TECHNICAL FIELD

This invention is in the field of compositions comprising bacterial strains isolated from the mammalian digestive tract and the use of such compositions in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be sterile in utero, but it is exposed to a large variety of maternal and environmental microbes immediately after birth. Thereafter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as delivery mode, environment, diet and host genotype, all of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently, the microbiota stabilizes and becomes adult-like [1] The human gut microbiota contains more than 1,500 different phylotypes dominated in abundance levels by two major bacterial divisions (phyla), the Bacteroidetes and the Firmi cutes [2-3] The successful symbiotic relationships arising from bacterial colonization of the human gut have yielded a wide variety of metabolic, structural, protective and other beneficial functions. The enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products providing an important nutrient source for the host and additional health benefits. Similarly, the immunological importance of the gut microbiota is well-recognized and is exemplified in germ-free animals which have an impaired immune system that is functionally reconstituted following the introduction of commensal bacteria [4-6]

A growing body of preclinical literature has demonstrated bidirectional signalling between the brain and the gut microbiome, involving multiple neurocrine and endocrine signalling systems. Indeed, increased levels of Clostridium species in the microbiome have been linked to brain disorders [7], and an imbalance of the Bacteroidetes and Firmicutes phyla has also been implicated in brain development disorders [8] Suggestions that altered levels of gut commensals, including those of Bifidobacterium, Lactobacillus, Sutterella, Prevotella and Ruminococcus genera and of the Alcaligenaceae family are involved in immune-mediated central nervous system (CNS) disorders, are questioned by studies suggesting a lack of alteration in the microbiota between patients and healthy subjects [9] This indicates that, at present, the practical effect of the link between the microbiome and human brain diseases is poorly characterised. Accordingly, more direct analytical studies are required to identify the therapeutic impact of altering the microbiome on CNS disorders.

In recognition of the potential positive effect that certain bacterial strains may have on the animal gut, various strains have been proposed for use in the treatment of various diseases (see, for example, [9-12]). Also, certain strains, including mostly Lactobacillus and Bifidobacterium strains, have been proposed for use in treating various inflammatory and autoimmune diseases that are not directly linked to the intestines (see [13] and [14] for reviews). In addition, a range of probiotics have been investigated in animal models to determine a role of the gut microbiome in modulating emotional behaviour, and Bifidobacterium and Lactobacillus are the main genera showing beneficial effects, reducing anxiety and repetitive behaviours, and increasing social interaction [15-17] However, the relationship between different diseases and different bacterial strains, and the precise effects of particular bacterial strains on the gut and at a systemic level and on any particular types of diseases, are poorly characterised, particularly for central nervous system diseases.

There is a growing body of evidence to suggest that the microbiota-gut-brain axis is affected in neurodevelopmental and neuropsychiatric disorders, such as autism spectrum disorders (ASD). Animal models have provided considerable insight into how the microbiota may be involved in ASD. Furthermore, preclinical studies have demonstrated that targeting the gut microbiota through administration of beneficial live biotherapeutics display efficacy in improving autistic- related behaviour in animal models.

Reference [18] discusses possible methods of treating neurodevelopmental disorders by administering a composition comprising a bacterial species selected from Bacteroides and/or Enterococcus, but provides data only for Bacteroides. Reference [19] discusses a similar use of Bacteroides and Enterococcus, with data limited to Bacteroides firagilis, Bacteroides vulgatus and Enterococcus faecalis. Reference [78] discusses use of Enterococcus faecium for treating and preventing central nervous system disorders and conditions. Reference [79] discloses the utilisation of bacteria to produce GABA. Table 10 of that document sets out a list of 2219 bacterial species which are capable of being engineered to produce GABA. The only reference to Enterococcus durans in Reference [79] is in Table 10; there is no suggestion at all that strains of the species E. durans are inherently capable of treating any CNS disorder.

There is a requirement in the art for new methods of treating central nervous system disorders.

SUMMARY OF THE INVENTION

The inventors have developed new therapies for treating or preventing central nervous system disorders and conditions, such as central nervous system disorders and conditions mediated by the microbiota-gut-brain axis. In particular, the inventors have identified that bacterial strains of the species Enterococcus durans can be effective for treating or preventing diseases and conditions mediated by the microbiota-gut-brain axis. As described in the examples, oral administration of compositions comprising Enterococcus durans may reduce symptoms associated with dysfunction of the microbiota-gut-brain axis.

Therefore, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing a central nervous system disorder or condition. The central nervous system disorder or condition may be mediated by the microbiota-gut-brain axis. The invention further provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing a neurodevelopmental disorder or a neuropsychiatric condition. For example, the inventors have identified that treatment with bacterial strains from this species can provide clinical benefits in mouse models of central nervous system disorders, in particular those mediated by the microbiota-gut-brain axis. In particular, treatment with bacterial strains from this species may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the hypothalamus-pituitary-adrenal (HPA) axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and/or may modulate the levels of commensal metabolites, inflammatory markers and/or gastrointestinal permeability of a subject.

In particular embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing a disorder or condition selected from the group consisting of: autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder (MDD); depression; seasonal affective disorder; anxiety disorders; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post-traumatic stress disorder; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; dementia; Alzheimer’s; Parkinson’s disease; epilepsy; chronic pain, such as central sensitisation or fibromyalgia; motor neuron disease; Huntington’s disease; Guillain-Barre syndrome and/or meningitis. The effects shown for the bacterial strains from the species Enterococcus durans on the microbiota-gut-brain axis and on disorders or conditions mediated by the microbiota-gut- brain axis suggest therapeutic benefits for other diseases and conditions mediated by the microbiota-gut-brain axis, such as those listed above.

In other embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating comorbidities that occur with disorders and conditions mediated by the microbiota-gut-brain axis, such as the diseases and conditions listed above. In particularly preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating gastrointestinal comorbidities associated with diseases and conditions mediated by the microbiota-gut-brain axis, such as the diseases and conditions listed above. The mouse model experiments used in this application for the assessment of the symptoms of autism spectrum disorders are known in the art to be applicable for the assessment of the symptoms of other central nervous system disorders including those listed above [20- 21]

In particularly preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing autism spectrum disorders, such as autism. For example, the inventors have identified that treatment with Enterococcus durans strains can reduce symptom severity in a mouse model of autism spectrum disorders and can reduce stereotyped, repetitive, compulsive and anxious behaviour. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans for use in reducing stereotyped, repetitive, compulsive or anxious behaviour, in particular in the treatment of autism spectrum disorders. The inventors have also identified that treatment with Enterococcus durans strains can enhance learning and memory function. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans for improving learning or memory function. The inventors have identified that treatment with Enterococcus durans can ameliorate feelings of despair and helplessness. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans for reducing feelings of despair or helplessness. The inventors have identified that treatment with Enterococcus durans can ameliorate neophobia and sociability deficit. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans for treating, preventing or ameliorating neophobia and / or sociability deficit. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in the treatment of behavioural symptoms of autism spectrum disorders. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans for use in the treatment of the gastrointestinal symptoms of autism spectrum disorders. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in the treatment of the behavioural and gastrointestinal symptoms of autism spectrum disorders. Treatment with Enterococcus durans strains may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and/or may modulate the levels of commensal metabolites, inflammatory markers and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of autism spectrum disorders. In certain embodiments, treatment with Enterococcus durans strains may modulate the levels of oxytocin and/or vasopressin hormones.

In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing obsessive compulsive disorder (OCD). In preferred embodiments, the invention provides a composition for use in reducing stereotyped, repetitive, compulsive or anxious behaviour, or improving learning or memory function in the treatment of OCD. In preferred embodiments, the invention provides a composition for reducing feelings of despair or helplessness in the treatment of OCD. In preferred embodiments, the invention provides a composition for treating or preventing neophobia and / or sociability deficit in the treatment of OCD. Treatment with Enterococcus durans strains may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and/or may modulate the levels of commensal metabolites and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of OCD.

In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing major depressive disorder (MDD). For example, treatment with Enterococcus durans strains may provide clinical benefits in a mouse model of depression. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in the treatment of depression. In preferred embodiments, the invention provides a composition for use in reducing stereotyped, repetitive, compulsive or anxious behaviour, or improving learning or memory function in the treatment of depression. In preferred embodiments, the invention provides a composition for reducing feelings of despair or helplessness in the treatment of depression. In preferred embodiments, the invention provides a composition for treating or preventing neophobia and / or sociability deficit in the treatment of depression. Treatment with Enterococcus durans strains may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may modulate the levels of commensal metabolites, inflammatory markers and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of MDD. In certain embodiments, treatment with Enterococcus durans strains may increase the level of oxytocin hormone and/or reduce the level of vasopressin hormone. In certain embodiments, treatment with Entrococcus durans strains may decrease gastrointestinal permeability.

In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing anxiety disorders. For example, treatment with Enterococcus durans strains reduces disease incidence and disease severity in a mouse model of anxiety, as shown in the examples. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in the treatment of anxiety disorder. Compositions using Enterococcus durans may be particularly effective for treating anxiety disorder. In preferred embodiments, the invention provides a composition for use in reducing stereotyped, repetitive, compulsive or anxious behaviour, or improving learning or memory function in the treatment of anxiety. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans for reducing feelings of despair or helplessness in the treatment of anxiety. In preferred embodiments, the invention provides a composition for treating or preventing neophobia and / or sociability deficit in the treatment of anxiety.

In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing stress disorders, such as post-traumatic stress disorder. For example, compositions comprising a bacterial strain of the species Enterococcus durans may reduce stress in mouse models of stress disorders. Treatment with Enterococcus durans strains may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may modulate the levels of commensal metabolites, inflammatory markers and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of stress disorders. In certain embodiments, treatment with Enterococcus durans strains may increase the level of oxytocin hormone and/or reduce the level of vasopressin hormone.

In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing schizophrenia spectrum and psychotic disorders, such as schizophrenia. Treatment with Enterococcus durans strains may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may modulate the levels of commensal metabolites and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of schizophrenia spectrum and psychotic disorders. In certain embodiments, treatment with Entrococcus durans strains may decrease gastrointestinal permeability.

In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing bipolar disorder. For example, compositions comprising a bacterial strain of the species Enterococcus durans may reduce occasions of mania and/or depression in mouse models of bipolar disorder. Treatment with Enterococcus durans strains may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may modulate the levels of commensal metabolites, inflammatory markers and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of bipolar disorder. In certain embodiments, treatment with Enterococcus durans strains may modulate the levels of oxytocin and/or vasopressin hormones.

In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing neurocognitive disorders, such as Alzheimer’s disease. For example, compositions comprising a bacterial strain of the species Enterococcus durans may improve cognitive and behavioural functioning in mouse models of neurocognitive disorders. Treatment with Enterococcus durans strains may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may modulate the levels of commensal metabolites and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of neurocognitive disorders.

In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing Parkinson’s disease. Treatment with Enterococcus durans strains may modulate signalling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may modulate the levels of commensal metabolites, inflammatory markers and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of Parkinson’s disease. In certain embodiments, treatment with Enterococcus durans strains may modulate the levels of oxytocin and/or vasopressin hormones.

In certain embodiments, the compositions of the invention are for use in a method of modulating the microbiota-gut-brain axis in the treatment or prevention of a disease or condition mediated by the microbiota-gut-brain axis. In particular, the compositions of the invention may be used in modulating the microbiota-gut-brain axis in the treatment or prevention of autism spectrum disorders; obsessive compulsive disorder; major depressive disorder; anxiety disorders; stress disorders; schizophrenia spectrum disorders; bipolar disorders; neurocognitive disorders and Parkinson’s disease.

In preferred embodiments, the bacterial strain in the compositions of the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO: 1 or 2. Preferably, the sequence identity is to SEQ ID NO:2. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:2. In these embodiments, the reference to a strain of the bacterial species Enterococcus durans is to be interpreted as any bacterial strain which has the sequence identity defined in this paragraph.

In preferred embodiments of the invention, the bacterial strain in the composition is of Enterococcus durans. The bacterial strain may have a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Enterococcus durans. Preferably, the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO:l or 2. Preferably, the sequence identity is to SEQ ID NO:2. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:2.

In certain embodiments, the compositions of the invention are for oral administration. Oral administration of the strains of the invention can be effective for treating central nervous system disorders and conditions, in particular those mediated by the microbiota-gut-brain axis. Also, oral administration is convenient for patients and practitioners and allows delivery to and / or partial or total colonisation of the intestine.

In certain embodiments, the composition of the invention comprises one or more pharmaceutically acceptable excipients or carriers.

The invention also provides a biologically pure culture of the Enterococcus durans strain deposited under accession number NCIMB 43456. A pure culture is a population of cells growing in the absence of other strains.

In certain embodiments, the composition of the invention has been lyophilised. The composition of the invention can also comprise a lyophilised bacteria strain of the species Enterococcus durans. Lyophilisation is an effective and convenient technique for preparing stable compositions that allow delivery of bacteria.

In certain embodiments, the invention provides a food product comprising a composition as described above.

In certain embodiments, the invention provides a vaccine composition comprising a bacterial strain of the species Enterococcus durans as described above, or an antigen from said bacterium.

Additionally, the invention provides a method of treating or preventing a disease or condition mediated by the microbiota-gut-brain axis, comprising administering a composition comprising a bacterial strain of the species Enterococcus durans, as described herein.

As those skilled in the art will recognise, the species Enterococcus durans is, relatively speaking, genetically similar to the bacterial species Enterococcus faecium. Despite this, however, these species are taxonomically and phenotypically distinct. There have been numerous articles published detailing their phenotypic differences, including references [30, 31 and 77] The organisms also have been reported as having different antibiotic resistance profiles and other functional properties. Further, if the results of the behavioural animal studies presented in the examples of the present application are compared to the corresponding studies in Reference [78], it can be observed that there are notable differences in the outcomes observed following the administration of strains of Enterococcus durans in the present application and Enterococcus faecium in Reference [78], demonstrating an unexpected yet advantageously complementary effect on CNS disorders.

In developing the above invention, the inventors have identified and characterised a bacterial strain that is particularly useful for therapy. The Enterococcus durans strain of the invention is shown to be effective for treating the diseases described herein, such as autism spectrum disorder. Therefore, in another aspect, the invention provides a cell of the Enterococcus durans strain deposited under accession number NCIMB 43456, or a biotype or derivative thereof. The invention also provides compositions comprising such cells, or biologically pure cultures of such cells. The invention also provides a cell of the Enterococcus durans strain deposited under accession number NCIMB 43456, or a biotype or derivative thereof, for use in therapy, in particular for the diseases described herein.

In especially preferred embodiments, the invention provides a composition comprising the strain deposited under accession number NCIMB 43456, for use in a method of treating or preventing a central nervous system disorder or condition. In especially preferred embodiments, the invention provides a composition comprising the strain deposited under accession number NCIMB 43456, for use in a method of treating or preventing a neurodevelopmental disorder or a neuropsychiatric condition. In especially preferred embodiments, the invention provides a composition comprising the strain deposited under accession number NCIMB 43456, for use in a method of treating or preventing autism spectrum disorder, or preferably autism. In especially preferred embodiments, the invention provides a composition comprising the strain deposited under accession number NCIMB 43456, for use in a method of reducing stereotyped, repetitive, compulsive and/or anxious behaviour, and / or improving learning and / or memory function, for example in the treatment of autism. In preferred embodiments, the invention provides a composition comprising the strain deposited under accession number NCIMB 43456 for reducing feelings of despair or helplessness for example in the treatment of depression and / anxiety. In preferred embodiments, the invention provides a composition comprising the straid deposited under accession number NCIMB 43456 for treating or preventing neophobia and / or sociability deficit.

Further numbered embodiments of the invention are provided below:

1. A composition comprising a bacterial strain of the species Enterococcus durans, for use in a method of treating or preventing a central nervous system disorder or condition.

2. The composition of embodiment 1, wherein the central nervous system disorder or condition is mediated by the microbiota-gut-brain axis.

3. The composition of embodiments 1 or 2, wherein the composition is for use in a method of treating or preventing a neurodevelopmental disorder or a neuropsychiatric condition.

4. The composition of embodiments 1 or 2, wherein the composition is for use in a method of treating or preventing a disorder or condition selected from the group consisting of autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder (MDD); depression; seasonal affective disorder; anxiety disorders; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post- traumatic stress disorder; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; dementia; Alzheimer’s; Parkinson’s disease; epilepsy; chronic pain, such as central sensitisation or fibromyalgia; motor neuron disease; Huntington’s disease; Guillain-Barre syndrome and meningitis. 5. The composition of embodiments 1 or 2, wherein the composition is for use in a method of treating or preventing autism spectrum disorder.

6. The composition of embodiment 5, wherein the composition is for use in a method of treating or preventing autism.

7. The composition of any preceding embodiment, wherein the composition prevents, reduces or alleviates stereotyped, repetitive, compulsive and/or anxious behaviour and / or improves learning and / or memory function.

8. The composition of embodiments 1 or 2, wherein the composition is for use in a method of treating or preventing obsessive compulsive disorder.

9. The composition of embodiment 8, wherein the composition prevents, reduces or alleviates repetitive, compulsive and/or anxious behaviour.

10. The composition of embodiments 1 or 2, wherein the composition is for use in in a method of treating or preventing MDD.

11. The composition of embodiment 10, wherein the composition treats or prevents acute major depressive episodes and/or prevents new episodes (recurrence prevention).

12. The composition of embodiments 10 or 11, wherein the composition prevents, reduces or alleviates the occurrence of mild, moderate or severe MDD episodes.

13. The composition of embodiments 1 or 2, wherein the composition is for use in in a method of treating or preventing anxiety disorders.

14. The composition of embodiment 13, wherein the anxiety disorder is generalised anxiety disorder (GAD); specific phobia; social anxiety disorder; separation anxiety disorder; agoraphobia; panic disorder and/or selective mutism.

15. The composition of embodiments 1 or 2, wherein the composition is for use in a method of treating or preventing neurocognitive disorders.

16. The composition of embodiment 15, wherein the neurocognitive disorder is vascular dementias; mixed form Alzheimer’s disease and vascular dementia; Lewy body disease; frontotemporal dementia; Parkinson’s dementia; Creutzfeldt- Jakob disease; Huntington’s disease; and Wemicke-Korsakoff syndrome.

17. The composition of embodiments 1 or 2, wherein the composition is for use in a method of treating epilepsy.

18. The composition of any preceding embodiment, wherein the composition is for use in a method of modulating the microbiota-gut-brain axis.

19. The composition of any preceding embodiments, wherein the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Enterococcus durans.

20. The composition of any preceding embodiments, wherein the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO: 1 or 2. 21. The composition of embodiment 20, wherein the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO:2, or wherein the bacterial strain has the 16s rRNA sequence represented by SEQ ID NO:2.

22. A composition comprising a bacterial strain, wherein the bacterial strain has a 16s rRNA sequence that is least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO:2, for use in a method of treating or preventing a central nervous system disorder or condition.

23. The composition of any preceding embodiment, wherein the composition is for oral administration.

24. The composition of any preceding embodiment, wherein the composition comprises one or more pharmaceutically acceptable excipients or carriers.

25. The composition of any preceding embodiment, wherein the bacterial strain is lyophilised.

26. The composition of any preceding embodiment, wherein the bacterial strain is viable and capable of partially or totally colonising the intestine.

27. The composition of any preceding embodiment, wherein the composition comprises a single strain of Enterococcus durans.

28. The composition of any preceding embodiment, which comprises the Enterococcus durans bacterial strain as part of a microbial consortium.

29. A food product comprising the composition of any preceding embodiment, for the use of any preceding embodiment.

30. A vaccine composition comprising the composition of any preceding embodiment, for the use in a method of preventing a central nervous system disorder or condition of any preceding embodiment.

31. A method of treating or preventing a central nervous system disorder or condition, comprising administering a composition comprising a bacterial strain of the species Enterococcus durans to a patient in need thereof.

32. A cell of the Enterococcus durans strain deposited under accession number NCIMB 43456, or a biotype or derivative thereof, for use in therapy, or for the use of any of embodiments 1- 28.

33. A composition comprising the cell of embodiment 32, for the use of embodiment 32.

34. The composition of embodiment 33, comprising a pharmaceutically acceptable carrier or excipient.

35. A biologically pure culture of the Enterococcus durans strain deposited under accession number NCIMB 43456, or a derivative thereof. BRIEF DESCRIPTION OF DRAWINGS

* denotes p < 0.05 relative to the vehicle control group; ** denotes p < 0.01 relative to the vehicle control group; *** denotes p < 0.001 relative to the vehicle control group; **** denotes p < 0.0001 relative to the vehicle control group.

Figure 1: Deposit receipt for NCIMB 43456.

Figure 2: Effect of treatment with strain NCIMB 43456 or vehicle on (A) occludin mRNA expression in the ileum and (B) occludin mRNA expression in the colon.

Figure 3: Effect of treatment with strain NCIMB 43456 or vehicle on IDOl mRNA expression in the ileum and (B) IDOl mRNA expression in the colon.

Figure 4: Effect of treatment with strain NCIMB 43456 or vehicle on TJP1 mRNA expression in the ileum and (B) TJP1 mRNA expression in the colon.

Figure 5: Effect of treatment with strain NCIMB 43456 or vehicle on Tphl mRNA expression in the ileum and (B) Tphl mRNA expression in the colon.

Figure 6: Effect of treatment with strain NCIMB 43456 or vehicle on short chain fatty acid (SCFA) production.

