Treatment of autism spectrum disorder and symptoms associated with autism spectrum disorder by probiotic supplementation in subjects with a diagnosis of autism spectrum disorder

Abstract

The present invention refers to the use of a composition comprising live Lactobacillus acidophilus, live Lacticaseibacillus paracasei and live Lacticaseibacillus rhamnosus in the treatment of Autism spectrum disorder (ASD) and in the treatment of multiple symptoms associated with ASD, selected from impaired communication, impaired sociability, impaired sensory awareness, impaired cognitive awareness and behaviors, gastrointestinal disorders or sleep disorders. The composition of the present invention is a nutritionally acceptable composition comprising the combination of live Lactobacillus Acidophilus, live Lacticaseibacillus paracasei and live Lacticaseibacillus rhamnosus selected from a group comprising a drinkable solution, a capsule, a tablet, a lozenge, gummies, powders, sachets, stick packs, fermented beverages, juices or yogurt.

Description

[0001] - 1 - P2771PC TITLE ”Treatment of autism spectrum disorder and symptoms associated with autism spectrum disorder by probiotic supplementation in subjects with a diagnosis of autism spectrum disorder” 5 Abstract The present invention refers to the use of a composition comprising live Lactobacillus acidophilus, live Lacticaseibacillus paracasei and live Lacticaseibacillus rhamnosus in the treatment of Autism spectrum disorder (ASD) 10 and in the treatment of multiple symptoms associated with ASD, selected from impaired communication, impaired sociability, impaired sensory awareness, impaired cognitive awareness and behaviors, gastrointestinal disorders or sleep disorders. The composition of the present invention is a nutritionally acceptable composition 15 comprising the combination of live Lactobacillus acidophilus, live Lacticaseibacillus paracasei and live Lacticaseibacillus rhamnosus selected from a group comprising a drinkable solution, a capsule, a tablet, a lozenge, gummies, powders, sachets, stick packs, , fermented beverages, juices or yogurt. 20 Background Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by stereotyped behavior and deficits in communication and social interaction. Worldwide, the prevalence of ASD is estimated to be between 1% and 2%. In Canada, 1 in 50 (2%) of children and youth aged 1 to 17 years were 25 diagnosed with ASD according to a report based on data from the 2019 Canadian Health Survey on Children and Youth. The etiology of ASD is probably due to multifactorial causes but has yet to be elucidated. Studies suggest a complex interaction between genetic, epigenetic, and environmental factors including a possible role for gut microbiota. Clinical features of ASD include the impairment - 2 - P2771PC of communication abilities and social development, the presence of repetitive / restrictive behaviors, language delay, learning disabilities and challenges with social interactions. Other symptoms are altered executive functions and organizational skills, hyperactivity, psychological comorbidities such as attention- 5 deficit hyperactivity disorder (ADHD), obsessive-compulsive disorder and anxiety, epilepsy and intellectual disability. In addition to the typical behavioral characteristics, symptoms which are associated with ASD are sleep disorders with subjects experiencing sleep duration issues, parasomnias, sleep-disordered breathing, daytime sleepiness, sleep disturbances 10 and circadian sleep alterations, including sleep resistance, prolonged sleep onset, long or frequent nocturnal awakenings and early morning awakenings. This can have negative consequences on the quality of life of children and parents, and could alter brain development and proper functioning. Of note, gastrointestinal (GI) disorders such as abdominal pain, diarrhea, 15 constipation, and bloating are symptoms which are associated with ASD. Gut dysbiosis is a symptom associated to ASD and its symptoms, suggesting a bidirectional communication through the microbiota-gut-brain axis. Nowdays no medication can improve the core signs of autism spectrum disorder and no medication can be useful for all the symptoms associated to ASD, but 20 specific medications can help to control some of the symptoms. For example, certain medications may be prescribed if the patient, in particular a child, is hyperactive; furthermore antipsychotic drugs are sometimes used to treat severe behavioral problems; and antidepressants may be prescribed for anxiety. Tipically when the patient is a child, the goal of known treatments is to maximize 25 the his / her ability to function by reducing autism spectrum disorder symptoms and supporting development and learning. Early intervention during the preschool years can help the child learn critical social, communication, functional and behavioral skills. However, till now there is no treatment available that is able to simultaneously - 3 - P2771PC reduce the severity of all the multiple symptoms associated with ASD, such as impaired communication, impaired sociability, impaired sensory awareness, impaired cognitive awareness and behaviors, gastrointestinal disorders or sleep disorders. 