacting with the adrenocorticotropic hormone, a 39-a.a. peptide containing the a- 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 CIpB and a-MSH.

Mouse IgG effects on MC4R signaling

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

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 ACIpB 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 (Figures 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 (Figures 3c and d). Remarkably, mice receiving ACIpB 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 (Figure 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 ACIpB E. coli groups (Figures 3f and g). Adsorption of plasma with α-MSH reduced anti-ClpB IgG levels in E. coli WT-gavaged mice (Figure 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 a-MSH caused dissociation of a-MSH IgM immune complexes crossreactive with ClpB that were increased in E. coli WT-gavaged mice (Figure 3f). Plasma levels of anti-a-MSH IgM were also increased by E. coli WT delivery as compared with all other groups (Figure 3h), while anti-a-MSHreactive IgG were only slightly increased without reaching significance (Figure 3i). Nevertheless, affinity kinetic analysis of a-MSH IgG revealed lower values of the dissociation equilibrium constants in E. coli-gavaged mice (Figure 3j), without significant changes of the association or dissociation rates (data not shown). These changes, including increased levels of the IgM class of a-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-a- MSH IgG associated with increased meal size and body weight gain, E. coli-gavaged mice showed increased production of both anti-a-MSH-reactive IgM and IgG with increased affinities associated with decreased meal size and body weight. Anti-ClpB antibodies in ED patients

As the ability of the E. coli CIpB protein to stimulate production of α-MSH crossreactive auto-Abs was validated, the relevance of bacterial CIpB to ED was next determined by studying anti-ClpB antibodies in patients with AN, BN or BED. It was found that both anti- CIpB IgG and IgM were readily detectable in plasma of ED patients as well as healthy subjects with no significant differences of their mean levels (Figures 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^"6M a-MSH, leading to significant reduction of anti-ClpB IgG and IgM detectable levels in all study groups (Figures 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 (Figure 4e). Elevated levels of a-MSH crossreactive anti-ClpB IgM were found in AN as compared with BN (Figure 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, CIpB IgG correlated inversely with the normal range of a few psychological traits, but in AN patients,

CIpB 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 CIpB IgM in the opposite way, being negative in AN but positive in BED (Table 1 ). However, in BED patients, CIpB 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 CIpB IgG or IgM and drive for thinness or interpersonal distrust, respectively, resembled closely the correlations between the same psychological traits and a-MSH-reactive IgG or IgM found in a different group of AN patients in a previous study.

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 lnventory-2. All Spearman's r ^*p<0.05, ^**/x0.01 , except Pearson's r^*p<0.05 for perfectionism. (n=65 Contr., n=27 AN, and n=14 BED).

CIpB Maturity fears Impulse regulation Social insecurity

igG r= -0.31 ^* r= -0.26 ^* r= -0.26 ^*

(Contr.)

CIpB Body dissatisfaction Drive for thinness Perfectionism

IgG (AN) r= 0.4 ^* r= 0.35 ^* r= 0.38 ^*

CIpB Ineffectiveness Interpersonal distrust Social insecurity Anhedonia

IgM (AN) r= -0.42 ^* r= -0.58 ^** r= -0.52 ^** r= -0.35 ^*

CIpB Bulimia Perfectionism Age

IgM r= 0.53 ^* r= 0.6 ^* r= -0.74 ^**

(BED)

Plasma concentrations of bacterial CIpB protein

CIpB protein was detected in plasma samples of all study subjects ranging from 10 pM to 180 pM with a mean level of about 30 pM in healthy controls. Mean plasma levels of CIpB were significantly elevated in all groups of patients with eating disorders, including AN, BN and BED (see Figure 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 CIpB.

Conclusion

The results reveal a molecular link between CIpB expressing gut bacteria and the regulation of motivated behavior and emotion via production of CIpB protein and anti-ClpB antibodies crossreactive with a-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 CIpB protein and anti-ClpB IgG crossreactive with a-MSH in ED patients and correlations of anti-ClpB antibodies with patient's psychopathological traits support the involvement of CIpB-expressing microorganisms in increased CIpB antibody production and establishment of abnormal feeding behavior and emotion.

In conclusion, the results identify CIpB as a protein responsible for the origin of auto-Abs crossreactive with a-MSH, associated with psychopathological traits in ED patients and, hence, that CIpB-expressing microorganisms as a novel specific target for diagnostics and treatment of ED.

Claims

Claims

1 . A composition comprising at least one antibiotic directed against at least one CIpB protein expressing bacterium for use in the treatment or prevention of eating disorders.

2. A non-therapeutic method of regulating appetite in a subject, comprising administering to said subject an effective amount of a composition comprising at least one antibiotic directed against at least one CIpB protein expressing bacterium.

3. A method of treating or reducing the chances of occurrence of eating disorders in a subject, said method comprising administering to a subject in need thereof a composition comprising at least one antibiotic directed against at least one CIpB expressing bacterium.

4. A composition comprising probiotics not expressing CIpB protein for use in the treatment or prevention of an eating disorder.

5. A non-therapeutic method of regulating appetite in a subject, comprising administering to said subject an effective amount of a composition comprising probiotics not expressing CIpB protein.

6. A composition comprising a CIpB protein for use as a vaccine or as an immunogenic composition.

7. The composition for use according to claim 5, for the immunization against eating disorders.

8. The composition for use according to claim 5 or 6, for preventing eating disorders.

9. An in vitro method for diagnosing an eating disorder in a subject, which method comprises:

a) measuring the level of CIpB protein and/or anti-ClpB antibodies in a biological sample from said subject;

b) comparing said measured level of CIpB protein and/or anti-ClpB antibodies to a reference value; and

c) deducing if the subject suffers from an eating disorder.

10. An in vitro method of selecting subjects suffering from an eating disorder as likely to respond to a treatment reducing the level of CIpB protein and/or anti-ClpB antibodies which comprises:

a) measuring the level of CIpB protein and/or anti-ClpB antibodies in a biological sample from said subject;

b) comparing said measured level of CIpB protein and/or anti-ClpB antibodies to a reference value; and

c) selecting the subject for a treatment reducing the level of CIpB protein and/or anti-ClpB antibodies, wherein said treatment reducing the level of CIpB protein and/or anti-ClpB antibodies comprises administering to said subject an effective amount of a composition comprising one antibiotic directed against at least one CIpB expressing bacterium and/or administering to said subject an effective amount of a composition comprising probiotics not expressing CIpB and/or a combination thereof.

1 1 . An in vitro method according to any of claim 9 to 10, in which the level CIpB protein in step a) is measured by quantifying the CIpB DNA in the stool of said subject.

12. The composition for use according to any of claims 1 , 4 or 6 to 8, the non-therapeutic method according to claim 2 or 5 or the in vitro method according to any of claims 9 to 10, wherein the eating disorder is selected from the group consisting of anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED), overeating, hyperphagia, wasting diseases such as cachexia.

13. A composition comprising probiotics overexpressing CIpB protein for use in the treatment or prevention of obesity.