Figure 7: Effect of treatment with strain NCIMB 43456 or vehicle on gene expression in the hippocampus.

Figure 8: Effect of treatment with strain NCIMB 43456 or vehicle on gene expression in the amygdala.

Figure 9: Effect of treatment with strain NCIMB 43456 or vehicle on gene expression in the pre frontal cortex.

Figure 10: Effect of treatment with strain NCIMB 43456 or vehicle on stereotype-related behaviour in the marble burying test for wild type mice and mouse models of autism.

Figure 11: Effect of treatment with strain NCIMB 43456 or vehicle on stereotype-related behaviour in the grooming test for wild type mice and mouse models of autism.

Figure 12: Effect of treatment with strain NCIMB 43456 or vehicle on anxiety-like behaviour in the elevated plus maze test for wild type mice and mouse models of autism.

Figure 13: Effect of treatment with strain NCIMB 43456 or vehicle on anxiety-like behaviour in the open field arena test for wild type mice and mouse models of autism.

Figure 14: Effect of treatment with strain NCIMB 43456 or vehicle on social behaviour in the three-chamber test for wild type mice and mouse models of autism. Figure 15: Effect of treatment with strain NCIMB 43456 or vehicle on depression-like behaviour in the novel object recognition test for wild type mice and mouse models of autism.

Figure 16: Effect of treatment with strain NCIMB 43456 or vehicle on depression-like behaviour in the forced swim test for wild type mice and mouse models of autism.

Figure 17: Effect of treatment with strain NCIMB 43456 or vehicle on intestinal motility for wild type mice and mouse models of autism.

Figure 18 Effect of treatment with strain NCIMB 43456 or vehicle on gut permeability for wild type mice and mouse models of autism.

Figure 19: Effect of treatment with strain NCIMB 43456 or vehicle on animal body weight for wild type mice and mouse models of autism.

Figure 20: Effect of treatment with strain NCIMB 43456 or vehicle on organ weight and colon length for wild type mice and mouse models of autism.

Figure 21: Effect of treatment with strain NCIMB 43456 or vehicle on brainstem monoamine levels for wild type mice and mouse models of autism.

Figure 22: Effect of treatment with strain NCIMB 43456 or vehicle on amygdalar gene expression for wild type mice and mouse models of autism.

DISCLOSURE OF THE INVENTION Bacterial strains

The invention provides a composition comprising a bacterial strain of the species Enterococcus durans for use in therapy, for example, for use in treating or preventing a central nervous system disorder or condition, in particular a central nervous system disorder or condition mediated by the microbiota-gut-brain axis. The mouse model experiments used in this application for the assessment of the symptoms of autism spectrum disorders are known in the art to be applicable for the assessment of the symptoms of other central nervous system disorders including those listed above.

In certain embodiments, the compositions of the invention comprise Enterococcus durans and do not contain any other bacterial species. In certain embodiments, the compositions of the invention comprise a single strain of Enterococcus durans and do not contain any other bacterial strains or species.

Enterococcus durans is a Gram-positive, coccus bacterium in the genus Enterococcus. Enterococcus durans is a facultative anaerobe. The type strain of Enterococcus durans is 98D = ATCC 19432 = DSM 20633 = CCM 5612 = NCDO 596 = NCTC 8307 = NCIMB 700596 [22] The GenBank accession number for the 16S rRNA gene sequence of Enterococcus durans strain 98D is NR_036922 (disclosed herein as SEQ ID NO:l). This exemplary Enterococcus durans strain is described in [23]

Other Enterococcus durans strains for use in the invention include: NCIMB 662 [24], NCIMB 8587 [24], NCIMB 8782 [24], NCIMB 11077 [25], NCIMB 701624 [26], NCIMB 701630 [26], NCIMB 701632 [26], NCIMB 701724 (NCD01724), NCIMB 701951 [27] In certain embodiments, the compositions of the invention comprise one of these strains, or a derivative or biotype thereof. The Enterococcus durans bacterium tested in the examples was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksbum, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 9 August 2019 as “ Enterococcus durans ” and was assigned accession number NCIMB 43456. A 16S rRNA sequence for strain NCIMB 43456 is provided in SEQ ID NO:2.

Prior to being deposited with NCIMB, Ltd, strain NCIMB 43456 was taxonomically identified as belonging to the Enterococcus durans species using techniques known to those skilled in the art and reported in the literature as being capable of taxonomically distinguishing the Enterococcus durans species from other species in the Enterococcus genus, including E. faecium mdE. faecalis, including 16S rRNA and MALDI-TOF analysis.

Further, while the use of strains of Enterococcus faecium as a probiotic has been known, particularly in animal feeds, to the inventors’ knowledge, the use of Enterococcus durans as a probiotic or in the treatment of disease has not been comprehensively explored.

Bacterial strains closely related to the strain tested in the examples, including biotypes of the bacterium deposited under accession number NCIMB 43456 are also expected to be effective for treating or preventing autism spectrum disorders and central nervous system disorders and conditions, in particular central nervous system disorders and conditions mediated by the microbiota-gut-brain axis.

In certain embodiments, the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Enterococcus durans. Preferably, the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO:l or 2. Preferably, the sequence identity is to SEQ ID NO:2. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:2.

A biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics. Strains that are biotypes of the bacterium deposited under accession number NCIMB 43456 and that are suitable for use in the invention may be identified by sequencing other nucleotide sequences for the bacterium deposited under accession number NCIMB 43456. For example, substantially the whole genome may be sequenced and a biotype strain for use in the invention may have at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity across at least 80% of its whole genome (e.g. across at least 85%, 90%, 95% or 99%, or across its whole genome). For example, in some embodiments, a biotype strain has at least 98% sequence identity across at least 98% of its genome or at least 99% sequence identity across 99% of its genome. Other suitable sequences for use in identifying biotype strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTGfy or REP [28] Biotype strains may have sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number NCIMB 43456. In some embodiments, a biotype strain has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the 16S rRNA sequence of SEQ ID NO: 2.

Alternatively, strains that are biotypes of the bacterium deposited under accession number NCIMB 43456 and that are suitable for use in the invention may be identified by using the accession number NCIMB 43456 deposit and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23s rDNA sequencing. In preferred embodiments, such techniques may be used to identify other Enterococcus durans strains.

In certain embodiments, strains that are biotypes of the bacterium deposited under accession number NCIMB 43456 and that are suitable for use in the invention are strains that provide the same pattern as the bacterium deposited under accession number NCIMB 43456 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example, [29]). Alternatively, biotype strains are identified as strains that have the same carbohydrate fermentation patterns as the bacterium deposited under accession number NCIMB 43456.

In some embodiments, strains that are biotypes of the bacterium deposited under accession number NCIMB 43456 and that are suitable for use in the invention are Enterococcus strains that do not produce acid from glycerol. In some embodiments, strains that are biotypes of the bacterium deposited under accession number NCIMB 43456 and that are suitable for use in the invention are Enterococcus strains that do not produce acid from mannitol. In some embodiments, strains that are biotypes of the bacterium deposited under accession number NCIMB 43456 and that are suitable for use in the invention are Enterococcus strains that do not produce acid from sucrose. In some embodiments, whether acid is produced from glycerol, mannitol and/or sucrose is determined with the API 50CH system, using bromocresol purple (0.002%, wt/vol) as the indicator in 1% (wt/vol) peptone water (see reference 23). The ability of enterococci to grow under particular conditions is widely used for their selective isolation ([30]). In some embodiments, strains that are biotypes of the bacterium deposited under accession number NCIMB 43456 and that are suitable for use in the invention are Enterococcus strains that are incapable of metabolising one or more of (e.g. more than 1, 2, 3, 4, 5, 6 of or all 7 of) glycerol, D-mannitol, D-sorbitol, L-arabinose, gluconate, melibiose and/or sucrose, preferably one or more of (e.g. more than 1, 2, 3 of or all 4 of) glycerol, D-mannitol, D-sorbitol and/or sucrose; such strains are in accordance with the phenotype of Enterococcus durans provided in references [30, 31 and 77]).

In some embodiments, strains that are biotypes of the bacterium deposited under accession number NCIMB 43456 and that are suitable for use in the invention are Enterococcus strains that are incapable of metabolising D-mannitol (for example in accordance with the phenotype of Enterococcus durans provided in references [30 and 31]).

In some embodiments, strains that are biotypes of the bacterium deposited under accession number NCIMB 43456 and that are suitable for use in the invention are Enterococcus strains that grow in tellurite (as is the case for Enterococcus durans, see references [30 and 31]).

In some embodiments, the biotype is not an E. casseliflavus strain. In some embodiments, the biotype is not an E. malodoratus strain. In some embodiments, the biotype is not an E. faecium strain.

In some embodiments, the biotype is an E. hirae strain.

Other Enterococcus durans strains that are useful in the compositions and methods of the invention, such as biotypes of the bacterium deposited under accession number NCIMB 43456, may be identified using any appropriate method or strategy, including the assays described in the examples. For instance, strains for use in the invention may be identified by culturing in anaerobic YCFA and/or administering the bacteria to an autism spectrum disorder mouse model and then assessing cytokine levels. In particular, bacterial strains that have similar growth patterns, metabolic type and/or surface antigens to the bacterium deposited under accession number NCIMB 43456 may be useful in the invention. In particular, a biotype strain will elicit comparable effects on the autism spectrum disorder models to the effects shown in the examples, which may be identified by using the culturing and administration protocols described in the examples.

A particularly preferred strain of the invention is the Enterococcus durans strain deposited under accession number NCIMB 43456. This is the exemplary Enterococcus durans strain tested in the examples and shown to be effective for treating disease. Therefore, the invention provides a cell, such as an isolated cell, of the Enterococcus durans strain deposited under accession number NCIMB 43456, or a derivative thereof. The invention also provides a composition comprising a cell of the Enterococcus durans strain deposited under accession number NCIMB 43456, or a derivative thereof. The invention also provides a biologically pure culture of the Enterococcus durans strain deposited under accession number NCIMB 43456. The invention also provides a cell of the Enterococcus durans strain deposited under accession number NCIMB 43456, or a derivative thereof for use in therapy, in particular for the diseases described herein.

A derivative of the strain deposited under accession number NCIMB 43456 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original. A derivative of a strain of the invention may be modified, for example at the genetic level, without ablating the biological activity. In particular, a derivative strain of the invention is therapeutically active. A derivative strain will have comparable immune modulatory activity to the original NCIMB 43456 strain. In particular, a derivative strain will elicit comparable effects on the autism spectrum disorder models to the effects shown in the examples, which may be identified by using the culturing and administration protocols described in the examples. For example, a derivative strain will elicit comparable effects on the BTBR mouse model and/or the maternal immune activation (MIA) mouse model, which may be identified by the marble burying test, the elevated plus maze test, the three-chamber social interaction test, the novel object recognition test, the forced swim test, brainstem monoamine levels and/or amygdalar gene expression as set out in example 2. A derivative of the NCIMB 43456 strain will generally be a biotype of the NCIMB 43456 strain.

The bacterial strain may also be a strain that has the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 43456, and such cells are encompassed by the invention.

In some embodiments, the bacterial strain in the compositions of the invention is an E. hirae strain.

In preferred embodiments, the bacterial strains in the compositions of the invention are viable and capable of partially or totally colonising the intestine.

In certain embodiments, the bacterial strain for use in the invention is resistant to one of more of amoxicillin, ampicillin, arbekacin and dibekacin, azlocillin, bacampicillin, carbenicillin, ceftobiprole, clarithromycin, doripenem, erythromycin, fusidic acid, gentamicin, grepafloxacin, imipenem, josamycin, meropenem, meziocillin, piperacillin, rifampin, rifaximin, rokitamycin, rosaramicin, roxithromycin, spiramycin, streptomycin, sulfamethoxazole/trimethoprim, telithromycin, ticarcillin, ticarcillin/clavulanate, tosufloxacin, trimethoprim and virginiamycin. In certain embodiments, the bacterial strain for use in the invention is susceptible to Quinopristin- dalfopristin.

In certain embodiments, the bacterial strain for use in the invention is resistant to b-lactam antibiotics. In certain embodiments, the bacterial strain for use in the invention is resistant to vancomycin. In certain embodiments, the bacterial strain for use in the invention is resistant to ampicillin. Therapeutic uses

Modulation of the microbiota-gut-brain axis

Communication between the gut and the brain (the microbiota-gut-brain axis) occurs via a bidirectional neurohumoral communication system. Recent evidence shows that the microbiota that resides in the gut can modulate brain development and produce behavioural phenotypes via the microbiota-gut-brain axis. Indeed, a number of reviews suggest a role of the microbiota-gut- brain axis in maintaining central nervous system functionality and implicate dysfunction of the microbiota-gut-brain axis in the development of central nervous system disorders and conditions [8], [9], [32]

The bidirectional communication between the brain and the gut (i.e. the-gut-brain axis) includes the central nervous system, neuroendocrine and neuroimmune systems, including the hypothalamus-pituitary-adrenal (HP A) axis, sympathetic and parasympathetic arms of the autonomic nervous system (ANS), including the enteric nervous system (ENS) and the vagus nerve, and the gut microbiota.

As demonstrated in the examples, the compositions of the present invention can modulate the microbiota-gut-brain axis and reduce the behavioural symptoms associated with a CNS disorder. Accordingly, the compositions of the invention may be useful for treating or preventing disorders of the central nervous system (CNS), in particular those disorders and conditions associated with dysfunction of the microbiota-gut-brain axis. The compositions of the invention may be useful for reducing behavioural symptoms associated with a CNS disorder.

The compositions of the invention may also be useful for treating or preventing neurodevelopmental disorders and/or neuropsychiatric conditions. Neurodevelopmental diseases and neuropsychiatric conditions are often associated with the microbiota-gut-brain axis. The compositions of the invention may be useful for treating or preventing neurodevelopmental diseases and/or neuropsychiatric conditions mediated by dysfunction of the microbiota-gut-brain axis. In further preferred embodiments, the compositions of the invention are for use in treating or preventing a neurodevelopmental disorder or a neuropsychiatric condition.

In particular embodiments, the compositions of the invention may be useful for treating or preventing a disease or condition selected from the group consisting of: autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder; depression; seasonal affective disorder; anxiety disorders; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post-traumatic stress disorder; dementia; Alzheimer’s; Parkinson’s disease; epilepsy; chronic pain (for example, central sensitisation or fibromyalgia); motor neuron disease; Huntington’s disease; Guillain-Barre syndrome and/or meningitis. The compositions of the invention may be particularly useful for treating or preventing chronic disease, treating or preventing disease in patients that have not responded to other therapies (such as treatment with anti-psychotics and/or anti-depressants), and/or treating or preventing the tissue damage and symptoms associated with dysfunction of the microbiota-gut-brain axis.

In certain embodiments, the compositions of the invention modulate the CNS. In some embodiments, the compositions of the invention modulate the autonomic nervous system (ANS). In some embodiments, the compositions of the invention modulate the enteric nervous system (ENS). In some embodiments, the compositions of the invention modulate the hypothalamic, pituitary, adrenal (HP A) axis. In some embodiments, the compositions of the invention modulate the neuroendocrine pathway. In some embodiments, the compositions of the invention modulate the neuroimmune pathway. In some embodiments, the compositions of the invention modulate the CNS, the ANS, the ENS, the HPA axis and/or the neuroendocrine and neuroimmune pathways. In certain embodiments, the compositions of the invention module the levels of commensal metabolites and/or the gastrointestinal permeability of a subject.

The signalling of the microbiota-gut-brain axis is modulated by neural systems. Accordingly, in some embodiments, the compositions of the invention modulate signalling in neural systems. In certain embodiments, the compositions of the invention modulate the signalling of the central nervous system. In some embodiments, the compositions of the invention modulate signalling in sensory neurons. In other embodiments, the compositions of the invention modulate signalling in motor neurons. In some embodiments, the compositions of the invention modulate the signalling in the ANS. In some embodiments, the ANS is the parasympathetic nervous system. In preferred embodiments, the compositions of the invention modulate the signalling of the vagus nerve. In other embodiments, the ANS is the sympathetic nervous system. In other embodiments, the compositions of the invention modulate the signalling in the enteric nervous system. In certain embodiments, the signalling of ANS and ENS neurons responds directly to luminal contents of the gastrointestinal tract. In other embodiments, the signalling of ANS and ENS neurons responds indirectly to neurochemicals produced by luminal bacteria. In other embodiments, the signalling of ANS and ENS neurons responds to neurochemicals produced by luminal bacteria or enteroendocrine cells. In certain preferred embodiments, the neurons of the ENS activate vagal afferents that influence the functions of the CNS. In some embodiments, the compositions of the invention regulate the activity of enterochromaffm cells.

In certain embodiments, the compositions of the invention modulate fear conditioning in an animal model. In certain embodiments, the compositions of the invention can be used to modulate the development of fear and/or anxiety, and/or modulate the extent to which the fear and/or anxiety becomes extinct in a subject. In certain embodiments, the compositions of the invention can be used to modulate the extent of stress-induced hyperthermia in an animal model. In certain embodiments, the compositions of the invention modulate the level of stress and/or anxiety in a subject.

Autism spectrum disorder (ASD)

Autism spectrum disorder is a set of heterogeneous neurodevelopmental conditions, characterised by early-onset difficulties in social interaction, communication and unusually restricted, repetitive behaviour and interests. Symptoms can be recognised from a very early age but ASD is often diagnosed in more able children starting mainstream education. Autism represents the primary type of ASD.

Historically, autism has been diagnosed on the basis of three core domains: impaired social interaction, abnormal communication, and restricted and repetitive behaviours and interests. In the International Classification of Diseases (ICD-10R, WHO 1993) and the Diagnostic and Statistical Manual (DSM-IV, American Psychiatric Association, 2000), autism comes under the umbrella term of Pervasive Developmental Disorder (PDD), with four possible diagnostic subtypes: Asperger Syndrome, Childhood Autism/Autistic Disorder, Atyptical Autism, and PDD-not otherwise specified. In DMS-5, these diagnostic subtypes are combined into a single category of autism spectrum disorder (ASD) and the previous use of three core domains of impairment has been reduced to two main areas, namely social communication and interaction, and repetitive behaviour, which include sensory integration dysfunctions.

ASD is a ‘spectrum disorder’ as it affects each person in a variety of different ways and can range from very mild to severe. The functioning of the affected individual varies substantially depending on language abilities, level of intelligence, co-morbidity, composition of symptoms and access to services. Cognitive functioning, learning, attention and sensory processing are usually impaired.

DSM-IV states that the diagnosis of autism requires the presence of at least six symptoms, including a minimum of two measures of qualitative impairment in social interaction, one symptom of qualitative impairment in communication, and one symptom of restricted and repetitive behaviour. DMS-5 redefines diagnosis of ASD into two symptom domains: (i) social interaction and social communication deficits; and (ii) restricted, repetitive patterns of behaviour, interests or activities.

Co-morbid medical conditions are highly prevalent in ASD. Co-morbid medical conditions include anxiety and depression, seizures, attention deficits, aggressive behaviours, sleep problems, gastrointestinal disorders, epilepsy, mental retardation, intellectual disabilities and/or feeding difficulties.

The examples demonstrate that the compositions of the invention achieve a reduction in disease incidence and disease severity in an animal model of autism spectrum disorder and that they are therefore useful in the treatment or prevention of ASD. ASD is a central nervous system disorder that is partially triggered by environmental factors. Therefore, dysfunction of the microbiota-gut-brain axis may be responsible for development and persistence of ASDs. Accordingly, in preferred embodiments, the compositions of the invention are for use in treating or preventing ASDs. In some embodiments, the compositions of the invention are for use in treating or preventing autism. In some embodiments, the autism is Pervasive Developmental Disorder (PDD). In another embodiment, the PDD is Asperger Syndrome, Childhood Autism/Autistic Disorder, Atyptical Autism and/or PDD-not otherwise specified. Accordingly, in some embodiments, the compositions of the invention are for use in treating or preventing autism spectrum disorders, autism, pervasive developmental disorder; Asperger Syndrome; Childhood Autism/Autistic Disorder, Atypical Autism and/or PDD-not otherwise specified.

The compositions of the invention may be useful for modulating the microbiota-gut-brain axis of a subject. Accordingly, in preferred embodiments the compositions of the invention are for use in preventing ASD in a patient that has been identified as at risk of ASD, or that has been diagnosed with ASD at a prenatal or an early developmental stage; in childhood and/or in adulthood. The compositions of the invention may be useful for preventing the development of ASD.

The compositions of the invention may be useful for managing or alleviating ASDs. Treatment or prevention of ASD may refer to, for example, an alleviation of the severity of symptoms or a reduction in the frequency of exacerbations or the range of triggers that are a problem for the patient.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate at least one core symptom of ASD.

In some embodiments, the compositions of the invention prevent, reduce or alleviate at least one of the two symptom domains of ASD classified in the DMS-5. In some embodiments, the compositions of the invention prevent, reduce or alleviate social interaction and/or social communication deficits. In some embodiments, the compositions of the invention prevent, reduce or alleviate restrictive, repetitive patterns of behaviour, interests or activities. In some embodiments, the compositions of the invention prevent, reduce or alleviate social interaction, social communication deficits and/or restrictive, repetitive patterns of behaviour, interests or activities.