5 There is therefore the need of an effective treatment that can be used to treat a patient affected by ASD and to reduce the severity of the multiple symptoms associated with ASD in adult and infant patients. Detailed Description of the Disclosure 10 The inventors have now surprisingly found that a drinkable solution, such as Bio- K+, that is a commercially available probiotic containing a combination of three stains, Lactobacillus acidophilus CL1285, Lacticaseibacillus paracasei LBC80R (previously named Lactobacillus casei before the signifficant bacterial taxonomy reclassification of 2020), and Lacticaseibacillus rhamnosus CLR2 (previously 15 named Lactobacillus rhamnosus before the bacterial taxonomy reclassification of 2020), was effective in the treatment of Autism Spectrum Disorder (ASD) and in the treatment of multiple sympoms related to ASD, in particular when the subject diagnosed with ASD is a child. As reported in the experimental data, the inventors have also demonstrated that the 20 combination of the three Lactobacillaceae strains of the present invention is able to inhibit the growth of Clostridioides difficile and that it also affects the production of their metabolites such as p-cresol, in particular they induce a decrease of the level of p-cresol, that is a metabolite that has been linked to autism. Products comprising a formulation of L. acidophilus CL1285, L. paracasei 25 LBC80R, and L. rhamnosus CLR2 strains have never shown or suggested to be effective in the treatment of Autism Spectrum Disorder (ASD) and in the treatment of the multiple sympoms associated with ASD. Surprisingly, the findings of the study disclosed in the experimental section confirm that the consumption of the composition according to the present invention by - 4 - P2771PC children diagnosed with ASD improved the symptoms associated with ASD, including GI symptoms and symptoms of sleep disorders. Notably, the obtained results clearly demonstrate that the capacity of the claimed composition to improve the quality of life of study participants, including 5 improving dysphoria, health anxiety, food avoidance disorder and social interaction, is mediated by restoration of gut microbiota. The inventors have further observed a beneficial impact of the probiotic supplementation comprising live L. acidophilus, live L. paracasei, and live L. rhamnosus, on the total Autism Treatment Evaluation Checklist (ATEC) score, its 10 4 sub-categories and on the GSI score in comparison with other studies evaluating the impact of probiotics on ATEC and GSI scores. As it can be observed from Table 4, the reduction of the ATEC scores following probiotics supplementation range from 7.5% to 32.4%. Clearly, the 42.8% reduction observed in the present study using live L. acidophilus, live L. paracasei, and live L. rhamnosus, indicates 15 a superior impact of the claimed composition on ASD symptoms over the prior art. The study data disclosed in the experimental section therefore clearly shows the efficacy of the composition of the invention in improving behavioural symptoms in children with ASD and in improving GI and sleep disorders associated with ASD. An embodiment of the present invention is therefore a composition comprising a 20 combination of live Lactobacillus acidophilus (L. acidophilus), live Lacticaseibacillus paracasei (L. paracasei) and live Lacticaseibacillus rhamnosus (L. rhamnosus), for use in the treatment of ASD and in the treatment of multiple symptoms associated with ASD. According to a preferred embodiment, said composition further comprises some 25 additional food additives, selected from flavour based ingriedients or additional protein, preferably said additional protein is a plant based protein source, more preferably is a pea based protein comprising pea isoleat. Preferably the plant based protein is vegan and gluten free. This is beneficial for subjects with ASD as it improves GI issues, by being non - 5 - P2771PC dairy and non gluten which inturn reduces inflammation in the gastroinsestional tract. According to an embodiment of the present invention said composition consists of a combination of live L. acidophilus, live L. paracasei and live L. rhamnosus. 5 Preferably said treatment is configured to decrease an Autism Treatment evaluation checklist score of a subject with ASD. According to a preferred embodiment, said Lactobacillus acidophilus is Lactobacillus acidophilus CL1285® deposited at the National Collection of Microorganisms Cultures (CNCM) in Paris (deposit No. CNCM I-40991), said 10 Lacticaseibacillus paracasei is Lacticaseibacillus paracasei LBC80R® deposited at the CNCM (deposit No. CNCM I-39892), and said Lacticaseibacillus rhamnosus is Lacticaseibacillus rhamnosus CLR2® deposited at the CNCM (deposit No. CNCM I-39903). According to a further preferred embodiment said multiple symptoms of ASD are 15 simultaneously treated. Preferably said symptoms include impaired communication, impaired sociability, impaired sensory awareness, impaired cognitive awareness and behaviors, gastrointestinal disorders or sleep disorders. Preferably said gastrointestinal disorders are selected from a group comprising 20 abdominal pain, abdominal discomfort, bloating, constipation, slow bowel movement, diarrhoea flatulence, and smelly stool. Preferably said sleep disorders are selected from a group comprising sleep duration, parasomnias, sleep disordered breathing and daytime sleepiness. According to a preferred embodiment the composition for use according to the 25 present invention is a nutritionally