In some embodiments, the compositions of the invention prevent, reduce or alleviate repetitive behaviour, stereotyped behaviour, compulsive behaviour, routine behaviour, sameness behaviour and restricted behaviour. In some embodiments, the compositions of the invention improve social awareness, learning, memory function, social information processing, capacity for social communication, social anxiety/avoidance, and autistic preoccupations and traits in a subject with ASD. In preferred embodiments, the compositions of the invention reduce feelings of despair or helplessness in a subject with ASD. In preferred embodiments, the invention provides a composition for treating or preventing neophobia and / or sociability deficit in a subject with ASD.

In some embodiments, the compositions of the invention prevent, reduce or alleviate additional symptoms associated with the core symptoms of ASD. In some embodiments, the compositions of the invention prevent, reduce or alleviate irritability (including aggression, deliberate self- injury and temper tantrums), agitation, crying, lethargy, social withdrawal, stereotypic behaviour, hyperactivity, non-compliance, inappropriate speech, anxiety, depression, and/or over or under controlled behaviour in a subject with ASDs. In some embodiments, the compositions of the invention improve cognitive functioning, learning, memory function, attention and/or sensory processing in a subject with ASD.

In other embodiments, the compositions of the invention improve secondary outcome measures in a subject with ASDs. In some embodiments, the secondary outcome measures include additional symptom and/or functional rating scales, behavioural scales and miscellaneous measures of interest.

In some embodiments, the compositions of the invention cause in a positive change in the diagnostic and/or symptomatic scale for the assessment of core symptoms of a subject with ASDs. In some embodiments, the diagnostic and/or symptomatic scale is the Autism Diagnostic Interview - Revised (ASI-R). In some embodiments, the diagnostic or symptomatic scale is the Autism Diagnostic Observation Schedule-Generic (ADOS-G) now ADOS-2. In other embodiments, the diagnostic or symptomatic scale is the Autism Diagnostic Interview Revised (ADI-R). In other embodiments, the diagnostic or symptomatic scale is the Diagnostic Interview for Social and Communication Disorders (DISCO). In yet other embodiments, the diagnostic or symptomatic scale is the Childhood Autism Rating Scale (CARS and CARS2).

In some embodiments, the compositions of the invention cause a positive change in generic measures of the efficacy endpoints of ASD. In certain embodiments, the generic measures include, but are not limited to the Aberrant Behaviour Checklist (ABC), the Child Behaviour Checklist (CBCL), the Vineland-II Adaptive Behaviour Scales (VABS), the Social Responsiveness Scale (SRS), and/or the Repetitive Behaviour Scale - Revised (RBS-R).

In some embodiments, the compositions of the invention improve the Clinical Global Impression - Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the compositions of the invention display a positive effect on global functioning of the subject with ASD. Additional scales would be known to a person skilled in the art. In some embodiments, the compositions of the invention would improve the outcome of diagnostic and/or symptomatic scales known to a person skilled in the art.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate the incidence of comorbidities of ASDs. In some embodiments, the compositions of the invention prevent, reduce or alleviate the incidence of anxiety and depression, seizures, attention deficits, aggressive behaviours, sleep problems, gastrointestinal disorders (including irritable bowel syndrome (IBS)), epilepsy, mental retardation, intellectual disabilities and/or feeding difficulties. In certain embodiments, the compositions of the invention prevent, reduce or alleviate gastrointestinal comorbidities, such as abdominal pain, diarrhoea and/or flatulence.

In some embodiments, the compositions of the invention prevent, reduce or alleviate the symptoms of certain psychiatric and behavioural disorders that may present clinically with similarities to autism. Accordingly, in some embodiments, the compositions of the invention, prevent, reduce or alleviate attention deficit disorder (ADHD); affective/anxiety disorders; attachment disorders; oppositional defiant disorder (ODD); obsessive compulsive disorder (OCD) and/or psychoses including schizophrenia (cognitive impairment).

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating ASD when used in combination with another therapy for treating ASD. Such therapies include anti-psychotic, anti-anxiety and anti-depressant drugs. Such drugs include risperidone (Risperdal®); olanzapine (Zyprexa®); fluoxetine (Prozac®); sertraline (Zoloft®); fluvoxamine (Luvox®); clomipramine (Anafranil®); haloperidol (Haldol®); thioridazine; fluphenazine; chlorpromazine; ziprasidone (Geogon®); carbamazepine (Tegretol®); lamotrigine (Lamictal®); topiramate (Topomax®); valproic acid (Depakote®); methylphenidate (Ritalin®); diazepam (Valium®) and lorazepam (Ativan®).

Obsessive compulsive disorder (OCD)

OCD is a heterogeneous, chronic and disabling disorder belonging to the anxiety disorders. According to the DSM-IV definition, the essential features of OCD are recurrent obsessions and/or compulsions (criterion A) that are severe and time consuming (more than one hour a day) or cause marked distress or significantly interfere with the subject’s normal routine, occupational functioning, usual social activities or relationships (criterion C). As some point during the course of the disorder, the person has recognised that the obsessions or compulsions are excessive or unreasonable (criterion B).

Obsessions are defined as recurrent and persistent thoughts, impulses or images that are experienced as intrusive and inappropriate and cause marked anxiety or distress. The thoughts, impulses or images are not simply excessive worries about real-life problems, they are recognised by the patient as a product of his own mind (e.g. fear for contamination, symmetry obsession). The person attempts to ignore, suppress or neutralise the obsessions with some other thoughts or actions.

Compulsions are defined as repetitive behaviours (e.g. hand washing, ordering, hoarding, checking) or mental acts (e.g. praying, counting, repeating words silently) that the person feels driven to perform in response to an obsession or according to rules that must be applied rigidly.

OCD is often associated with co-morbidity rates of other psychiatric diseases including major depressive disorder, other anxiety disorders (generalised anxiety disorder, social anxiety disorder, panic disorder), substance abuse and eating disorders (anorexia and bulimia).

OCD is a psychiatric disorder that may develop or persist due to dysfunction of the microbiota- gut-brain axis. Accordingly, in preferred embodiments, the compositions of the invention are for use in treating or preventing OCD in a subject.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate the essential symptomatic features of OCD. In certain embodiments, the compositions of the invention prevent, reduce or alleviate recurrent obsessions and/or compulsions in a subject. In certain embodiments, the obsessions are recurrent or persistent thoughts, impulses or images that are experienced as intrusive and inappropriate and cause marked anxiety or distress. In certain embodiments, the compulsions are repetitive behaviours that the subject feels driven to perform in response to an obsession or according to rules that must be applied rigidly.

In certain embodiments, the compositions of the invention improve symptoms of OCD in a subject accordingly to the Y-BOCS and/or the NIMH-OC diagnostic and/or symptomatic scales. In some embodiments, the Y-BOCS scale is used to monitor improvement of primary endpoints. In some embodiments, the NIMH-OC scale is used to monitor improvement of secondary parameters.

In some embodiments, the compositions of the invention improve the Clinical Global Impression - Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the compositions of the invention display a positive effect on global social functioning (relationships, work, etc.) of the subject with ASDs. In some embodiments, the global scale is the Sheehan disability scale.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate at least one comorbidity of OCD. The comorbidities of OCD include major depressive disorder, other anxiety disorders (generalised anxiety disorder, social anxiety disorder, panic disorder), substance abuse and eating disorders (anorexia and bulimia) Gilles de la Tourette syndrome, ADHD (Attention-Deficit/Hyperactivity Disorder) and developmental disorders.

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating OCD when used in combination with another therapy for treating OCD. Such therapies include serotonin and dopamine reuptake inhibitors; clomipramine and anti- psychotics.

Major depressive disorder (MDD)

MDD is associated with substantial psychosocial dysfunction and high individual mental strain as well as with excess morbidity and mortality (the risk of suicide is considerable). The term major depressive disorder encompasses clinical depression, major depression, unipolar depression, unipolar disorder, recurrent depression and simply depression. The term major depressive disorder covers mood disorders; dysthymia; chronic depression; seasonal affective disorder and borderline personality disorder.

According to the DMS-5 criteria, MDD symptoms include a depressed mood, or loss of interest or pleasure in daily activities for more than two weeks; and impaired social, occupational and educational function. Specific symptoms, at least five of the following nine, present nearly every day: depressed mood or irritable most of the day; decreased interest or pleasure in most activities, most of each day; significant weight change or change in appetite; change in sleep (insomnia or hypersomnia); change in activity (psychomotor agitation or retardation); fatigue or loss of energy; guilt or worthlessness (feelings of worthlessness or excessive or inappropriate guilt); reduced concentration (diminished ability to think or concentrate, or more indecisiveness; and suicidality (thoughts of death or suicide, or subject has a suicide plan). In addition, MDD is associated with anxiety symptoms including irrational worry; preoccupation with unpleasant worries; trouble relaxing and/or feeling tense. MDD episodes can be mild, moderate or severe.

MDD episodes are often associated with comorbidity with other psychiatric disorders or with somatic disorders like Parkinson’s disease, Alzheimer’s disease, cerebrovascular disorders, cancer and chronic pain syndromes. MDD is frequently associated with a wide spectrum of other mental disorders as comorbidities including generalised anxiety disorder; anxiety disorder; substance use disorders; post-traumatic stress disorder (PTSD); personality disorders; pain; stress; irritable bowel syndrome; insomnia; headaches and interpersonal problems.

Major depressive disorder is a psychiatric disorder that may develop or persist due to dysfunction of the microbiota-gut-brain axis. Accordingly, in preferred embodiments, the compositions of the invention are for use in treating or preventing MDD in a subject.

In certain embodiments, the compositions of the invention are for use in treating or preventing acute major depressive episodes and/or the prevention of new episodes (recurrence prevention). In certain embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of mild, moderate or severe MDD episodes.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the symptoms of MDD as classified by the DMS-5 criteria listed herein. In a preferred embodiment, the compositions of the invention prevent, reduce or alleviate a depressed mood in a subject. In a preferred embodiment, the compositions of the invention prevent, reduce or alleviate a decreased interest or pleasure in most activities in a subject. In some embodiments, the compositions of the invention reduce the occurrence of symptoms of MDD within a 2-week period.

In some embodiments, the compositions of the invention improve the symptoms of MDD according to a symptomatic or diagnostic scale. Such scales for assessing symptomatic improvement include the Hamilton Rating Scale of Depression (HAMD) and the Montgomery Asberg Depression Rating Scale. In addition, the Zung Self-Rating Depression Scale (SDS) and Zung Self-Rating Anxiety Scale (SAS) are also suitable symptomatic improvement scales.

In some embodiments, the compositions of the invention improve the Clinical Global Impression - Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the compositions of the invention display a positive effect on global social and occupational functioning of the subject with MDD.

In certain embodiments, the compositions of the invention are for use in treating or preventing treatment resistant MDD.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate at least one comorbidity of MDD. The comorbidities of MDD include generalised anxiety disorder; anxiety disorder; substance use disorders; post-traumatic stress disorder (PTSD); personality disorders; pain; stress; IBS; insomnia, and headaches.

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating MDD when used in combination with another therapy for treating MDD. Such therapies include antidepressants, augmentation strategies (e.g. combination therapy, lithium and other mood stabilizers, thyroid hormones and atypical antipsychotics) or even second generation antipsychotics.

Anxiety disorders

Anxiety disorders are psychiatric disorders that may develop or persist due to dysfunction of the microbiota-gut-brain axis. Accordingly, in preferred embodiments, the compositions of the invention are for use in treating or preventing anxiety disorders in a subject. In certain embodiments, the anxiety disorder is generalised anxiety disorder (GAD); specific phobia; social anxiety disorder; separation anxiety disorder; agoraphobia; panic disorder and selective mutism.

GAD is diagnosed according to DMS-5 in six criteria. The first criterion is too much anxiety or worry over more than six months wherein the anxiety or worry is present most of the time in regards to many activities. The second criterion is that the subject is unable to manage the symptoms of the first criterion. The third criterion is that at least three (one in children) of the following occurs: restlessness; tires easily; problems concentrating; irritability; muscle tension and problems with sleep. The final three criterion are that the symptoms results in significant social, occupational and functional impairment; the symptoms are not due to medications, drugs, or other physical health problems; and the symptoms do not fit better with another psychiatric problem such as panic disorder. All other anxiety disorders may be considered as differential diagnoses of GAD.

GAD is frequently associated with a wide spectrum of other mental disorders as comorbidities including depression; substance use disorders; stress; IBS; insomnia; headaches; pain; cardiac events; interpersonal problems and ADHD.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the symptoms of GAD in a subject as classified by the DMS-5 criteria listed herein. According to DMS-5, the same symptoms are associated with other anxiety disorders. Therefore, in certain embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the symptoms of anxiety disorders in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate the anxiety or worry of the subject. In certain embodiments, the compositions of the invention reduce the occurrence of symptoms within a six month period. In certain embodiments, the composition of the invention prevents, reduces or alleviates restlessness; fatigue; loss of concentration; irritability; muscle tension; and/or problems with sleep. In some embodiments, the compositions of the invention prevent, reduce or alleviate social, occupational and functional impairment associated with anxiety disorders.

In some embodiments, the compositions of the invention improve the symptoms of anxiety disorders according to a symptomatic or diagnostic scale. In certain embodiments, the scale for assessing symptomatic improvement includes the Hamilton Anxiety Rating Scale (HAM-A). In some embodiments, the HAM-A total scale is used to assess primary endpoint. In other embodiments, the HAM-A psychic anxiety factor may be useful as a secondary endpoint.

In some embodiments, the compositions of the invention improve the Clinical Global Impression - Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the compositions of the invention display a positive effect on global social, occupational and functional impairment of the subject with anxiety disorder. In some embodiments, the global scale is the Sheehan disability scale.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate at least one comorbidity of GAD and anxiety disorders. The comorbidities of GAD include depression; substance use disorders; stress; IBS; insomnia; headaches; pain; cardiac events, and ADHD.

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating anxiety disorders when used in combination with another therapy for treating anxiety disorders. Such therapies include selective serotonin reuptake inhibitors (venlafaxine, duloxetine, escitalopram and paroxetine); benzodiazepines (alprazolam, lorazepam and clonazepam); pregabalin (Lyrica®) and gabapentin (Neurontin ®); serotonin receptor partial agonists (buspirone and tandospirone); atypical serotonergic antidepressants (such as imipramine and clomipramine); monoamine oxidase inhibitors (MAOIs) (such as moclobemide and phenelzine); hydroxyzine; propranolol; clonidine; guanfacine and prazosin.

Post-traumatic stress disorder (PTSD)

PTSD is a severe and disabling disorder, an essential feature of which is the inclusion of a traumatic event as a precipitating factor of this disorder.

The symptoms of PTSD are grouped into four main clusters according to the DMS-V criteria: (i) intrusion: examples include nightmares, unwanted thoughts of the traumatic events, flashbacks, and reacting to traumatic reminders with emotional distress or physiological reactivity; (ii) avoidance: examples include avoiding triggers for traumatic memories including places, conversations, or other reminders; (iii) negative alterations in cognitions and mood: examples include distorted blame of self or others for the traumatic event, negative beliefs about oneself or the world, persistent negative emotions (e.g., fear, guilt, shame), feeling alienated, and constricted affect (e.g., inability to experience positive emotions); (iv) alterations in arousal and reactivity: examples include angry, reckless, or self-destructive behaviour, sleep problems, concentration problems, increased startle response, and hypervigilance.

Symptoms that resolve within 4 weeks of the traumatic event meet the criteria for an Acute Stress Disorder. The DSM distinguishes between acute (duration of symptoms for less than three months) and chronic PTSD (duration of symptoms longer than 3 months). If the symptoms begin more than 6 months after the stressor, the disorder is defined as delayed onset PTSD.

PTSD carries high comorbidities with major depressive disorder and substance use disorders.

PTSD is a psychiatric disorder that may develop or persist due to dysfunction of the microbiota-gut-brain axis. Accordingly, in preferred embodiments, the compositions of the invention are for use in treating or preventing PTSD in a subject. According to a similar pathogenesis, in certain embodiments, the compositions of the invention are for use in treating or preventing stress disorders. In certain embodiments, the compositions of the invention treat acute stress disorder. In some embodiments, the compositions of the invention treat acute and/or chronic PTSD. In some embodiments, the compositions of the invention treat delayed onset PTSD.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the symptoms of PTSD (or stress disorder) in a subject as classified by the DMS-5 criteria listed herein. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate intrusive thoughts in a subject with PTSD. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate avoidance behaviour in a subject with PTSD. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate negative alterations in cognitions and mood in a subject with PTSD. In preferred embodiments, the compositions of the invention prevent alterations in arousal and reactivity in a subject with PTSD.

In some embodiments, the compositions of the invention improve the symptoms of PTSD and stress disorders according to a symptomatic or diagnostic scale. In certain embodiments, the scale for assessing symptomatic improvement is the Clinical-Administered PTSD (CAPS) scale.

In some embodiments, the compositions of the invention improve the Clinical Global Impression - Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the compositions of the invention display a positive effect on global social, occupational and functional impairment of the subject with PTSD and stress disorders. In some embodiments, the global scale is the Sheehan disability scale.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate at least one comorbidity of PTSD and stress disorders. The comorbidities of PTSD and stress disorders include MDD, substance use disorders; stress and anxiety.

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating PTSD and stress disorders when used in combination with another therapy for treating PTSD and stress disorders. Such therapies include serotoninergic agents, tricyclic antidepressants, mood stabilisers, adrenergic inhibiting agents, antipsychotics, benzodiazepines, sertraline (Zoloft®), fluoxetine (Prozac®) and/or paroxetine (Paxil®).

Schizophrenia spectrum and psychotic disorders

These diseases affect a subject’s ability to think clearly, make good judgements, respond emotionally, communicate effectively, understand reality, and behave appropriately. Psychotic diseases include schizophrenia (symptoms listed below); schizoaffective disorder (the subject has symptoms of both schizophrenia and a mood disorder, such as depression or bipolar disorder); schizophreniform disorder (displays the symptoms of schizophrenia, but the symptoms last for a shorter time: between 1 and 6 months); brief psychotic disorder (subjects display a sudden, short period of psychotic behaviour, often in response to a very stressful event, such as a death in the family - recovery is usually less than a month); delusional disorder (delusions last for at least 1 month); shared psychotic disorder; substance-induced psychotic disorder; psychotic disorder due to another medical condition; paraphrenia (displaying symptoms similar to schizophrenia and starting late in life, when people are elderly). The most well-known psychotic disorder is schizophrenia and the majority of psychotic disorders display similar symptoms to schizophrenia.

Schizophrenia is a severe psychiatric disease with a heterogeneous course and symptom profile. Schizophrenia presents clinically with so-called positive and negative symptoms. The positive symptoms include delusions, hallucinations, disorganised speech, and disorganised or catatonic behaviours. Negative symptoms include affective flattening, restriction in the fluency and productivity of thought and speech and in the initiation of goal directed behaviour. The positive symptoms appear to reflect an excess or distortion of normal functions, whereas negative symptoms appear to reflect a diminution or loss of normal function. In addition, cognitive deficits (defects of working memory, information processing, attention/vigilance, learning, reasoning and social cognition) are common. Cognitive deficits generally show poor improvement with current antipsychotic treatment. Schizophrenic patients also suffer from mood symptoms. Besides these predominant symptoms, schizophrenia is associated with a comorbidity with other psychiatric symptoms such as manic and depressive symptoms, anxiety or obsessive-compulsive symptoms, substance abuse and dependence, and personality disorder.

According to the DMS-5, for the diagnosis of schizophrenia, a subject must have at least two of the following symptoms: delusions; hallucinations; disorganised speech; disorganised or catatonic behaviour and negative symptoms. At least one of the symptoms must be the presence of delusions, hallucinations or disorganised speech. Continuous signs of disturbance must persist for at least 6 months, during which the subject must experience at least 1 month of active symptoms, with social or occupational deterioration problems occurring over a significant amount of time.

Schizophrenia spectrum and psychotic disorders are psychiatric disorders that may develop or persist due to dysfunction of the microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing schizophrenia spectrum and/or psychotic disorders in a subject. In certain embodiments, the schizophrenia spectrum and psychotic disorder is selected from schizophrenia; schizoaffective disorder; schizophreniform disorder; brief psychotic disorder; delusional disorder; shared psychotic disorder; substance-induced psychotic disorder; psychotic disorder due to another medical condition and paraphrenia. In preferred embodiments, the compositions of the invention are for use in treating or preventing schizophrenia. In certain embodiments, the schizophrenia is selected from paranoid, disorganised, catatonic, undifferentiated and residual schizophrenia.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the symptoms of schizophrenia in a subject as classified by the DMS-5 criteria listed herein. These embodiments apply to the prevention, reduction or alleviation of symptoms of other schizophrenia spectrum and psychotic disorders. In certain embodiments, the compositions of the invention prevent, reduce or alleviate negative symptoms of schizophrenia. In certain embodiments, the compositions of the invention prevent, reduce or alleviate positive symptoms of schizophrenia. Positive symptoms are those that most people do not normally experience, but are present in people with schizophrenia, such as delusions, hallucinations, disordered speech. In certain embodiments, the compositions of the invention prevent, reduce or alleviate negative and positive symptoms of schizophrenia. Negative symptoms are deficits of normal emotional responses or of other thought processes, such as affective flattening and catatonic behaviour. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate delusions, hallucinations, disorganised speech, and disorganised or catatonic behaviours in a subject with schizophrenia. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate affective flattening, restriction in the fluency and productivity of thought and speech and in the initiation of goal directed behaviour in a subject with schizophrenia. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate the cognitive defects and/or mood disorders in a subject with schizophrenia.

In certain embodiments, the compositions of the invention reduce the occurrence of positive and/or negative symptoms of schizophrenia in a subject within a 6 month period. In certain embodiments, the compositions of the invention improve social and/or occupational functionality in a subject with schizophrenia spectrum or psychotic disorder.

In some embodiments, the compositions of the invention improve the symptoms of schizophrenia spectrum or psychotic disorders according to a symptomatic or diagnostic scale. In certain embodiments, the scale for assessing symptomatic improvement is the Positive and Negative Symptom Scale (PANSS) and Brief Psychiatric Rating Scale (BPRS). In certain embodiments, the Scale for Assessment of Negative Symptoms (SANS) is used.

In some embodiments, the compositions of the invention improve the Clinical Global Impression - Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the compositions of the invention display a positive effect on global social and occupational impairment of the subject with schizophrenia spectrum or psychotic disorders.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate at least one comorbidity of schizophrenia spectrum or psychotic disorder. In certain embodiments, the comorbidity is/are: manic and/or depressive symptoms, anxiety or obsessive-compulsive symptoms, substance abuse and dependence, and personality disorder.

In certain embodiments, the compositions of the invention are for use in treating or preventing treatment resistant of refractory schizophrenia.

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating schizophrenia spectrum or psychotic disorders when used in combination with another therapy for treating PTSD and stress disorders. In certain embodiments, such therapies include first generation antipsychotics including chlorpromazine, fluphenazine, haloperidol and/or perphenazine. In certain embodiments, such therapies include second generation therapies including aripiprazole (Abilify®); asenapine (Saphris®); brexpiprazole (Rexulti®); cariprazine (Vraylar®); clozapine (Clozaril®); iloperidone (Fanapt®); lurasidone (Latuda®); olanzapine (Zyprexa®); paliperidone (Invega); quetiapine (Seroquel®); risperidone (Risperdal®); ziprasidone (Geodon®). Bipolar disorder

Bipolar disorder in general is a chronic disease. Mania is the cardinal symptom of bipolar disorder. There are several types of bipolar disorder based upon the specific duration and pattern of manic and depressive episodes. In DMS-5, a distinction is made between bipolar I disorder, bipolar II disorder, cyclothymic disorder, rapid-cycling bipolar disorder and bipolar disorder NOS.

According to the DSM, mania is a distinct period of abnormally and persistently elevated, expansive, or irritable mood. The episode must last a week, and the mood must have at least three of the following symptoms: high self-esteem; reduced need for sleep; increase rate of speech; rapid jumping of ideas; easily distracted; an increased interest in goals or activities; psychomotor agitation; increased pursuit of activities with a high risk of danger.

Bipolar I disorder involves one or more manic or mixed (mania and depression) episodes and at least one major depressive episode (see above for symptoms of MDD episodes). Bipolar II disorder has one or more major depressive episodes accompanied by at least one hypomanic episode. There are no manic or mixed episodes. Hypomania is a lesser form of mania. The symptoms are responsible for significant social, occupational and functional impairments. Cyclothymia is characterized by changing low-level depression along with periods of hypomania. The symptoms must be present for at least two years in adults or one year in children before a diagnosis can be made. Symptom free periods in adults and children last no longer than two months or one month, respectively. Rapid cycling bipolar disorder is a severe form of bipolar disorder. It occurs when a person has at least four episodes of major depression, mania, hypomania, or mixed states within a year. Not-otherwise specified (NOS) bipolar disorder classified bipolar symptoms that do not clearly fit into other types. NOS is diagnosed when multiple bipolar symptoms are present but not enough to meet the label for any of the other subtypes.

Bipolar disorder is associated with the following comorbidities: ADHD; anxiety disorders; substance disorders; obesity and metabolic syndrome.

Bipolar disorder is a psychiatric disorder that may develop or persist due to dysfunction of the microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing bipolar disorder in a subject. In certain embodiments, the bipolar disorder is bipolar I disorder. In certain embodiments, the bipolar disorder is bipolar II disorder. In certain embodiments, the bipolar disorder is cyclothymic disorder. In certain embodiments, the bipolar disorder is rapid-cycling bipolar disorder. In certain embodiments, the bipolar disorder is bipolar disorder NOS.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the symptoms of bipolar disorder in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of manic episodes in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of an abnormally and persistently elevated, expansive, or irritable mood. In certain embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the following symptoms: high self-esteem; reduced need for sleep; increase rate of speech; rapid jumping of ideas; easily distracted; an increased interest in goals or activities; psychomotor agitation; increased pursuit of activities with a high risk of danger. In certain embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of one or more manic or mixed episodes in a subject. In certain embodiments, the compositions of the invention reduce the occurrence of at least one major depressive episode in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of at least one major depressive episode accompanied by at least one hypomanic episode.

In preferred embodiments, the compositions of the invention treat the acute phase of bipolar disorder and/or prevent the occurrence of further episodes. In certain embodiments, the compositions of the invention treat the acute phase of manic/depressive episodes in a subject with bipolar disorder and prevent occurrence of further manic/depressive episodes.

In some embodiments, the compositions of the invention improve the symptoms of bipolar disorder according to a symptomatic or diagnostic scale. In certain embodiments, the scale for assessing symptomatic improvement of manic episodes is the Manic State Rating Scale and the Young Mania Rating Scale. In certain embodiments, the scale is the Bech-Rafaelsen Mania Scale (BRMAS). In certain embodiments, scales for assessing symptomatic improvement of the switch from manic to depressive episodes include the Hamilton Depression Rating Scale, the Montgomery-Asberg Rating Scale, and the Bech-Rafaelsen Depression Scale.

In some embodiments, the compositions of the invention improve the Clinical Global Impression - Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the compositions of the invention display a positive effect on global social, occupational and functional impairments of the subject with bipolar disorder.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate at least one comorbidity of bipolar disorder. In certain embodiments, the comorbidity is selected from ADHD, anxiety disorders, substance disorder, obesity and metabolic syndrome.

In certain embodiments, the compositions of the invention are for use in treating or preventing manic-depressive illness and bipolar disorder unresponsive to lithium and divalproex.

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating bipolar disorder when used in combination with another therapy for treating bipolar disorder. In certain embodiments, such therapies include lithium carbonate, anticonvulsant drugs (including valproate, divalproex, carbamazepine and lamotrigine) and antipsychotic drugs (including aripiprazole, olanzapine, quetiapine and risperidone).

Neurocognitive disorders and Alzheimer’s disease

In DSM-5, the term dementia was replaced with the terms major neurocognitive disorder and mild neurocognitive disorder. Neurocognitive disorder is a heterogeneous class of psychiatric diseases. The most common neurocognitive disorder is Alzheimer’s disease, followed by vascular dementias or mixed forms of the two. Other forms of neurodegenerative disorders ( e.g . Lewy body disease, frontotemporal dementia, Parkinson’s dementia, Creutzfeldt- Jakob disease, Huntington’s disease, and Wemicke-Korsakoff syndrome) are accompanied by dementia.

The symptomatic criteria for dementia under DSM-5 are evidence of significant cognitive decline from a previous level of performance in one or more cognitive domains selected from: learning and memory; language; executive function; complex attention; perceptual-motor and social cognition. The cognitive deficits must interfere with independence in everyday activities. In addition, the cognitive deficits do not occur exclusively in the context of a delirium and are not better explained by another mental disorder (for example MDD or schizophrenia).

In addition to the primary symptom, subjects with neurocognitive disorders display behavioural and psychiatric symptoms including agitation, aggression, depression, anxiety, apathy, psychosis and sleep-wake cycle disturbances.

Neurocognitive disorders are psychiatric disorders that may develop or persist due to dysfunction of the microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing neurocognitive disorders in a subject. In preferred embodiments, the neurocognitive disorder is Alzheimer’s disease. In other embodiments, the neurocognitive disorder is selected from vascular dementias; mixed form Alzheimer’s disease and vascular dementia; Lewy body disease; frontotemporal dementia; Parkinson’s dementia; Creutzfeldt-Jakob disease; Huntington’s disease; and Wemicke-Korsakoff syndrome.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the symptoms of neurocognitive disorders in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of cognitive decline in a subject. In certain embodiments, the compositions of the invention improve the level of performance of a subject with neurocognitive disorders in one or more cognitive domains selected from: learning and memory; language; executive function; complex attention; perceptual-motor and social cognition. In some embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of one or more behavioural and psychiatric symptoms associated with neurocognitive disorders selected from agitation, aggression, depression, anxiety, apathy, psychosis and sleep-wake cycle disturbances. In certain embodiments, the compositions of the invention prevent, reduce or alleviate symptomatic disease by intervention in suspected pathogenic mechanisms at a preclinical stage. In certain embodiments, the compositions of the invention improve disease modification, with slowing or arrest of symptom progression. In some embodiments, the slowing or arrest of symptom progression correlates with evidence in delaying the underlying neuropathological process. In preferred embodiments, the compositions of the invention improve symptoms of neurocognitive disorders comprising enhanced cognitive and functional improvement. In preferred embodiments, the compositions of the invention improve the behavioural and psychiatric symptoms of dementia (BPSD). In preferred embodiments, the compositions of the invention improve the ability of a subject with neurocognitive disorder to undertake everyday activities.

In preferred embodiments, the compositions of the invention improve both cognition and functioning in a subject with Alzheimer’s disease. In some embodiments, the composition of the invention improve the cognitive endpoint in a subject with Alzheimer’s disease. In some embodiments, the compositions of the invention improve the functional endpoint in a subject with Alzheimer’s disease. In preferred embodiments, the compositions of the invention improve the cognitive and functional endpoint in a subject with Alzheimer’s disease. In yet further preferred embodiments, the compositions of the invention improve the overall clinical response (the global endpoint) in a subject with Alzheimer’s disease.

In some embodiments, the compositions of the invention improve the symptoms of neurocognitive disorders according to a symptomatic or diagnostic test. In certain embodiments, the tests for assessing symptomatic improvement of Alzheimer’s disease (and other neurocognitive disorders) are selected from objective cognitive, activities of daily living, global assessment of change, health related quality of life tests and tests assessing behavioural and psychiatric symptoms of neurocognitive disorders.

In certain embodiments, the objective cognitive tests for assessment of symptomatic improvement use the Alzheimer’s disease Assessment Scale cognitive subscale (ADAS-cog) and the classic ADAS scale. In certain embodiments, symptomatic improvement of cognition is assessed using the Neurophysiological Test Battery for Use in Alzheimer’s Disease (NTB).

In some embodiments, the global assessment of change test uses the Clinical Global Impression - Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the global scale is the Clinician's Interview Based Impression of Change plus (CIBIC-plus). In some embodiments, the global scale is the Alzheimer’s Disease Cooperative Study Unit Clinician’s Global Impression of Change (ADCS-CGIC).

In certain embodiments, the health related quality of life measures are the Alzheimer’s Disease- Related QOL (ADRQL) and the QOL- Alzheimer’s Disease (QOL-AD). In certain embodiments, the tests assessing behavioural and psychiatric symptoms of neurocognitive disorders are selected from the Behavioural pathology in Alzheimer’s Disease Rating Scale (BEHAVE-AD); the Behavioural Rating Scale for Dementia (BRSD); the Neuropsychiatric Inventory (NPI); and the Cohen-Mansfield Agitation Inventory (CMAI).

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating neurocognitive disorders when used in combination with another therapy for treating neurocognitive disorders. In certain embodiments, such therapies include acetylcholinesterase inhibitors including donepezil (Aricept®), galantamine (Razadyne®) and rivastigmine (Exelon ®), and memantine.

Parkinson ’s disease

Parkinson’s disease is a common neurodegenerative disease neuropathologically characterised by degeneration of heterogeneous populations of neural cells (dopamine-producing cells). The clinical diagnosis of Parkinson’s disease requires bradykinesia and at least one of the following core symptoms: resting tremor; muscle rigidity and postural reflex impairment. Other signs and symptoms that may be present or develop during the progression of the disease are autonomic disturbances (sialorrhoea, seborrhoea, constipation, micturition disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances in the sense of smell or sense of temperature. Depressive symptoms and cognitive dysfunction comorbidities develop in many Parkinson’s disease patients, as well as neurocognitive disorders related to Lewy Bodies.

Parkinson’s disease is a psychiatric disorder that may develop or persist due to dysfunction of the microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing Parkinson’s disease in a subject.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the symptoms of Parkinson’s disease in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate one or more core symptoms of Parkinson’s disease in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate bradykinesia in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate resting tremor; muscle rigidity and/or postural reflex impairment in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate one or more symptoms associated with Parkinson’s disease progression selected from autonomic disturbances (sialorrhoea, seborrhoea, constipation, micturition disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances in the sense of smell or sense of temperature.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate depressive symptoms comorbid with Parkinson’s disease. In certain embodiments, the compositions of the invention improve verbal memory and/or executive functions. In certain embodiments, the compositions of the invention improve attention, working memory, verbal fluency and/or anxiety.

In other preferred embodiments, the compositions of the invention prevent, reduce or alleviate cognitive dysfunctions comorbid with Parkinson’s disease.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate Parkinson’s disease progression. In certain embodiments, the compositions of the invention prevent, reduce or alleviate later motor complications. In certain embodiments, the compositions of the invention prevent, reduce or alleviate late motor fluctuations. In certain embodiments, the compositions of the invention prevent, reduce or alleviate neuronal loss. In certain embodiments, the compositions of the invention improve symptoms of Parkinson’s disease dementia (PDD). In certain embodiments, the compositions of the invention prevent, reduce or alleviate impairment of executive function, attention and/or working memory. In certain embodiments, the compositions of the invention improve dopaminergic neurotransmission. In certain embodiments, the compositions of the invention prevent, reduce or alleviate impaired dopaminergic neurotransmission.

In some embodiments, the compositions of the invention improve the symptoms of Parkinson’s disease according to a symptomatic or diagnostic scale. In certain embodiments, the tests for assessing symptomatic improvement of motor function in Parkinson’s disease is the Unified Parkinson’s Disease Rating Scale. In particular, UPDRS II considers the activity of daily life and UPDRS IP considers motor-examination.

In some embodiments, the compositions of the invention improve the symptoms associated the PDD according to a symptomatic or diagnostic test and/or scale. In certain embodiments, the test or scale is selected from the Hopkins Verbal Learning Test - Revised (HVLT-R); the Delis- Kaplan Executive Function System (D-KEFS) Color-Word Interference Test; the Hamilton Depression Rating Scale (HAM-D 17; depression); the Hamilton Anxiety Rating Scale (HAM- A; anxiety) and the Unified Parkinson’s Disease Rating Scale (UPDRS; PD symptom severity).

In some embodiments, the compositions of the invention improve the Clinical Global Impression - Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the compositions of the invention display a positive effect on global social and occupational impairment of the subject with Parkinson’s disease.

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating neurocognitive disorders when used in combination with another therapy for treating neurocognitive disorders. In certain embodiments, such therapies include dopamine agonists (including L-Dopa+); monoamine oxidase inhibitors, catecholamine-O-methyl transferase inhibitors; anticholinergics and glutamate modulators. Other central nervous system disorders

In preferred embodiments, the compositions of the invention are for use in treating or preventing a central nervous system disorder associated with dysfunction of the microbiota-gut-brain axis. In addition to the embodiments above, the compositions of the invention are for use in treating or preventing psychosis; chronic fatigue syndrome (myalgic encephalomyelitis) and/or chronic pain. In further embodiments, the compositions of the invention may be useful for treating or preventing motor neuron disease; Huntington’s disease; Guillain-Barre syndrome and/or meningitis.

Neurochemical factors, neuropeptides and neurotransmitters and the microbiota-gut-brain axis As outlined above, the microbiota-gut-brain axis is modulated by a number of different physiological systems. The microbiota-gut-brain axis is modulated by a number of signalling molecules. Alterations in the levels of these signalling molecules results in defects in central nervous system development and/or functionality. Indeed, many of the molecules disclosed in this section have been implicated in the functionality of the microbiota-gut-brain axis and the pathogenesis of central nervous system disorders or conditions ([33], [9], [32], [34]). The experiments performed by the inventors indicate that behavioural changes can be triggered by administration of Enterococcus durans. This effect may be mediated by an effect on levels of the signalling molecules, in particular those listed in this section. These alterations may be responsible for the therapeutic benefits associated with Enterococcus durans. Accordingly, due to the fact that the central nervous system disorders and conditions disclosed herein display a similar fundamental biochemical and physiological pathogenesis (i.e. via the microbiota-gut- brain axis), a similar therapeutic benefit of Enterococcus durans may be also achieved for these disorders and conditions.

The signalling of the microbiota-gut-brain axis is modulated by levels of neurochemical factors, neuropeptides and neurotransmitters. Accordingly, in certain embodiments, the compositions of the invention modulates levels of neurochemical factors, neuropeptides and neurotransmitters. Accordingly, in certain preferred embodiments, the compositions of the invention directly alter CNS biochemistry. In preferred embodiments, the compositions of the invention modulate the levels of brain-derived neurotrophic factor (BDNF). In certain embodiments, the compositions of the invention modulate the levels of monoamines. In certain embodiments, the monoamines are serotonin (5-hydroxytryptamine (5-HT)), dopamine, norepinephrine and/or epinephrine. In certain embodiments, the monoamines are catecholamines. In certain embodiments, the catecholamines are dopamine, norepinephrine and epinephrine. In certain embodiments, the monoamines are tryptamines. In certain embodiments, the tryptamines are serotonin and melatonin. In certain embodiments, the compositions of the invention modulate the levels of acetylcholine. In certain preferred embodiments, the compositions of the invention modulate the levels of oxytoxin. Oxytocin is associated with emotional, social, cognitive and neuroendocrine physiologies as well as autoregulation. In particular, oxytocin release is involved in anxiolysis; positive mood; maternal behaviour, pair bonding; sexual behaviour; social memory; olfactory memory; anorexiant effects; attenuation of the HPA axis response to stress; autoexcitation during birth and suckling as well as other physiological and psychological processes. In certain embodiments, the compositions of the invention increase the levels of oxytocin. In certain embodiments, the compositions of the invention decrease the levels of oxytocin. In certain embodiments, the compositions of the invention increase or decrease oxytocin signalling. In certain embodiments, the compositions of the invention modulate the levels of oxytocin receptors. In certain embodiments, the compositions of the invention modulate the flux of calcium ions into or out of neuronal, muscle and gastrointestinal cells. In preferred embodiments, the compositions of the invention treat and prevent neurodevelopmental and neuropsychiatric disorders and diseases associated with the microbiota-gut-brain axis by modulating the levels of oxytocin.

In certain embodiments, the compositions of the invention modulate the levels of brain monoamines and metabolites thereof. In preferred embodiments, the monoamine is serotonin. In certain embodiments, the compositions of the invention modulate the serotonergic and/or kynurenine routes of tryptophan metabolism. In certain embodiments, the compositions of the invention modulate the levels of serotonin metabolites, such as 5-Hydroxyindoleacetic acid (5- HIAA). In certain embodiments, the compositions of the invention modulate the levels of dopamine metabolites, such as Homovanillic acid (HVA). Modulation of these neurotransmitters and neurochemical factors is useful for treating stress, depression and anxiety-related disorders.

The signalling of the microbiota-gut-brain axis is modulated by levels of g-aminobutyric acid (GABA). Accordingly, in preferred embodiments, the compositions of the invention modulate the levels of GABA. GABA is an inhibitory neurotransmitter that reduces neuronal excitability. In certain embodiments, the compositions of the invention increase the levels of GABA. In certain embodiments, the compositions of the invention decrease the levels of GABA. In certain embodiments, the compositions of the invention alter GABAergic neurotransmission. In certain embodiments, the compositions of the invention modulate the level of GABA transcription in different regions of the central nervous system. In certain embodiments, the commensal derived GABA crosses the blood-brain barrier and affects neurotransmission directly. In certain embodiments, the compositions of the invention lead to a reduction of GABA in the hippocampus, amygdala and/or locus coeruleus. In certain embodiments, the compositions of the invention lead to an increase of GABA in cortical regions.

Compositions which modulate the levels of GABA have been found to be useful in the treatment of epilepsy. Consequently, in some embodiments, the compositions of the invention are for use in the treatment of epilepsy. In some embodiments, the treatment reduces the frequency and/or the intensity of epileptic seizures in a subject. The frequency and/or intensity of epileptic seizures can vary greatly in different subjects. In some embodiments, the treatment comprises reducing the frequency and/or intensity of seizures by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%.

Modes for measuring the reduction in frequency and/or intensity of epileptic seizures are known in the art. For example, the intensity of a seizure may be assessed using the Chalfont Seizure Severity Scale.

In some subjects it may be appropriate to measure the reduction in frequency and/or intensity over the course of one day. In some subjects it may be appropriate to measure the reduction in frequency and/or intensity over either a day or a longer period, e.g. two days, a week, a month, six months, a year or longer. Thus, in some embodiments, the percentage reduction in the frequency of the seizures is measured over a period of one day, two days, three days, a week, two weeks, a month, three months, six months, a year, two years or longer than two years. In some embodiments, the treatment comprises reducing the frequency and/or intensity of epileptic seizures whilst the subject is awake. In some embodiments, the treatment comprises reducing the frequency and/or intensity of epileptic seizures whilst the subject is asleep. In some embodiments, the treatment comprises reducing the frequency and/or intensity of epileptic seizures whilst the subject is awake but not whilst the subject is asleep, or whilst the subject is asleep but not whilst the subject is awake. In some embodiments, the treatment comprises eliminating epileptic seizures. In some embodiments, the frequency of epileptic seizures is reduced to once a day or less, once every two days or less, once every week or less, once every two weeks or less, once a month or less, once every three months or less, once every six months or less, once a year or less, once every two years or less, or once every three years or less. In some embodiments, the treatment results in the subject experiencing an epileptic seizure free period of at least a day, at least two days, at least a week, at least a month, at least three months, at least six months, at least a year, at least two years, or at least three years.

In some embodiments, the compositions of the invention are particularly effective at treating epilepsy when used in combination with another therapy for treating epilepsy. In some embodiments, the other therapy for use in combination with the composition of the invention is selected from one or more of: Acetazolamide, Brivaracetam, Carbamazepine, Clobazam, Clonazepam, Eslicarbazepine acetate, Ethosuximide, Everolimus, Gabapentin, Lacosamide, Lamotrigine, Levetiracetam, Oxcarbazepine, Perampanel, Phenobarbital, Phenytoin, Piracetam, Pregabalin, Primidone, Rufmamide, Sodium valproate, Stiripentol, Tiagabine, Topiramate, Valproic acid, Vigabatrin, and Zonisamide. In some embodiments, the treatment results in the subject being able to reduce the dose or stop taking one or more other anti-epileptic drugs without seeing a deterioration in symptoms. For example, in some embodiments, the dose of a GABA-mediator anti-epileptic drug may be reduced or the GABA-mediator anti-epileptic drug may no longer be taken. In some embodiments, the GABA-mediator anti-epileptic drug is selected from valproic acid, sodium valproate and pregabalin.

The levels of neuroactive molecules, such as serotonin, melatonin, GABA, histamines and acetylcholine are linked to the pathophysiology of central nervous system diseases such as dementia, Alzheimer’s disease, Huntington’s disease and epilepsy.

The signalling of the microbiota-gut-brain axis is modulated by levels of histamines. Accordingly, in certain embodiments, the compositions of the invention modulate the levels of histamines. In certain embodiments, the histamines has an immunoregulatory effect. In certain embodiments, histamine levels enable translocation of bacteria from the lumen into systemic circulation. Therefore, in some embodiments, the compositions of the invention alter gastrointestinal tract permeability and/or barrier function. In certain other embodiments, the histamine acts as a neurotransmitter linked to central processes.

The signalling of the microbiota-gut-brain axis is modulated by the HPA axis. Accordingly, in certain embodiments, the compositions of the invention modulate HPA activity. In certain embodiments, the compositions of the invention attenuate the HPA stress response. In certain preferred embodiments, the compositions of the invention modulate inflammatory responses associated with HPA activity. In certain embodiments, the compositions of the invention modulate the levels of glucocorticoids. In certain preferred embodiments, the compositions of the invention modulate the levels of corticosterone and adrenaline. In certain embodiments, the compositions of the invention modulate the levels of corticotrophin-rel easing factor and/or vasopressin. In certain embodiments, the compositions of the invention modulate the levels of vasopressin and/or other neurohypophysial or antidiuretic hormones. Alterations in HPA axis activity are associated with anxiety and stress disorders.

The signalling of the microbiota-gut-brain axis is modulated by alterations in the immune response and inflammatory factors and markers. Accordingly, in certain embodiments, the compositions of the invention may modulate the immune response. In certain embodiments, the compositions of the invention modulate the systemic levels of circulating neuroimmune signalling molecules. In certain preferred embodiments, the compositions of the invention modulate pro-inflammatory cytokine production and inflammation. In certain embodiments, the compositions of the invention modulate the inflammatory state. In certain embodiments, the compositions of the invention modulate the splenocyte proliferative response. In certain embodiments, the compositions of the invention modulate the systemic and/or plasma levels of C-reactive protein; IL-1 family cytokines; IL-Ib; IL-2; IL-4; IL-6; IL-8; IL-10; IL-12p40; IL-17; IL-17A; IL-21; IL-23; TNF- a and IFN-g. In some embodiments the compositions of the invention module the levels of anti-inflammatory cytokines, for example IL-10. In preferred embodiments, the compositions of the invention increase the levels of IL-10. In some embodiments, the compositions of the invention modulate the levels of TNF-a. In preferred embodiments, the compositions of the invention modulate the levels of IFN-g. In some embodiments, the compositions of the invention modulate the IFN-y:IL- 10 ratio. In certain preferred embodiments, the compositions of the invention decrease the IFN-y:IL- 10 ratio. In preferred embodiments, the compositions of the invention decrease the levels of the pro- inflammatory cytokines TNF-a and IFN-g. Increased circulating levels of cytokines are closely associated with various neuropsychiatric disorders, including depression, anxiety, schizophrenia and ASD. Evidence of inflammatory state alteration is highlighted in disorders such as schizophrenia, major depressive disorder and bipolar disorder.

In certain embodiments, the compositions of the invention modulates the levels of tolerance- mediating dendritic cells and reciprocally regulate pro and anti-inflammatory cytokine responses. In certain embodiments, the compositions of the invention decrease the systemic level of myeloperoxidase (a marker for inflammation and oxidation). Therapeutic modulators of the immune system and of inflammatory responses are useful for treating autism spectrum disorders and mood disorders.

In certain embodiments, the compositions of the invention modulate the immune response to an infection or vaccination. In certain embodiments, the compositions of the invention modulate the level of inflammation in response to infection or vaccination. In certain preferred embodiments, the compositions of the invention modulate maternal immune activation in response to an infection or vaccination during pregnancy. Accordingly, the compositions of the invention can be administered during pregnancy in order to treat or prevent a central nervous system disorder in the offspring.

The signalling of the microbiota-gut-brain axis is modulated by levels commensal metabolites. Accordingly, in certain embodiments, the compositions of the invention modulate the systemic levels of microbiota metabolites. In certain preferred embodiments, the compositions of the invention modulate the level of short chain fatty acids (SCFAs). In certain embodiments the level of SCFAs is increased or decreased. In some embodiments, the SCFA is butyric acid (BA) (or butyrate). In some embodiments, the SCFA is propionic acid (PPA). In some embodiments, the SCFA is acetic acid. In certain embodiments, the compositions of the invention modulate the ability of SCFAs to cross the blood-brain barrier. In certain embodiments, the compositions of the invention modulate the level of Polysaccharide A (PSA). In certain embodiments, the compositions of the invention modulate the levels of the potent pro-inflammatory endotoxin lipopolysaccharide (LPS). LPS leads to the production of inflammatory cytokines that alter physiological brain activity and modulate neuropeptide synthesis. LPS has an important influence on the modulation of the CNS, increasing the activity of areas devoted to the control of emotions ( e.g . the amygdala). In certain embodiments, the compositions of the invention modulate the level of tryptophan and/or its metabolites. In certain embodiments, the compositions of the invention modulate the levels of 4-ethylphenylsulphate (4EPS; a uremic toxic associated with ASD-related behavioural abnormalities). In preferred embodiments, the compositions of the invention decrease the levels of 4-ethylphenylsulphate in a subject. The signals generated by the stimulation of neuronal signalling pathways caused by intraluminal gut stimuli strongly modulate brain activity, including pain perception, immune-response modulation, emotional control and other homeostatic functions. Accordingly, a composition able to modulate levels of these factors would have broad therapeutic applications for treating or preventing CNS disorders.

The signalling of the microbiota-gut-brain axis is modulated by levels gastrointestinal permeability. Accordingly, in some embodiments, the compositions of the invention alter the integrity of the gastrointestinal tract epithelium. In certain embodiments, the compositions of the invention modulate the permeability of the gastrointestinal tract. In certain embodiments, the compositions of the invention modulate the barrier function and integrity of the gastrointestinal tract. In certain embodiments, the compositions of the invention modulate gastrointestinal tract motility. In certain embodiments, the compositions of the invention modulate the translocation of commensal metabolites and inflammatory signalling molecules into the bloodstream from the gastrointestinal tract lumen.

The signalling of the microbiota-gut-brain axis is modulated by microbiome composition in the gastrointestinal tract. Accordingly, in certain embodiments, the compositions of the invention modulates the microbiome composition of the gastrointestinal tract. In certain embodiments, the compositions of the invention prevents microbiome dysbiosis and associated increases in toxic metabolites (e.g. LPS). In certain embodiments, the compositions of the invention modulate the levels of Clostridium in the gastrointestinal tract. In preferred embodiments, the compositions of the invention reduce the level of Clostridium in the gastrointestinal tract. In certain embodiments, the compositions of the invention reduce the levels of Campylobacter jejuni. In certain embodiments, the compositions of the invention modulate the proliferation of harmful anaerobic bacteria and the production of neurotoxins produced by these bacteria. In certain embodiments, the compositions of the invention modulate the microbiome levels of Lactobacillus and/or Bifidobacterium. In certain embodiments, the compositions of the invention modulate the microbiome levels of Sutterella, Prevotella, Ruminoccucs genera and/or the Alcaligenaceae family. In certain embodiments, the compositions of the invention increase the level of Lactobacillus plantarum and/or Saccharomyces boulardii.

In certain embodiments, the compositions of the invention prevent the dysregulation of the composition of the microbiome by extensive antibiotic use. In certain preferred embodiments, the compositions of the invention maintain a functional maternal microbiome composition upon administration of antibiotics during pregnancy. Accordingly, the compositions of the invention can be administered during pregnancy in order to treat or prevent a central nervous system disorder in the offspring.

Modulation of the microbiome has been shown to be effective at improving psychiatric disorder- related behaviours, including anxiety, depression, autism spectrum disorder, obsessive- compulsive disorder and memory abilities (including spatial and non-spatial memory), as well as other CNS-related disorders including Parkinson’s disease. Certain studies have suggested that probiotics can reduce psychological stress, somatisation, depression and anger-hostility. The levels of Lactobacillus are associated with depression and have been implicated in pain signalling associated with gastrointestinal discomfort.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate at least one of the behavioural symptoms associated with a central nervous system disorder described herein. In preferred embodiments, the compositions of the invention improve the overall clinical response in a subject.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate stereotyped, repetitive behaviour in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of unusually restrictive behaviours and/or interests. In certain embodiments, the compositions of the invention prevent, reduce or alleviate recurrent obsessions and/or compulsions in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate deficits in social behaviour in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate avoidance behaviour in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate deficits in communication behaviour in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate neophobia and / or sociability deficit.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate negative alterations in cognitions and mood in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate anxiety-related behaviour in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate stress-related behaviour in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate depression-related behaviour in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate aggressive behaviour in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of an abnormally and persistently elevated, expansive, or irritable mood. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate intrusive thoughts in a subject. In preferred embodiments, the compositions of the invention prevent alterations in arousal and reactivity in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate delusions, hallucinations, disorganised speech, and disorganised or catatonic behaviours in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate affective flattening, restriction in the fluency and productivity of thought and speech and in the initiation of goal directed behaviour in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the following symptoms: despair, helplessness, high self-esteem; reduced need for sleep; increase rate of speech; rapid jumping of ideas; easily distracted; an increased interest in goals or activities; psychomotor agitation; increased pursuit of activities with a high risk of danger.

In preferred embodiments, the compositions of the invention improve memory function, for example spatial and/or non-spatial memory deficits in a subject. In preferred embodiments, the compositions of the invention improve learning in a subject. In preferred embodiments, the compositions of the invention improve both cognition and functioning in a subject. In preferred embodiments, the compositions of the invention improve locomotor activity in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate bradykinesia in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate resting tremor; muscle rigidity and/or postural reflex impairment in a subject.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate at least one comorbidity associated with a CNS disorder disclosed herein.

In preferred embodiments, the compositions of the invention improve the scores of a subject on at least one of the symptomatic and/or diagnostic scales for CNS disorders described herein. In certain other embodiments, the symptomatic and/or diagnostic scale is selected from the General Health Questionnaire (GHQ); the Depression Anxiety and Stress Scale (DASS); the Leiden Index of Depression Sensitivity-Revised (LEIDS-r); the Positive and Negative Symptom Scale (PANSS); the State-Trait Anxiety Inventory (STAI); the Development Behavior Checklist (DBC); the Beck Depression Inventory (BDI); the Beck Anxiety Inventory (BAI); the Hopkins Symptom Checklist (HSCL-90); the Hospital Anxiety and Depression Scale (HADS); the Perceived Stress Scale (PSS); the Coping Checklist (CCL) (also used to counter the stress of daily life); and the questionnaire-based Profile of Mood State (POMS).

In certain embodiments, the compositions of the invention may improve the symptomatic and/or diagnostic scale when assessing therapeutic efficacy in other animal models of CNS disorders known to a person skilled in the art. In addition to the behavioural assays disclosed in the examples, the compositions of the invention may improve reciprocal social interactions; olfactory communication; ultrasonic vocalisation; motor stereotypes (such as circling and vertical jumping), repetitive behaviour such as self-grooming and diffing; and perseverance in spatial tasks.

In addition, the compositions of the invention will be useful in treating and/or preventing CNS disorders in other animal models of CNS disorders. Other mouse models include inbred mice strains (including BALB/cJ and C58/J) and also genetically modified mice strains (including NEUREXIN1, NEUROLIGIN3, NEUROLIGIN4, SHANK2, SHANK3, CNTNAP2, Tscl/2 and Fmrl gene mutant mice strains).

In certain embodiments, the compositions of the invention improve social behaviour of a subject. In preferred embodiments, the compositions of the invention improve the recognition of social novelty in a subject. In preferred embodiments, the compositions of the invention improve sociability deficit. In preferred embodiments, the compositions of the invention improve the ability to discriminate between familiar and novel objects and familiar and novel subjects. In preferred embodiments, the composition of the invention improve ability to recognise other subjects.

In certain embodiments, the compositions of the invention regulate plasma levels of amino acids. In certain embodiments, the compositions of the invention regulate the biosynthesis or catabolism of amino acids. In preferred embodiments, the compositions of the invention regulate plasma levels of proline. In preferred embodiments, the compositions of the invention reduce the plasma levels of proline. Elevated proline is known to negatively affect brain function by an increase in dopamine in the prefrontal cortex [35] In addition, proline is considered to be a neurotransmitter that modulates glutamatergic neurotransmission in the hippocampus, and neurotransmission elsewhere in the brain. Accordingly, proline has been implicated in CNS disorders and psychiatric disorders, in particular psychosis. In preferred embodiments, the reduction in plasma levels of proline treats or prevents CNS disorders, in particular, ADHD, OCD, mood disorders, autism spectrum disorder, psychosis and schizophrenia.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate the symptoms of psychiatric disorders, for example schizophrenia and bipolar disorder, associated with 22qll.2 deletion syndrome (22qllDS) [35] In certain embodiments, the compositions of the invention improve the social behavioural and social cognitive problems in subjects with 22ql IDS. In preferred embodiments, the compositions of the invention modulate the associated cognitive and behavioural outcomes in 22qllDS subjects. In preferred embodiments, the modulation of these outcomes is a consequence of reduced plasma levels of proline. In certain embodiments, the compositions of the invention modulate the activity of proline hydrogenase.

In certain embodiments, the compositions of the invention modulate the levels of NMDA receptors and/or the subunits thereof. In preferred embodiments, the compositions of the invention modulate the levels of the NMDA receptor 2B. In certain embodiments, the compositions of the invention increase the levels of the NMD A receptor 2B. In preferred embodiments, the compositions of the invention decrease the levels of the NMDA receptor 2B. Dysregulation of NMDA receptors have been associated with CNS disorders, in particular ASD and schizophrenia. There have been suggestions that NMDA receptor antagonists may be effective in treating ASD [36] In addition, suppression of NMDA receptor function has been demonstrated to improve social deficits and reduce repetitive behaviour in valproic acid induced models of ASD [37] In certain embodiments, the compositions of the invention cause hypofunction of the NMDA receptor 2B. In certain embodiments, the compositions of the invention cause hyperfunction of the NMDA receptor 2B. In certain embodiments, the compositions of the invention prevent, reduce or alleviate the symptoms of CNS disorders, for example ASD or schizophrenia as a consequence of the modulation of NMDA receptor 2B activity. In preferred embodiments, the compositions of the inventions suppress NMDA receptor activity and reduce social deficits and stereotypical behaviour in subjects with CNS disorders.

In certain embodiments, the compositions of the invention modulate the levels of BDNF. In preferred embodiments, the compositions of the invention reduce the levels of BDNF. In certain embodiments, the reduction in BDNF is localised to the amygdala. Meta-analyses of ASD populations have shown that higher levels of BDNF are detected in ASD subjects compared to controls [38] In preferred embodiments, the compositions of the invention prevent, reduce or alleviate the symptoms of CNS disorders, in particular ASD, as a consequence of the reduction in levels of BDNF. Altered levels of BDNF have been associated with a number of neurodevelopmental disorders, as well as psychosis and schizophrenia. In certain embodiments, the compositions of the invention modulate levels of BDNF in order to prevent, reduce or alleviate the symptoms of neurodevelopmental and psychiatric disorders.

In certain embodiments, the compositions of the invention modulate the levels of inflammatory markers produced in response to an antigen challenge. In preferred embodiments, the compositions of the invention increase the levels of IL-Ib in response to a viral antigen challenge. In certain embodiments, the compositions of the invention modulate the innate immune response. In certain embodiments, the compositions of the invention modulate the adaptive immune response. In certain embodiments, the compositions of the invention modulate the inflammatory response.

Modes of administration

Preferably, the compositions disclosed herein are to be administered to the gastrointestinal tract in order to enable delivery to and / or partial or total colonisation of the intestine with the bacterial strain of the invention. In other words, the bacteria may have colonised some or all of the gastrointestinal tract and / or such colonisation may be transient or permanent. More specifically, in some embodiments, the “total colonisation of the intestine” means that bacteria have colonised all parts of the intestine (i.e. the small intestine, large intestine and rectum). Additionally or alternatively, the term “total colonisation” means that the bacteria engraft permanently in the some or all parts of the intestine.

In some embodiments, “partial colonisation of the intestine” means that bacteria have colonised some but not all parts of the intestine. Additionally or alternatively, the term “partial colonisation” means that the bacteria engraft transiently in some or all parts of the intestine.

The transience of engraftment can be determined by assessing (e.g. in a fecal sample) the abundance of the bacterial strain of the invention periodically (e.g. daily) following the end of a dosing interval to determine the washout period, i.e. the period between conclusion of the dosing interval and there being no detectable levels of the bacterial strain of the invention present. In embodiments of the invention, the washout period is 14 days or less, 12 days or less, 10 days or less, 7 days or less, 4 days or less, 3 days or less, 2 days or less or 1 day or less.

In embodiments of the invention, the bacteria of the present invention engraft transiently in the large intestine.

Generally, the compositions of the invention are administered orally, but they may be administered rectally, intranasally, or via buccal or sublingual routes.

In some embodiments, the compositions of the invention are administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract.

Pharmaceutical formulations suitable for oral administration include solid plugs, solid microparticulates, semi-solid and liquid (including multiple phases or dispersed systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids (e.g. aqueous solutions), emulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.

In certain embodiments, the compositions of the invention may be administered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may be administered as a suppository, such as a rectal suppository, for example in the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.

In certain embodiments, the composition of the invention is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports. The compositions of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions of the invention are to be administered daily (either once or several times).

In certain embodiments, the compositions of the invention are administered regularly, such as daily, every two days, or weekly, for an extended period of time, such as for at least one week, two weeks, one month, two months, six months, or one year.

In some embodiments the compositions of the invention are administered for 7 days, 14 days, 16 days, 21 days or 28 days or no more than 7 days, 14 days, 16 days, 21 days or 28 days. For example, in some embodiments the compositions of the invention are administered for 16 days.

In certain embodiments of the invention, treatment according to the invention is accompanied by assessment of the patient’s gut microbiota. Treatment may be repeated if delivery of and / or partial or total colonisation with the bacterial strain is not achieved such that efficacy is not observed, or treatment may be ceased if delivery and / or partial or total colonisation is successful and efficacy is observed.

In certain embodiments, the composition of the invention may be administered to a pregnant animal, for example a mammal such as a human in order to prevent an inflammatory or autoimmune disease developing in her child in utero and / or after it is bom.

The compositions of the invention may be administered to a patient that has been diagnosed with a central nervous system disorder or condition, in particular a central nervous system disorder or condition mediated by the microbiota-gut-brain axis, or that has been identified as being at risk of a central nervous system disorder or condition, in particular central nervous system disorder or condition mediated by the microbiota-gut-brain axis. The compositions may also be administered as a prophylactic measure to prevent the development of central nervous system disorders or conditions, in particular central nervous system disorders or conditions mediated by the microbiota-gut-brain axis in a healthy patient.

The compositions of the invention may be administered to a patient that has been identified as having an abnormal gut microbiota. For example, the patient may have reduced or absent colonisation by Enterococcus durans.

The compositions of the invention may be administered as a food product, such as a nutritional supplement.

Generally, the compositions of the invention are for the treatment or prevention of disease in humans, although they may be used to treat or prevent disease in animals including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits. The compositions of the invention may be useful for enhancing the growth and performance of animals. If administered to animals, oral gavage may be used. In some embodiments, the subject to whom the composition is to be administered is a human. The human may be an adult, a child or an infant.

Compositions

The compositions of the invention comprise bacteria. In preferred embodiments of the invention, the composition is formulated in freeze-dried form. The composition of the invention may comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilised bacteria. Lyophilisation of bacteria is a well-established procedure and relevant guidance is available in, for example, references [39-41]

Alternatively, the composition of the invention may comprise a live, active bacterial culture.

In some embodiments, the bacterial strain in the composition of the invention has not been inactivated, for example, has not been heat-inactivated. In some embodiments, the bacterial strain in the composition of the invention has not been killed, for example, has not been heat- killed. In some embodiments, the bacterial strain in the composition of the invention has not been attenuated, for example, has not been heat-attenuated. For example, in some embodiments, the bacterial strain in the composition of the invention has not been killed, inactivated and/or attenuated. For example, in some embodiments, the bacterial strain in the composition of the invention is live. For example, in some embodiments, the bacterial strain in the composition of the invention is viable. For example, in some embodiments, the bacterial strain in the composition of the invention is capable of partially or totally colonising the intestine. For example, in some embodiments, the bacterial strain in the composition of the invention is viable and capable of partially or totally colonising the intestine.

In some embodiments, the composition comprises a mixture of live bacterial strains and bacterial strains that have been killed.

In preferred embodiments, the composition of the invention is encapsulated to enable delivery of the bacterial strain to the intestine. Encapsulation protects the composition from degradation until delivery at the target location through, for example, rupturing with chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier surfaces, self-aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule. Guidance on encapsulation that may be useful for preparing compositions of the invention is available in, for example, references [42-43] The composition may be administered orally and may be in the form of a tablet, capsule or powder. Encapsulated products are preferred.

A composition of the invention includes a therapeutically effective amount of a bacterial strain of the invention. A therapeutically effective amount of a bacterial strain is sufficient to exert a beneficial effect upon a patient. A therapeutically effective amount of a bacterial strain may be sufficient to result in delivery to and / or partial or total colonisation of the patient’s intestine.

A suitable daily dose of the bacteria, for example for an adult human, may be from about 1 x 10 ³ to about 1 x 10 ¹¹ colony forming units (CFU); for example, from about 1 x 10 ⁷ to about 1 x 10 ¹⁰ CFU; in another example from about 1 x 10 ⁶ to about 1 x 10 ¹⁰ CFU; in another example from about 1 x 10 ⁷ to about 1 x 10 ¹¹ CFU; in another example from about 1 x 10 ⁸ to about 1 x 10 ¹⁰ CFU; in another example from about 1 x 10 ⁸ to about 1 x 10 ¹¹ CFU.

In certain embodiments, the dose of the bacteria is at least 10 ⁹ cells per day, such as at least 10 ¹⁰ , at least 10 ¹¹ , or at least 10 ¹² cells per day.

A dose of the composition may comprise the bacterial strain from about 1 x 10 ⁶ to about 1 x 10 ¹¹ colony forming units (CFU) /g, respect to the weight of the composition. The dose may be suitable for an adult human. For example, the composition may comprise the bacterial strain from about 1 x 10 ³ to about 1 x 10 ¹¹ CFU/g; for example, from about 1 x 10 ⁷ to about 1 x 10 ¹⁰ CFU/g; in another example from about 1 x 10 ⁶ to about 1 x 10 ¹⁰ CFU/g; in another example from about 1 x 10 ⁷ to about 1 x 10 ¹¹ CFU/g; in another example from about 1 x 10 ⁸ to about 1 x 10 ¹⁰ CFU/g; in another example from about 1 x 10 ⁸ to about 1 x 10 ¹¹ CFU/g, from about 1 x 10 ⁸ to about 1 x 10 ¹⁰ CFU/g. The dose may be, for example, 1 g, 3g, 5g, and lOg.

The composition may be formulated as a probiotic. A probiotic is defined by the FAO/WHO as a live microorganism that, when administered in adequate amounts, confers a health benefit on the host.

Typically, a probiotic, such as the composition of the invention, is optionally combined with at least one suitable prebiotic compound. In certain embodiments, the probiotic composition of the present invention includes a prebiotic compound in an amount of from about 1 to about 30% by weight, respect to the total weight composition, (e.g. from 5 to 20% by weight). Known prebiotics include commercial products such as inulin and transgalacto-obgosaccharides.

A prebiotic compound is usually a non-digestible carbohydrate such as an oligo- or polysaccharide, or a sugar alcohol, which is not degraded or absorbed in the upper digestive tract. The carbohydrates may be selected from the group consisting of: fructo- oligosaccharides (or FOS), short-chain fructo-obgosaccharides, inulin, isomalt-oligosaccharides, pectins, xylo- obgosaccharides (or XOS), chitosan-obgosaccharides (or COS), beta-glucans, arable gum modified and resistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers. In one aspect, the prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible carbohydrates, generally obtained by the conversion of the beet sugar and including a saccharose molecule to which three glucose molecules are bonded.

Other prebiotic compounds (such as vitamin C, for example), may be included as oxygen scavengers and to improve the delivery and / or partial or total colonisation and survival in vivo. Alternatively, the probiotic composition of the invention may be administered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical product.

In certain embodiments, the compositions of the invention are used in combination with another therapeutic compound for treating or preventing the central nervous system disorder. In some embodiments, the compositions of the invention are administered with nutritional supplements that modulate central neurotransmitters and neuropeptides. In preferred embodiments, the nutritional supplements comprise or consist of nutritional vitamins. In certain embodiments, the vitamins are vitamin B6, magnesium, dimethylglycine (vitamin B16) and vitamin C. In certain embodiments, the compositions of the invention are administered in combination with another probiotic. In certain preferred embodiments, the probiotic comprises or consists of Trichuris suis ova.

The compositions of the invention may comprise pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients may be found in reference [44] Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [45] Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, com sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. A further example of a suitable carrier is saccharose. A further example of a preservative is cysteine. The compositions of the invention may be formulated as a food product. For example, a food product may provide nutritional benefit in addition to the therapeutic effect of the invention, such as in a nutritional supplement. Similarly, a food product may be formulated to enhance the taste of the composition of the invention or to make the composition more attractive to consume by being more similar to a common food item, rather than to a pharmaceutical composition. In certain embodiments, the composition of the invention is formulated as a milk-based product. The term "milk-based product" means any liquid or semi-solid milk- or whey- based product having a varying fat content. The milk-based product can be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products. Another important group includes milk beverages, such as whey beverages, fermented milks, condensed milks, infant or baby milks, flavoured milks, ice cream, milk-containing food such as sweets.

In some embodiments, the compositions of the invention comprise one or more bacterial strains of the species Enterococcus durans and do not contain bacteria from any other species, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another species. Thus, in some embodiments, the invention provides a composition comprising one or more bacterial strains of the species Enterococcus durans, which does not contain bacteria from any other species or which comprises only de minimis or biologically irrelevant amounts of bacteria from another species, for use in therapy.

In certain embodiments, the compositions of the invention contain a single bacterial species and do not contain any other bacterial species. In certain embodiments, the compositions of the invention contain a single bacterial strain and do not contain any other bacterial strains. Such compositions may comprise only de minimis or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture that is substantially free from other species of organism. In some embodiments, such compositions may be a lyophilisate that is substantially free from other species of organism.

In some embodiments, the invention provides a composition comprising a single bacterial strain of the species Enterococcus durans, which does not contain bacteria from any other strains or which comprises only de minimis or biologically irrelevant amounts of bacteria from another strain for use in therapy.

In embodiments of the invention, the compositions of the invention do not contain bacteria from the genus Bacteroides or comprise only de minimis or biologically irrelevant amounts of bacteria from the genus Bacteroides for use in therapy.

In some embodiments, the compositions of the invention comprise more than one bacterial strain or species. For example, in some embodiments, the compositions of the invention comprise more than one strain from within the same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or 45 strains), and, optionally, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise less than 50 strains from within the same species (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or 3 strains), and, optionally, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1- 6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 strains from within the same species and, optionally, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise more than one species from within the same genus (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 23, 25, 30, 35 or 40 species), and, optionally, do not contain bacteria from any other genus. In some embodiments, the compositions of the invention comprise less than 50 species from within the same genus (e.g. less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or 3 species), and, optionally, do not contain bacteria from any other genus. In some embodiments, the compositions of the invention comprise 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2- 30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 species from within the same genus and, optionally, do not contain bacteria from any other genus. In some embodiments, the composition comprises fewer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 bacterial strains. The invention comprises any combination of the foregoing.

In some embodiments, the composition comprises a microbial consortium. For example, in some embodiments, the composition comprises the Enterococcus durans bacterial strain as part of a microbial consortium. For example, in some embodiments, the Enterococcus durans bacterial strain is present in combination with one or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterial strains from the genus Enterococcus and/or other genera with which it can live symbiotically in vivo in the intestine. For example, in some embodiments, the composition comprises a bacterial strain of Enterococcus durans in combination with a bacterial strain from a different genus. In another example, the composition comprises a bacterial strain of Enterococcus durans in combination with a bacterial strain from the genus Enterococcus or the composition comprises a bacterial strain of Enterococcus in combination with a bacterial strain from the genus Enterococcus and a bacterial strain from a different genus. In some embodiments, the microbial consortium comprises two or more bacterial strains obtained from a faeces sample of a single organism, e.g. a human. In some embodiments, the microbial consortium is not found together in nature. For example, in some embodiments, the microbial consortium comprises bacterial strains obtained from faeces samples of at least two different organisms. In some embodiments, the two different organisms are from the same species, e.g. two different humans. In some embodiments, the two different organisms are an infant human and an adult human. In some embodiments, the two different organisms are a human and a non-human mammal. In some embodiments, the composition of the invention additionally comprises a bacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 43456, but which is not strain NCIMB 43456, or which is not Enterococcus durans.

In some embodiments, the composition of the invention does not comprise a bacterial strain of the genus Bacillus. In some embodiments, the composition of the invention does not comprise Bacillus subtilis and/or does not comprise Bacillus coagulans. In some embodiments, the CNS disorder to be treated by the composition of the invention is not bipolar disorder. In some embodiments, the patient to be treated by the composition of the invention does not have a fungal infection. In some embodiments, the patient to be treated by the composition of the invention does not suffer from candidiasis. In some embodiments, the patient to be treated by the composition of the invention has not been diagnosed as having a fungal infection and/or has not been diagnosed as suffering from candidiasis. In preferred such embodiments, the patient to be treated by the composition of the invention has never been diagnosed as having a fungal infection and/or has never been diagnosed as suffering from candidiasis.

In some embodiments, the composition of the invention does not comprise a bacterial strain of the species Enterococcus faecium and/or of the species Enterococcus faecalis.

In some embodiments in which the composition of the invention comprises more than one bacterial strain, species or genus, the individual bacterial strains, species or genera may be for separate, simultaneous or sequential administration. For example, the composition may comprise all of the more than one bacterial strain, species or genera, or the bacterial strains, species or genera may be stored separately and be administered separately, simultaneously or sequentially. In some embodiments, the more than one bacterial strains, species or genera are stored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the invention is obtained from human adult faeces. In some embodiments in which the composition of the invention comprises more than one bacterial strain, all of the bacterial strains are obtained from human adult faeces or if other bacterial strains are present they are present only in de minimis amounts. The bacteria may have been cultured subsequent to being obtained from the human adult faeces and being used in a composition of the invention.

In some embodiments, the one or more Enterococcus durans bacterial strains is/are the only therapeutically active agent(s) in a composition of the invention. In some embodiments, the bacterial strain(s) in the composition is/are the only therapeutically active agent(s) in a composition of the invention.

The compositions for use in accordance with the invention may or may not require marketing approval. In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised. In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is spray dried. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is live. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is capable of partially or totally colonising the intestine. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable and capable of partially or totally colonising the intestine.

In some cases, the lyophilised or spray dried bacterial strain is reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.

The compositions of the invention can comprise pharmaceutically acceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceutical composition comprising: a bacterial strain as discussed earlier; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of: autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder; depression; seasonal affective disorder; anxiety disorders; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post- traumatic stress disorder; dementia; Alzheimer’s; Parkinson’s disease; epilepsy; chronic pain (for example, central sensitisation or fibromyalgia); motor neuron disease; Huntington’s disease; Guillain-Barre syndrome and/or meningitis.

In certain embodiments, the invention provides pharmaceutical composition comprising: a bacterial strain as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a central nervous system disorder or condition, in particular central nervous system disorder or condition mediated by the microbiota-gut-brain axis. In preferred embodiments, said disease or condition is selected from the group consisting of: autism spectrum disorders (ASDs); child developmental disorder; obsessive compulsive disorder (OCD); major depressive disorder; depression; seasonal affective disorder; anxiety disorders; schizophrenia spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder; chronic fatigue syndrome (myalgic encephalomyelitis); stress disorder; post-traumatic stress disorder; dementia; Alzheimer’s; Parkinson’s disease; epilepsy; chronic pain (for example, central sensitisation or fibromyalgia); motor neuron disease; Huntington’s disease; Guillain-Barre syndrome and/or meningitis.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1 x 10 ³ to about 1 x 10 ¹¹ colony forming units per gram with respect to a weight of the composition.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered at a dose of 1 g, 3 g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol and sorbitol.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, com sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.

In certain embodiments, the invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphere having 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units, remains after a period of at least about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years. In some embodiments, the composition of the invention is provided in a sealed container comprising a composition as described herein. In some embodiments, the sealed container is a sachet or bottle. In some embodiments, the composition of the invention is provided in a syringe comprising a composition as described herein.

The composition of the present invention may, in some embodiments, be provided as a pharmaceutical formulation. For example, the composition may be provided as a tablet or capsule. In some embodiments, the capsule is a gelatine capsule (“gel-cap”). The capsule can be a hard or a soft capsule. In some embodiments, the formulation is a soft capsule. Soft capsules are capsules which may, owing to additions of softeners, such as, for example, glycerol, sorbitol, maltitol and polyethylene glycols, present in the capsule shell, have a certain elasticity and softness. Soft capsules can be produced, for example, on the basis of gelatine or starch. Gelatine- based soft capsules are commercially available from various suppliers. Depending on the method of administration, such as, for example, orally or rectally, soft capsules can have various shapes, they can be, for example, round, oval, oblong or torpedo-shaped. Soft capsules can be produced by conventional processes, such as, for example, by the Scherer process, the Accogel process or the droplet or blowing process.

In some embodiments the pharmaceutical formulation is an enteric formulation, i.e. a gastro- resistant formulation (for example, resistant to gastric pH) that is suitable for delivery of the composition of the invention to the intestine by oral administration. Enteric formulations may be particularly useful when the bacteria or another component of the composition is acid- sensitive, e.g. prone to degradation under gastric conditions.

In some embodiments, the enteric formulation comprises an enteric coating. In some embodiments, the formulation is an enteric-coated dosage form. For example, the formulation may be an enteric-coated tablet or an enteric-coated capsule, or the like. The enteric coating may be a conventional enteric coating, for example, a conventional coating for a tablet, capsule, or the like for oral delivery. The formulation may comprise a film coating, for example, a thin film layer of an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is intrinsically enteric, for example, gastro- resistant without the need for an enteric coating. Thus, in some embodiments, the formulation is an enteric formulation that does not comprise an enteric coating. In some embodiments, the formulation is a capsule made from a thermogelling material. In some embodiments, the thermogelling material is a cellulosic material, such as methylcellulose, hydroxymethylcellulose or hydroxypropylmethylcellulose (HPMC). In some embodiments, the capsule comprises a shell that does not contain any film forming polymer. In some embodiments, the capsule comprises a shell and the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [46]). In some embodiments, the formulation is an intrinsically enteric capsule (for example, Vcaps® from Capsugel).

Culturing methods

The bacterial strains for use in the present invention can be cultured using standard microbiology techniques as detailed in, for example, references [47] -[49]

The solid or liquid medium used for culture may be YCFA agar or YCFA medium. YCFA medium may include (per 100ml, approximate values): Casitone (1.0 g), yeast extract (0.25 g), NaHCOs (0.4 g), cysteine (0.1 g), K ₂ HP0 ₄ (0.045 g), KH ₂ P0 ₄ (0.045 g), NaCl (0.09 g), (NH ₄ ) ₂ S0 ₄ (0.09 g), MgS0 ₄ · 7H ₂ 0 (0.009 g), CaCl ₂ (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotin (1 pg), cobalamin (1 pg), /;-aminobenzoic acid (3 pg), folic acid (5 pg), and pyridoxamine

(15 pg).

Bacterial strains for use in vaccine compositions

The inventors have identified that the bacterial strains of the invention are useful for treating or preventing central nervous system disorders or conditions, in particular central nervous system disorders or conditions mediated by the microbiota-gut-brain axis. This is likely to be a result of the effect that the bacterial strains of the invention have on the host central, autonomic and/or enteric nervous system; the activity of the HPA pathway; the neuroimmune and neuroendocrine pathways; and the level of commensal metabolites in the host gastrointestinal tract and/or gastrointestinal permeability of the host. Therefore, the compositions of the invention may also be useful for preventing central nervous system disorders or conditions, in particular central nervous system disorders or conditions mediated by the microbiota-gut-brain axis, when administered as vaccine compositions. In certain such embodiments, the bacterial strains of the invention are viable. In certain such embodiments, the bacterial strains of the invention are capable of partially or totally colonising the intestine. In certain such embodiments, the bacterial strains of the invention are viable and capable of partially or totally colonising the intestine. In other certain such embodiments, the bacterial strains of the invention may be killed, inactivated or attenuated. In certain such embodiments, the compositions may comprise a vaccine adjuvant. In certain embodiments, the compositions are for administration via injection, such as via subcutaneous injection.

General

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references [50] and [51 ]-[57], etc.

The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y. The term “about” in relation to a numerical value x is optional and means, for example, x+10%.

In certain embodiments the term “modulate” means increase or activate. In alternative embodiments, the term “modulate” means decrease or suppress.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

References to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. [58] A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 5 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. [59]

Treatment or prevention may refer to, for example, an alleviation of the severity of symptoms or a reduction in the frequency of exacerbations or the range of triggers that are a problem for the subject or a prevention of relapse.

Unless specifically stated, a process or method comprising numerous steps may comprise additional steps at the beginning or end of the method, or may comprise additional intervening steps. Also, steps may be combined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will be appreciated that the features specified in each embodiment may be combined with other specified features, to provide further embodiments. In particular, embodiments highlighted herein as being suitable, typical or preferred may be combined with each other (except when they are mutually exclusive).

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Any reference to a method for treatment comprising administering an agent to a patient, also covers that agent for use in said method for treatment, as well as the use of the agent in said method for treatment, and the use of the agent in the manufacture of a medicament.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

MODES FOR CARRYING OUT THE INVENTION

Example 1- ex vivo screening for effects on the microbiota gut-brain axis

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by deficits in social behaviour, increased repetitive behaviour, anxiety and gastrointestinal symptoms. The aetiology of autism is complex and most likely involves both genetic and environmental factors[60]. Recent evidence suggests that the microorganisms resident to the gastrointestinal system play a fundamental role in regulating brain health status, behaviour and mood via the microbiota-gut-brain axis [61] Emerging preclinical and clinical studies have documented a potential role for gut bacteria in ASD, and consequently, the microbiota represents a potential target in the development of novel therapeutics for this neurodevelopmental disorder.

Additional behavioural complications include depression, anxiety, cognitive impairments and seizures in autistic individuals. Moreover, non-neurological symptoms such as gastrointestinal dysfunction are becoming increasingly recognised as a major comorbidity of this neurodevelopmental disorder. For instance, several studies have documented pronounced gastrointestinal distress such as abdominal pain, gaseousness, diarrhoea, flatulence and constipation in patients with ASD [62,63,64,65] . These gastrointestinal disturbances correspond with reports of alterations to the gut microbiota of individuals with ASD. For instance, several studies have documented an increased abundance of potentially pathogenic strains, such as Clostridia and Desulfovibrio, along with a reduction in the presence of beneficial genera such as Bifidobacteria [66,67,68,69] ;. In light of these pronounced alterations to the microbiota in ASD, emerging clinical evidence has shown that targeting the microbiota through prebiotics or microbiota transfer therapy can improve both the gastrointestinal and behavioural symptoms of children with autism [70,71] These preliminary studies suggest that targeting the microbiota may represent a novel therapeutic strategy for the treatment of autism.

BAFB/c mice display an inherent anxiety-like phenotype and are thus a useful model for screening the beneficial effects of live biotherapeutic strains on the gut-brain axis.

Example la - Materials and methods

Animals

BAFBc (Envigo, UK) adult male mice were group housed under a 12 h light-dark cycle; standard rodent chow and water were available ad libitum. All experiments were performed in accordance with European guidelines following approval by University College Cork Animal Ethics Experimentation Committee. Animals were 8 weeks old at the start of the experiment.

Dosins and strain

The strain used was Enterococcus durans bacterium deposited under accession number NCIMB 43456.

Biotherapeutic was provided in glycerol stock. Live biotherapeutics were grown in the facility in anaerobic conditions.

Animals were allowed to habituate to their holding room for one week after arrival into the animal unit. They received oral gavage (200pL dose) of strain NCIMB 43456 at a dose of 1 X 10 ⁹ CFU or PBS (vehicle control) for 6 consecutive days between 15:00 and 17:00. On day 7, the animals are decapitated and tissues are harvested for experimentation.

Tissue collection

Animals were sacrificed in a random fashion regarding treatment and testing condition; sampling occurred between 9.00 a.m. and 2:30 p.m. Trunk blood was collected in potassium EDTA (Ethylene Diamine Tetra Acetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolated and stored at -80 °C for further analysis. The brain was quickly excised, dissected and each brain region was snap-frozen on dry ice and stored at -80 °C for further analysis. Spleen was removed, collected in 5 mL RPMI media (with L-glutamine and sodium bicarbonate, R8758 Sigma + 10 % FBS (F7524, Sigma) + 1% Pen/Strep (P4333, Sigma)) and processed immediately after culls for ex-vivo immune stimulation. Intestinal tissue (2 cm segments of ileum and colon closest to the caecum were excised, and the furthest 1cm of tissue from the caecum were used) were mounted into the Ussing chambers for intestinal permeability assay. A further 1cm of ileum and colon tissue was taken for tight junction gene expression analysis. The caecum was removed, weighed and stored at -80 °C for SCFAs analysis.

Statistical Analysis

Normally distributed data are presented as mean ± SEM; non-parametric datasets are presented as median with inter-quartile range. Unpaired two-tailed t-test were applied to analyse parametric data and Mann-Whitney test was used for non-parametric. Spearman's rank correlation coefficient was employed for the correlation analysis in the pooled datasets. A p value < 0.05 was deemed significant in all cases.

Example lb — Tight junction protein mRNA expression

Method and rationale

Total RNA was extracted using the mirVana™ miRNA Isolation kit (Ambion/Llife technologies, Paisley, UK) and DNase treated (Turbo DNA-free, Ambion/life technologies) according to the manufacturers’ recommendations. RNA was quantified using NanoDrop™ spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) according to the manufacturer's instructions. RNA quality was assessed using the Agilent Bioanalyzer (Agilent, Stockport, UK) according to the manufacturer's procedure and an RNA integrity number (RIN) was calculated. RNA with RIN value >7 was used for subsequent experiments. RNA was reverse transcribed to cDNA using the Applied Biosystems High Capacity cDNA kit (Applied Biosystems, Warrington, UK) according to manufacturer's instructions. Briefly, Multiscribe Reverse Transcriptase (50 U/pL) (1)(2)(1)(10) was added as part of RT master mix, incubated for 25°C for 10 min, 37°C for 2 h, 85°C for 5 min and stored at 4°C. Quantitative PCR was carried out using probes (6 carboxy fluorescein - FAM) designed by Applied Biosystems to mouse specific targeted genes, while using b-actin as an endogenous control. Amplification reactions contained 1 mΐ cDNA, 5 mΐ of the 2X PCR Master mix (Roche), 900 nM of each primer and were brought to a total of 10 mΐ by the addition of RNase-free water. All reactions were performed in triplicate using 96-well plates on the LightCycler®480 System. Thermal cycling conditions were as recommended by the manufacturer (Roche) for 55 cycles. To check for amplicon contamination, each run contained no template controls in triplicate for each probe used. Cycle threshold (Ct) values were recorded. Data was normalized using b-actin and transformed using the 2-AACT method and presented as a fold change vs. control group.

Tight junction proteins including TJP1 and occludin help regulate the permeability of the gut epithelium. An increase in these genes would be a desirable trait. Indoleamine 2,3 dioxygenase- 1 (IDOl) is an enzyme that initiates tryptophan catabolism along a pathway that generates several bioactive kynurenine-based metabolites. Tryptophan hydroxylase-1 (TPH-1) catalyze the formation of 5-hydroxy-L-tryptophan (5-HTP) from L-tryptophan, the first and rate-limiting step in the biosynthesis of 5-HT. Thus the relationship between these genes is important in the regulation of tryptophan/kynurenine system.

Results

No difference was observed between strain NCIMB 43456-treated and vehicle-treated mice for occludin mRNA expression in the ileum or colon (Figure 2).

IDOl mRNA expression in the colon was increased in the strain NCIMB 43456-treated mice relative to the vehicle-treated mice (P=0.062) (Figure 3A). No difference was observed between strain NCIMB 43456-treated and vehicle-treated mice for IDOl mRNA expression in the ileum (Figure 3B).

TJP1 mRNA expression in the ileum was increased in the strain NCIMB 43456-treated mice relative to the vehicle-treated mice (Figure 4A). No difference was observed between strain NCIMB 43456-treated and vehicle-treated mice for TJP1 mRNA expression in the colon (Figure 4B).

Tphl mRNA expression in the ileum was increased in the strain NCIMB 43456-treated mice relative to the vehicle-treated mice (Figure 5A). No difference was observed between strain NCIMB 43456-treated and vehicle-treated mice for Tphl mRNA expression in the colon (Figure 5B).

Conclusions

Daily treatment with strain NCIMB 43456 leads to an increase in TJP1 and Tphl mRNA expression in the ileum, and IDOl mRNA expression in the colon. Chronic treatment with strain NCIMB 43456 can therefore be expected to improve regulation of permeability of the gut epithelium. Example lc- Short chain faty acid production

Method and rationale

Caecum content was mixed and vortexed with MilliQ water and incubated at room temperature for 10 min. Supernatants were obtained by centrifugation (10000 g, 5 min, 4 °C) to pellet bacteria and other solids and filtration by 0.2 pm. It was transferred to a clear GC vial and 2-Ethylbutyric acid (Sigma) was used as the internal standard. The concentration of short-chain fatty acids (SCFAs) was analyzed using a Varian 3500 GC flame-ionization system, fitted with a with a ZB-FFAP column (30 m x 0.32 mm x 0.25 mm; Phenomenex). A standard curve was built with different concentrations of a standard mix containing acetate, propionate, iso-butyrate, n-butyrate, isovalerate and valerate (Sigma). Peaks were integrated by using the Varian Star Chromatography Workstation version 6.0 software. All SCFA data are expressed as pmol/g.

SCFAs are microbial byproducts of dietary fibre. An increase in acetate, propionate, valerate, butyrate, isobutyrate or isovalerate suggest an increase in productivity of the microbiota and a desirable trait.

Results

Mice treated with strain NCIMB 43456 had significantly increased caecum levels of acetate and butyrate relative to vehicle-treated mice (Figure 6).

Conclusions

Treatment with strain NCIMB 43456 results in increased levels of acetate and butyrate in the caecum, suggesting increased productivity of the microbiota.

Example Id- Screening for effects of administration of live biotherapeutics on central gene expression

Methods and rationale

The mRNA levels for markers for the oxytocinergic system (oxytocin receptor and vasopressin receptor), endocrine system (mineralocorticoid (Nr3cl); glucocorticoid receptor (Nr3c2); corticosterone releasing hormone (CRH) and receptors; Brain derived neurotrophic factor (BDNF)), immune system (11-6); and neurotransmitter systems (NMDA receptor 2A (Grin2A); NMDA receptor 2B (Grin2B); GABAA receptor subunit A2; GABAB receptor subunit Bl; serotonin 2C) were assessed in the amygdala, hippocampus and prefrontal cortex - key brain regions of the limbic system involved in emotional response.

RNA was extracted and quantified as described in example lb above.

Results Expression of oxytocin receptor mRNA, CRH mRNA, IL-6 mRNA, Grin2a mRNA and GABA B1R in the hippocampus was significantly increased in strain NCIMB 43456-treated mice relative to vehicle-treated mice (Figure 7).

Expression of oxytocin receptor mRNA in the amygdala was also significantly increased in strain NCIMB 43456-treated mice relative to vehicle-treated mice (Figure 8).

Expression of BDNF mRNA in the prefrontal cortex was significantly increased in strain NCIMB 43456-treated mice relative to vehicle-treated mice (Figure 9).

Conclusions

Treatment with strain NCIMB 43456 results in increased mRNA expression for stress markers (CRH), immune proteins (IL-6), markers of the oxytocinergic system (oxytocin receptor) and neurotransmitters (Grin2A, GABA B1R, BDNF) in regions of the brain involved in emotional response.

These data suggest that treatment strain NCIMB 43456 can be used to treat diseases and conditions of the brain, for example, those which have a gastrointestinal component.

Overall conclusions for ex vivo screening experiments

The ex vivo screening data show that daily treatment with strain NCIMB 43456 led to an increase in productivity of the microbiota, demonstrated by increased production of SCFA microbial by products of dietary fibre. The treatment with strain NCIMB 43456 led to increased expression in the tight junction protein TJP1 in the ileum and a concomitant improvement in ileal permeability (reduced leakiness). Expression of catabolic enzymes IDOl and Tphl was increased in the colon and ileum respectively.

These changes in the gut microbiota were accompanied by increased mRNA expression for various neurological markers, suggesting that treatment with strain NCIMB 43456 will be effective for the treatment of CNS disorders with a gastrointestinal component, such as autism and Parkinson’s disease.

Example 2 - Assessing anxiety, depression, cognitive and social domains of behaviour in mouse models of autism spectrum disorders

The present study aimed to assess the effect of strain NCIMB 43456 treatment on psychiatric and neurological disorders in two different mouse models that display behavioural characteristics associated with neurodevelopmental and psychiatric disorders. In particular, the study focused on autistic-related behaviour in (i) C57BL/6 wt mouse model, (ii) a BTBR inbred, genetically modified mouse model and (iii) a maternal immune activation (MIA) mouse model. The effects of chronic strain NCIMB 43456 versus vehicle treatment across anxiety, depression, and cognitive and social domains of behaviour in the three mouse models were investigated. In addition, physiological and anatomical analyses were performed as well as analysis of neurotransmitter concentrations in brainstem tissue.

The BTBR mouse model uses inbred, genetically modified mice that display a robust autistic- like phenotype. Deficits in social behaviours, increased repetitive behaviours and increased anxiety-related behaviours have been reported in this strain [72] Due to this robust behavioural phenotype, the BTBR mouse is an ideal animal model to assess the efficacy of novel therapeutic agents for the treatment of autistic-related behaviours. Alleviation of such symptoms by a live biotherapeutic can also be indicative of efficacy of the biotherapeutic in the treatment of other psychiatric or neurological diseases.

The MIA mouse model uses an environmental immune challenge in pregnant mice in order to trigger the core symptoms of autism spectrum disorder in the offspring. MIA mice typically display stereotyped behaviour (as shown by the grooming and marble burying tests) and deficits in social communication (as shown by the social play, 3 -chamber social interaction, and social transmission of food preference tests). The offspring display the three core symptoms of autism (reduced communication; reduced sociability; and increased repetitive or stereotyped behaviour) and therefore provide a suitable model in which to determine whether administration of a therapeutic can alleviate the behavioural phenotypes associated with autistic spectrum disorders and indeed in a number of other neurological disorders. It is well established that alteration of behavioural phenotypes in animal models is indicative of a potentially clinically relevant intervention, irrespective of an understanding of the underlying biological or physiological mechanism [73]

The EMA Guidelines on the clinical development of medicinal products for the treatment of autism spectrum disorder state that, due to the heterogeneity of the diseases, it may not be possible to achieve a significant effect on all core symptoms with a single compound, and so short term efficacy has to be demonstrated on at least one core symptom. The strain NCIMB 43456 live biotherapeutic tested in the Examples has shown effective treatment of at least one core symptom of autistic spectrum disorder, so this strain and related Enterococcus strains are expected to be effective against human disease. Similarly, other central nervous system disorders or conditions display complex pathology with multiple different symptoms, and have few approved treatments. Therefore, it is understood that an effective treatment does not need to treat all symptoms of a central nervous system disorder or condition. A treatment would be considered therapeutically useful if it treated one of the symptoms associated with a central nervous system disorder or condition. However, as is demonstrated in the examples that follow, Enterococcus durans was advantageously able to elicit improvements in a range of symptoms.

Example 2a Materials and methods

Mice and dosins BTBR animals were bred in house with brother-sister mating. The male offspring from these animals were separated from their mothers at 3 weeks old and daily administration of the live biotherapeutic or control commenced at 8 weeks of age. Behaviour assessment started at 11 weeks old. A control age-matched C57BL/6 group was included as a reference control group.

For the maternal immune activation studies, female C57B/6 mice (8 weeks old) and age matched males were purchased from Harlan UK. After 1-week habituation these animals were mated. At embryonic day 12.5, females received either an injection of the viral mimetic poly-IC to activate the maternal immune system, or a saline vehicle injection. The male offspring from these animals were separated from their mothers at 3 weeks old and daily administration of the live biotherapeutic or control commenced at 8 weeks of age. Behaviour started at 11 weeks old.

For dosing, animals received daily oral gavage of PBS or the live biotherapeutics prepared to 10 ⁹ cfu/mL in PBS. Dosing continued daily throughout the behavioural paradigm.

All experiments were conducted in accordance with the European Directive 2010/63/EEC, the requirements of the S.I No 543 of 2012, and approved by the Animal Experimentation Ethics Committee of University College Cork.

Strain

The strain used was Enterococcus durans bacterium deposited under accession number NCIMB 43456.

Biotherapeutic was provided in glycerol stock. Live biotherapeutics were grown in the facility in anaerobic conditions.

Administration schedule

The treatment groups for the study are shown below. The vehicle for oral administration is PBS. Daily oral administration occurs via oral gavage. For the MIA and BTBR animals, once they reached 8 weeks they started getting a daily gavage of the live biotherapeutic of interest. This they got from 8 weeks old until they were culled. At week 11 (3 weeks later) the animals began undergoing behaviour testing. There were 10-12 animals per group. Both the MIA and BTBR were run over 2 cohorts each - each animal within each cohort underwent the exact same procedures in the exact same order for the same length of time. Animals were always randomised such that not all one group was run on one individual day.

Experimental design and methods

As outlined above, dosing with strain NCIMB 43456 commenced when the mice were 8 weeks old. The initial dosing took place for 3 weeks before the behavioural experiments. The behavioural battery occurred in the following order: marble burying and grooming tests at week 4; the elevated plus maze and three chamber tests at week 5; the open field and novel object recognition tests, and the in vivo intestinal motility assay at week 6; and the forced swim test at week 7. Finally, in week 8, the mice were culled and dissected for brain, proximal and distal colon, and spleen regions, along with plasma samples.

Graphical design and statistical analysis

Normally distributed data are presented as mean ± SEM; non-parametric datasets are presented as median with inter-quartile range. Unpaired two-tailed /-test were applied to analyse parametric data and Mann-Whitney test was used for non-parametric. Spearman's rank correlation coefficient was employed for the correlation analysis in the pooled datasets. A p value < 0.05 was deemed significant in all cases.

Example 2b - Assessing stereotype-related behaviour the marble burying test

Rationale

The marble burying test is a useful model of neophobia, anxiety and obsessive compulsive behaviour. It is also used to test novel antidepressants, anxiolytics and antipsychotics. Mice pre-treated with pharmacological agents such as anxiolytics show decreased marble burying behaviour, compared to the control mice. A higher number of marbles buried represents a heightened state of stereotype behaviour or higher levels of anxiety (neophobia).

Methods

Mice were individually placed in a novel plexiglas cage (35 ^c 28 ^c 18.5 cm, L ^c W ^c H), filled up with sawdust (5-10 cm) and 20 marbles on top of it (five rows or marbles regularly spaced 2 cm away from the walls and 2 cm apart). Thirty minutes later, the number of marbles buried for more than 2/3 of their surface was counted.

Results

The number of marbles buried during the test was reduced for strain NCIMB 43456-treated BTBR mice and strain NCIMB 43456-treated MIA mice relative to the respective vehicle-treated controls (Figure 10).

Conclusions

Chronic treatment with strain NCIMB 43456 reduces anxiety-like behaviour in BTBR and MIA mice in the marble burying test.

Example 2c -Assessing stereotype-related behaviour - the grooming test

Rationale

This test is used as an index for stereotyped and repetitive behaviour. An increase in time spent grooming is indicative of increased stereotyped or repetitive behaviour.

Methods

Self-grooming is evaluated in a 6.5 cm diameter ^c 10 cm tall, clear glass beaker covered with a filter top. Experimental animals will be brought to the test room to habituate up to 1 hour prior to the test. The test is approximately 20 minutes in duration. The investigator records the cumulative time spent by the test animal grooming. Between tests the beakers are cleaned thoroughly for next use.

Results

Treatment with strain NCIMB 43456 did not have a significant effect on the time spent grooming by BTBR or MIA mice (Figure 11).

Conclusions

Chronic treatment with strain NCIMB 43456 did not observably alter repetitive behaviours in BTBR or MIA mice in the grooming test.

Example 2d - Assessment of anxiety-like behaviours - the elevated plus maze

Rationale

The elevated plus maze (EPM) is a widely used test to assess anxiety-like behaviours in rodents. The EPM assesses general anxiety behaviour, with less anxious mice spending more time in the open arms of the maze. An increase in time or number of entries into an open arm is an index of reduced anxiety.

Methods

The set up was made of a grey plastic cross-shaped maze 1 m elevated from the floor, comprising two open (fearful) and two closed (safe) arms (50 ^c 5 ^c 15 cm walls or 1 cm no wall). Experiments occurred under red light (~5 lux). Mice were individually placed into the center of the maze facing an open arm (to avoid direct entrance into a closed one) and were allowed 5 -min free exploration. Experiments were videotaped using a ceiling camera for further parameters analysis using Ethovision software (3.1 version, Noldus, TrackSys, Nottingham, UK). The time spent in the open arms and the number of entries in each arm were measured (entrance in an arm was defined as all four paws inside the arm).

Results

Treatment with strain NCIMB 43456 led to a significant increase in the number of entries into the open arms of the maze (OA entries) for MIA mice relative to vehicle-treated mice (Figure 12). This increase corresponds to a full recovery of the deficit in OA entries for the vehicle- treated MIA mice relative to the wild type controls. Treatment with strain NCIMB 43456 did not have a significant effect on the time spent by MIA mice in the open arms of the maze (OA duration), nor did it have a significant effect on the OA entries or OA duration for BTBR mice (Figure 12).

Conclusions

Chronic treatment with strain NCIMB 43456 significantly reduces anxiety-like behaviour in MIA mice in the elevated plus maze. Chronic treatment with strain NCIMB 43456 has no effect on anxiety-like behaviour in BTBR mice in the elevated plus maze.

Example 2e - Assessment of anxiety-like behaviours the open field arena

Rationale

The open field arena is used to assess the response of exposure to a novel stressful environment and locomotor activity. Naive mice naturally spend most of their time alongside the walls of the arena, as it is less exposed than the centre of the arena. An increase in duration of time spent in the centre represents a decrease in anxiety-like behaviour.

Methods

To assess the response to a novel stressful environment and locomotor activity, mice were placed into open arena (40 ^c 32 ^c 23 cm, L ^c w x h) with ~60 lux lighting and allowed to explore for 10-mins. Experiments were videotaped using a ceiling camera for further parameter analysis using Ethovision software (3.1 version, Noldus, TrackSys, Nottingham, UK). The distance travelled and the latency to enter a virtual central zone (defined at 50% away from the edges) was scored.

Results

There was no significant difference in the total distance travelled or time spent in the centre of the area between strain NCIMB 43456-treated mice and the vehicle treated controls (Figure 13). Conclusions

Chronic treatment with strain NCIMB 43456 did not observably alter anxiety-like behaviour in BTBR or MIA mice in the open field arena test.

Example 2f - Assessment of social behaviours - the three-chamber social interaction test

Rationale

The 3-Chamber Social Interaction Test (3-CSIT) is a well validated ethologically relevant model that assesses social interaction between sex-matched conspecifics and allows for readouts of social novelty and social preference in mice. The test allows mice to freely explore between an inanimate object or sex-matched conspecific mice. Control animals naturally are more interested in a conspecific mouse more than an inanimate object (sociability). Similarly, control animals spend more time interacting with a novel unfamiliar mouse then one they have already had interactions with.

Methods

The social testing apparatus was a rectangle, three-chambered box. Each chamber was 20 cm L x 40 cm W x 22 cm H. Dividing walls were made with small circular openings (5 cm in diameter) allowing access into each chamber. Two identical wire cup-like cages, with a bottom diameter of 10 cm, 13 cm in height and bars spaced 1.2 cm, allowing nose contact between the bars, but prevented fighting, were placed inside each side chamber in bilaterally symmetric positions. The test has three phases of 10 min each: 1) habitation 2) mouse versus object 3) novel mouse versus familiar mouse. Experiments were videotaped using a ceiling camera for further parameters analysis using Ethovision software (3.1 version, Noldus, TrackSys, Nottingham, UK). For the first phase the test mouse was placed into the middle chamber and allowed to explore the entire box with empty small wire cages inside for a 10-min habituation session. After the habituation period, the test mouse is removed from the testing box for a short interval while an object is placed in one side chamber and an unfamiliar conspecific male mouse (no prior contact with the test subject) in the other side chamber, both enclosed in a wire cup-like cage. During phase two, the test mouse is placed in the middle chamber and allowed to explore the entire box for 10 min. The amount of time spent exploring the object or mouse in each chamber and the number of entries into each chamber were evaluated. The location of the unfamiliar mouse in the left vs right side chamber was systematically alternated between trials. An entry was defined as all four paws in one chamber. During the third phase an object was replaced with an unfamiliar mouse serving as a novel mouse and in the other chamber the mouse used in phase two was kept the same, now serving as familiar mouse. After every trial, all chambers and cup-like wire cages were cleaned with 10% ethanol, dried and ventilated for a few minutes to prevent olfactory cue bias and to ensure proper disinfection. Lack of innate side preference was confirmed during the initial 10 min of habituation to the entire arena.

Results

For both BTBR and MIA mice, treatment with strain NCIMB 43456 led to an increase in the time spent with an unfamiliar mouse relative to an inanimate object, and with an unfamiliar mouse relative to a familiar mouse. In each case, treatment with strain NCIMB 43456 led to a complete recovery of the wild type behavioural phenotype (Figure 14).

Conclusions

Chronic treatment with strain NCIMB 43456 increases social novelty and social cognition of BTBR mice and MIA mice in the three chamber test.

Example 2g — Assessment of depression-like behaviour - the novel object recognition test

Rationale

The novel object recognition test is used to test recognition memory and learning. Improved memory is a reflection of reduced depression-like behaviour. Control animals will discriminate between an object they have had time to explore and a new object.

Methods

Mice were placed in the middle of a grey plastic rectangular box (40 ^c 32 ^c 23 cm, L ^c W ^c H) under a dimly light, 60 lux at the level of the arena, for 10 min. 24 h after mice were placed in the box with the two identical objects for a total time of 10 min (acquisition phase). After a 24 h, one of the two identical objects were substituted with a novel object and mice were placed in the middle of the box at the mid-point of the wall opposite the sample objects for a total time of 10 min (retention phase). Animals were acclimatized to the testing room for 30 min prior each experiment. Box and objects were cleaned with alcohol 10% to avoid any cue smell between each trial. Experiments were videotaped using a ceiling camera for further parameter analysis. Directed contacts with the objects, include any contact with mouth, nose or paw or minimal defined distance were counted as an interaction.

Results

Vehicle-treated BTBR mice spent more time interacting with the familiar object than with the novel object. Treatment with strain NCIMB 43456 did not have an observable effect on the time spent interacting with a novel object versus time spent interacting with a familiar object in BTBR mice (Figure 15 A).

Vehicle-treated MIA mice also spent more time interacting with the familiar object than with the novel object. However, in strain NCIMB 43456-treated MIA mice this trend was reversed: strain NCIMB 43456-treated MIA mice spent more time interacting with the novel object (Figure 15B).

Conclusions

Chronic treatment with strain NCIMB 43456 led to a reduction of depression-like behaviour in MIA mice in the novel object recognition test. Chronic treatment with strain NCIMB 43456 has no effect on cognitive behaviour for BTBR mice in the novel object recognition test.

Example 2h - Assessment of depression-like behaviour the forced swim test

Rationale

The forced swim test (FST) is the most widely used experimental paradigm to assess antidepressant activity. Naive animals will display escape behaviour in the form of swimming, climbing and diving before adapting an immobile floating posture. The duration of immobility is indicative of behavioural despair. An increase in immobility means an increase in depressive- like behaviour as the animal has resigned itself to its situation (learned helplessness). Antidepressant drugs decrease the time spent immobile in this test.

Methods

Mice were individually placed in a clear glass cylinder (24 ^c 21 cm diameter), containing 15-cm- depth water (25 ± 0.5 °C). The water was changed between each animal. The test lasted 6 min and experiments were videotaped using a numeric tripod-fixed camera; data were further scored twice using the videos (Video Media Player software) and averaged by an experimenter blind to conditions. The latency to immobility was scored. The time of immobility (s) was measured for the last 4 min of the test, with immobility being defined as a total absence of movement except slight motions to maintain the head above the water.

Results

For both BTBR and MIA mice, treatment with strain NCIMB 43456 led to a significant decrease in immobility time relative to vehicle-treated mice (Figure 16).

Conclusions

Chronic treatment with strain NCIMB 43456 results in a reduction in depression-like behaviour for both BTBR mice and MIA mice in the forced swimming test. Example 2i in vivo intestinal motility assay

Rationale

The MIA model has been reported to lead to changes in gut barrier function. Therefore, it was important to ascertain whether chronic treatment with the biotherapeutic alters intestinal motility.

Methods

This procedure involves the oral administration of a given amount of a non-toxic, coloured marker (Carmin Red) to determine motility of the gut. Time to excretion of the first coloured faecal bolus is recorded as ‘time of whole gut transit’ and is used as an index of peristaltic motility in the whole intestine. Mice are single housed for 3h prior to the assay to allow habituation to a new cage. Carmin red dye (100-200 ul of 6% Carmin red in 0.5% methylcellulose per mouse) is given orally by gavage. Each cage is visually inspected every 10 min. The time of the first coloured bolus (red) is recorded. Following the appearance of the first coloured bolus mice are returned to normal housing conditions.

Results

Treatment with strain NCIMB 43456 did not lead to a significant difference in intestinal motility time relative to vehicle-treated controls for BTBR or MIA mice (Figure 17).

Conclusions

Chronic treatment with strain NCIMB 43456 did not affect intestinal motility compared to the control or vehicle groups for BTBR mice or MIA mice.

Example 2j ex vivo gastrointestinal permeability assay

Rationale

MIA mice exhibit increases in intestinal permeability which corresponded with reduced expression of tight junction proteins and inflammation in the colon [74]

Methods

The permeability of the ileum and colon was assessed ex vivo using Ussing chambers. An increase in FITC concentration represents an undesired effect as it indicates an increase in the ‘leakiness’ of the intestinal barrier.

Mice were euthanized by cervical dislocation, and the distal ileum and colon were removed, placed in chilled Krebs solution opened along the mesenteric line and carefully rinsed. Preparations were then placed in Ussing chambers (Harvard Apparatus, Kent, UK, exposed area of 0.12 cm ² ) as described previously [75] with oxygenated (95% 02, 5% CO2) Krebs buffer maintained at 37°C. 4 kDa FITC-dextran was added to the mucosal chamber at a final concentration of 2.5 mg/mL; 200 pL samples were collected from the serosal chamber every 30 min for the following 3 h.

Results

A significant reduction in in FITC concentration in the serosal chamber was observed for ileum preparations from strain NCIMB 43456-treated MIA mice relative to ileum preparations of vehicle-treated MIA mice. No significant difference was observed for colon preparations from strain NCIMB 43456-treated MIA mice versus vehicle-treated MIA mice, and no significant difference was observed for colon or ileum preparations from strain NCIMB 43456-treated BTBR mice versus vehicle-treated BTBR mice (Figure 18).

Conclusions

Chronic treatment with strain NCIMB 43456 reduced leakiness of the ileal intestinal barrier in MIA mice. Chronic treatment with strain NCIMB 43456 did not affect colonic intestinal barrier permeability in MIA mice, and did not affect colonic or ileal intestinal barrier permeability in BTBR mice.

Example 2k Weight monitoring

Animal body weights were assessed weekly throughout the study to determine whether chronic treatment with strain NCIMB 43456 influences this particular parameter.

Conclusions

Chronic treatment with strain NCIMB 43456 does not influence body weight in BTBR mice or MIA mice (Figure 19).

Example 21 Organ weight and colon length

Methods

Animals were sacrificed in a random fashion regarding treatment and testing condition; sampling occurred between 9.00 a.m. and 2:30 p.m. Trunk blood was collected in potassium EDTA (Ethylene Diamine Tetra Acetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolated and stored at -80 °C for further analysis. The brain was quickly excised, dissected and each brain region was snap-frozen on dry ice and stored at -80 °C for further analysis. Spleen was removed, collected in 5 mL RPMI media (with L-glutamine and sodium bicarbonate, R8758 Sigma + 10 % FBS (F7524, Sigma) + 1% Pen/Strep (P4333, Sigma)) and processed immediately after culls for ex-vivo immune stimulation. Intestinal tissue (2 cm segments of ileum and colon closest to the caecum were excised, and the furthest 1cm of tissue from the caecum were used) were mounted into the Ussing chambers for intestinal permeability assay (see Example 2j above). A further 1cm of ileum and colon tissue was taken for tight junction gene expression analysis. The caecum was removed, weighed and stored at -80 °C for SCFAs analysis. Results

For MIA mice, treatment with strain NCIMB 43456 did not result in a significant difference in colon length, caecum weight/body weight or spleen weight/body weight relative to vehicle- treated controls.

For BTBR mice, treatment with strain NCIMB 43456 did not result in a significant difference in colon length or spleen weight/body weight relative to vehicle-treated controls. Caecum weight/body weight was increased for strain NCIMB 43456-treated BTBR mice relative to vehicle-treated BTBR mice (p=0.06) (Figure 20).

Conclusions

Treatment with strain NCIMB 43456 did not affect colon length or spleen weight/body weight for BTBR or MIA mice. Treatment with strain NCIMB 43456 resulted in a non-significant increase in caecum weight/body weight in BTBR mice, but did not have an effect on caecum weight/body weight in MIA mice.

Example 2m Brainstem monoamine levels

Rationale

Serotonin and noradrenaline levels, and 5-HIAA/5-HT turnover were assessed in the brainstem. The brainstem encompasses a number of cell bodies for all the major neurotransmitter systems of the brain including serotonin and noradrenaline. 5-HIAA is the main metabolite of serotonin (5HT). Elevated 5HT and 5-HIAA levels and reduced 5-HIAA/5HT turnover were found in brainstem samples from suicides relative to those from non-psychiatric, sudden death controls, and may be linked to major depressive disorder (MDD) [76]

Methods

Neurotransmitter concentration was analysed by HPLC on samples from the brainstem. Briefly, brainstem tissue was sonicated in 500 mΐ of chilled mobile phase spiked with 4 ng/40 mΐ of N- Methyl 5-HT (Sigma Chemical Co., UK) as internal standard. The mobile phase contained 0.1 M citric acid, 5.6 mM octane- 1-sulphonic acid (Sigma), 0.1 M sodium dihydrogen phosphate, 0.01 mM EDTA (Alkem/Reagecon, Cork) and 9% (v/v) methanol (Alkem/Reagecon), and was adjusted to pH 2.8 using 4 N sodium hydroxide (Alkem/Reagecon). Homogenates were then centrifuged for 15 min at 22,000 ^c g at 4 °C and 40 mΐ of the supernatant injected onto the HPLC system which consisted of a SCL 10-Avp system controller, LECD 6A electrochemical detector (Shimadzu), a LC-10AS pump, a CTO-IOA oven, a SIL-10A autoinjector (with sample cooler maintained at 40 C) and an online Gastorr Degasser (ISS, UK). A reverse-phase column (Kinetex 2.6 u Cl 8 100 x 4.6 mm, Phenomenex) maintained at 30 °C was employed in the separation (Flow rate 0.9 ml/min). The glassy carbon working electrode combined with an Ag/AgCl reference electrode (Shimdazu) operated a +0.8 V and the chromatograms generated were analyzed using Class-VP 5 software (Shimadzu). The neurotransmitters were identified by their characteristic retention times as determined by standard injections, which run at regular intervals during the sample analysis. The ratios of peak heights of analyte versus internal standard were measured and compared with standard injection. Results were expressed as ng of neurotransmitter per g fresh weight of tissue.

Results

The vehicle-treated BTBR mice had significantly reduced brainstem noradrenaline levels and 5- HIAA/5HT turnover relative to wild type C57BL/6 controls. Treatment of BTBR mice with strain NCIMB 43456 resulted in a significant increase in 5-HIAA/5HT turnover and a significant decrease in serotonin (5HT) in the brainstem relative to vehicle-treated BTBR mice. Levels of noradrenaline were also increased in strain NCIMB 43456-treated BTBR mice relative to vehicle-treated controls (p=0.061) (Figure 21).

The vehicle-treated MIA mice had significantly elevated brainstem 5HT levels and significantly reduced 5-HIAA/5HT turnover relative to wild type C57BL/6 controls. Treatment of MIA mice with strain NCIMB 43456 also led to a significant decrease in brainstem 5HT levels relative to vehicle-treated controls, but did not affect brainstem noradrenaline levels or 5-HIAA/5HT turnover (Figure 21).

Conclusions

Chronic treatment with strain NCIMB 43456 resulted in a reduction in 5HT levels in both BTBR and MIA mice and an increase in 5-HIAA/5HT turnover, indicating that strain NCIMB 43456 may be effective in the treatment of MDD. Chronic treatment with strain NCIMB 43456 also led to an increase in noradrenaline levels in BTBR mice, but did not affect noradrenaline levels or 5- HIAA/5HT turnover in MIA mice.

Example 2n - Amygdalar gene expression

Methods

Total RNA was extracted using the mirVana™ miRNA Isolation kit (Ambion/Llife technologies, Paisley, UK) and DNase treated (Turbo DNA-free, Ambion/life technologies) according to the manufacturers recommendations. RNA was quantified using NanoDrop™ spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) according to the manufacturer's instructions. RNA quality was assessed using the Agilent Bioanalyzer (Agilent, Stockport, UK) according to the manufacturer's procedure and an RNA integrity number (RIN) was calculated. RNA with RIN value >7 was used for subsequent experiments. RNA was reverse transcribed to cDNA using the Applied Biosystems High Capacity cDNA kit (Applied Biosystems, Warrington, UK) according to manufacturer's instructions. Briefly, Multiscribe Reverse Transcriptase (50 U/pL) (1)(2)(1)(10) was added as part of RT master mix, incubated for 25°C for 10 min, 37°C for 2 h, 85°C for 5 min and stored at 4°C. Quantitative PCR was carried out using probes (6 carboxy fluorescein - FAM) designed by Applied Biosystems to mouse specific targeted genes, while using b-actin as an endogenous control. Amplification reactions contained 1 mΐ cDNA, 5 mΐ of the 2X PCR Master mix (Roche), 900 nM of each primer and were brought to a total of 10 mΐ by the addition of RNase-free water. All reactions were performed in triplicate using 96-well plates on the LightCycler®480 System. Thermal cycling conditions were as recommended by the manufacturer (Roche) for 55 cycles. To check for amplicon contamination, each run contained no template controls in triplicate for each probe used. Cycle threshold (Ct) values were recorded. Data was normalized using b-actin and transformed using the 2-AACT method and presented as a fold change vs. control group. mRNA levels for markers for the oxytocinergic system (oxytocin receptor), endocrine system (mineralocorticoid (Nr3cl); glucocorticoid receptor (Nr3c2); corticosterone releasing factor (CRF) and receptors; Brain derived neurotrophic factor (BDNF)); and neurotransmitter systems (NMDA receptor 2A (Grin2A); NMDA receptor 2B (Grin2B); GABAA receptor subunit A2; GABAB receptor subunit Bl; serotonin 1A receptor (5HT1AR)) were assessed in the amygdala.

RNA was extracted and quantified as described in example lb above.

Results

In MIA mice, chronic treatment with strain NCIMB 43456 led to a significant increase in oxtocin receptor, Grin2A and GABA A2 mRNA expression relative to vehicle-treated controls. No significant difference was observed in amygdalar expression for any other of the mRNAs tested (Figure 22).

In BTBR mice, chronic treatment with strain NCIMB 43456 led to a significant decrease in GABA A2 mRNA expression relative to vehicle-treated controls. No significant difference was observed in amygdalar expression for any other of the mRNAs tested (Figure 22).

Conclusions

Treatment with strain NCIMB 43456 resulted in increased amygdala mRNA expression for oxytocin receptor and the neurotransmitters, Grin2A GABA A2 for BTBR mice, but resulted in decreased amygdala GABA A2 mRNA expression in MIA mice.

Overall conclusions regarding strain NCIMB 43456 in the treatment of autistic spectrum disorders

Chronic treatment with strain NCIMB 43456 led to a reduction in the number of marbles buried during the marble burying test in BTBR and MIA mice, but did not affect the time spent grooming. Treatment with strain NCIMB 43456 also led to a significant increase in the number of entries made into the open arms of the maze by MIA mice in the elevated plus maze test, but did not have an effect on time spent in the open arms of the maze for MIA mice, and did not affect the behaviour of BTBR mice in the elevated plus maze test. The ability of strain NCIMB 43456 to improve anxiety related behaviours in mouse models of ASD is promising and indicates that it may be an effective therapeutic.

In both BTBR and MIA mice, chronic treatment with strain NCIMB 43456 led to a complete recovery of wild type social behaviour phenotypes in the three-chamber social interaction test. This strongly suggests that strain NCIMB 43456 treatment may be effective in the treatment of diseases characterised by impaired social behaviour.

Chronic treatment with strain NCIMB 43456 led to a reduction in depression-like behaviour for both BTBR and MIA mice in the forced swim test. Treatment with strain NCIMB 43456 also led to a reduction of depression-like behaviour in MIA mice in the novel object recognition test, but did not significantly affect the behaviour of BTBR mice in this test. This is concomitant with an increase in brainstem 5-HIAA/5HT turnover following strain NCIMB 43456 treatment in BTBR mice, and a decrease in brainstem serotonin levels following strain NCIMB 43456 treatment in both BTBR and MIA mice, i.e. an improvement in markers that may be linked with major depressive disorder. These data strongly suggest that strain NCIMB 43456 treatment may be effective in the treatment of depression and depressive disorders.

Chronic treatment with strain NCIMB 43456 did not significantly affect the majority of the physiological parameters measured. Treatment with strain NCIMB 43456 did not affect the weight of BTBR or MIA mice. Intestinal motility was also unaffected, as was colon length and spleen weight/body weight. Caecum weight/body weight was unaffected by strain NCIMB 43456 treatment in MIA mice, but was non-significantly increased in BTBR mice. A reduction in ileum permeability (i.e. reduced leakiness) was observed following strain NCIMB 43456 treatment in MIA mice, but no effect was observed for colon permeability. Treatment with strain NCIMB 43456 did not affect intestinal barrier permeability in BTBR mice.

The EMA Guidelines on the clinical development of medicinal products for the treatment of autism spectrum disorder state that, due to the heterogeneity of the diseases, it may not be possible to achieve a significant effect on all core symptoms with a single compound, and so short term efficacy has to be demonstrated on at least one core symptom. The strain NCIMB 43456 live biotherapeutic has shown effective treatment of at least one core symptom or autistic spectrum disorder, so it and related Enterococcus durans strains are expected to be effective against human disease.

SEQUENCES

SED ID NO:l - Enterococcus durans type strain 98D, 16S rRNA gene, GenBank accession NR_036922

1 tcctggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgta cgcttctttt 61 tccaccggag cttgctccac cggaaaaaga agagtggcga acgggtgagt aacacgtggg

121 taacctgccc atcagaaggg gataacactt ggaaacaggt gctaataccg tataacaatc

181 gaaaccgcat ggttttgatt tgaaaggcgc tttcgggtgt cgctgatgga tggacccgcg

241 gtgcattagc tagttggtga ggtaacggct caccaaggcc acgatgcata gccgacctga

301 gagggtgatc ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt

361 agggaatctt cggcaatgga cgaaagtctg accgagcaac gccgcgtgag tgaagaaggt

421 tttcggatcg taaaactctg ttgttagaga agaacaagga tgagagtaac tgttcatccc

481 ttgacggtat ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt

541 aggtggcaag cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg tttcttaagt

601 ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactggga gacttgagtg

661 cagaagagga gagtggaatt ccatgtgtag cggtgaaatg cgtagatata tggaggaaca

721 ccagtggcga aggcggctct ctggtctgta actgacgctg aggctcgaaa gcgtggggag

781 caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttggag

841 ggtttccgcc cttcagtgct gcagctaacg cattaagcac tccgcctggg gagtacgacc

901 gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt

961 aattcgaagc aacgcgaaga accttaccag gtcttgacat cctttgacca ctctagagat

1021 agagcttccc cttcgggggc aaagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg

1081 tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta ttgttagttg ccatcattta

1141 gttgggcact ctagcaagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa

1201 tcatcatgcc ccttatgacc tgggctacac acgtgctaca atgggaagta caacgagtcg

1261 cgaagtcgcg aggctaagct aatctcttaa agcttctctc agttcggatt gtaggctgca

1321 actcgcctac atgaagccgg aatcgctagt aatcgcggat cagcacgccg cggtgaatac

1381 gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg

1441 gtgaggtaac cttttggagc cagccgccta aggtgggata gatgattggg gtgaagtcgt

1501 aacaaggtag ccgtatcgga aggtgcggct ggat

SED ID NO:2 - 16srRNA sequence for Enterococcus durans strain NCIMB 43456 assembled from 2 reads using Geneious

AAAAGGCGGCTGGCTCCAAAGGTTACCTCAMCGACTTCGGGTGTTACAAACTCTCG

TGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTG

ATCCGCGATTACTAGCGATTCCGGCTTCATGTAGGCGAGTTGCAGCCTACAATCCG

AACTGAGAGAAGCTTTAAGAGATTAGCTTAGCCTCGCGACTTCGCGACTCGTTGTA

CTTCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGT

CATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTRAAT

GATGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCAC

GACACGAGCTGACGACAACCATGCACCACCTGTCACTTTGCCCCCGAAGGGGAAG

CTCTATCTCTAGAGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGC

TTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGT

TTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCAC

TGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCGTGGACTACCA

GGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACC

AGAGAGCCGCCTTCGCCACTGGTGTTCCTCCATATATCTACGCATTTCACCGCTACA

CATGGAATTCCACTCTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCC CCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCTCGCTTTA CGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCAC GTAGTTAGCCGTGGCTTTCTGGTTAGATACCGTCAAGGGATGAACAGTTACTCTCA TCCTTGTTCTTCTCTAACAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGC GGCGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGCCTCCCG TAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTCGGC TATGCATCGTGGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCACCGCGGG TCCATCCATCAGCGACACCCGAAAGCGCCTTTCAAATCAAAACCATGCGGTTTCGA TTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTTCTGATGGGCAGGTT ACCCACGTGTTACTCACCCGTTCGCCACTCTTCTTTTTCCGGTGGAGCAAGCTCCGG

T GAA A AAGA AGCGT ACGACTT GC AT GT AT

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