acceptable composition selected from a group comprising a drinkable solution, a capsule, a tablet, a lozenge, gummies, sachets, 1 Depositor: Mr. Francois M. Luquet. Rue Aristide Briand- 91400 Orsei (France) 2 Depositor: Bio-K Plus International Inc.495 Ball- Armand-Frappier. Laval (Quebec). Canada 3 Depositor: Bio-K Plus International Inc.495 Ball- Armand-Frappier. Laval (Quebec). Canada - 6 - P2771PC stick packs or powders, preferably said drinkable solution is selected from the group comprising fermented beverages, juices or yogurt, more preferably said nutritionally acceptable composition is a probiotic drinkable solution comprising the combination of live L. acidophilus, live L. paracasei and live L. rhamnosus of 5 the present invention. According to a further preferred embodiment the composition for use according to the present invention comprises a combination of live L. acidophilus, live L. paracasei and live L. rhamnosus ranging from at least 10 billion colony forming units (CFU) to 200 billion colony forming units (CFU) of said combination of live 10 L. acidophilus, live L. para-casei and live L. rhamnosus. Preferably, the composition for use according to the present invention comprises at least 10 billion colony forming units (CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus, more preferably said composition comprises at least 50 billion colony forming units (CFU) of said 15 combination of live L. acidophilus, live L. paracasei and live L. rhamnosus, even more preferably said composition comprises at least 100 or 150 billion colony forming units (CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus. According to a preferred embodiment the composition for use according to the 20 present invention comprises 0.1 to 5% of L. acidophilus, 65 -99% of L. paracasei and 0.5-10% of L. rhamnosus of the colony forming units (CFU) of said combination of live L. acidophilus, live L. para-casei and live L. rhamnosus, preferably said composition comprises 0.2-5% of L. acidophilus, 70-99% of L. paracasei and 1-10% of L. rhamnosus of the colony forming units (CFU) of said 25 combination of live L. acidophilus, live L. para-casei and live L. rhamnosus, more preferably said composition comprises 0.5-5% of L. acidophilus, 75-99% of L. paracasei and 2-10% of L. rhamnosus of the colony forming units (CFU) of said combination of live L. acidophilus, live L. para-casei and live L. rhamnosus. According to an more preferred embodiment the composition for use according to - 7 - P2771PC the present invention comprises 1-5% L. acidophilus, 75-85% L. paracasei and 5- 10% L. rhamnosus of the colony forming units (CFU) of said combination of live L. acidophilus, live L. para-casei and live L. rhamnosus. Preferably the combination of live L. acidophilus, live L. paracasei and live L. 5 rhamnosus for use according to the present invention is administered at least once a day, more preferably a least four or five times a week. Preferably the combination of live L. acidophilus, live L. paracasei and live L. rhamnosus for use according to the present invention is administered for a period of at least 7 weeks, more preferably for a period of at least 14 weeks, even more 10 preferably for a period of at least 20 weeks, and even more preferably for a period of at least 30 weeks. According to a further preferred embodiment the subject diagnosed with ASD is under the age of 18 years old or it is an adult subject over the age of 18 years old, preferably said subject is aged between 4 and 17 years old, more preferably said 15 subject is aged between 4 and 11 years old. A further embodiment is the use of the composition comprising a combination of live L. acidophilus, live L. paracasei and live L. rhamnosus for inhibiting the growth of Clostridioides difficile strains in a subject affected by ASD. A further embodiments is the use of the composition comprising a combination of 20 live L. acidophilus, live L. paracasei and live L. rhamnosus for inducing a decrease of the level of p-cresol, preferably said level of p-cresol is reduced of about 80% in a subject affected by ASD. In a further preferred embodiment the composition for use according to the present invention inhibits the growth of Clostridium difficile strains in a subject affected by 25 ASD. In a further preferred embodiment the composition for use according to the present invention decrease of the level of p-cresol, preferably said level of p-cresol is reduced of about 80% in a subject affected by ASD. A further embodiment of the present invention is a method for treatment of Autism - 8 - P2771PC spectrum disorder (ASD) and for the treatment of multiple symptoms associated with ASD in a subject diagnosed with ASD comprising administering to said subject a composition comprising live Lactobacillus acidophilus, live Lacticaseibacillus paracasei and live Lacticaseibacillus rhamnosus. 5 A further embodiment is the present invention is the use of a composition comprising live Lactobacillus acidophilus, live Lacticaseibacillus paracasei and live Lacticaseibacillus rhamnosus for the manufacture of a medicament for the treatment of ASD and for the treatment of multiple symptoms associated with ASD. 10 Brief Description of the Drawings The present disclosure will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, figures 1 to 15. The figures are provided only by way of example and not by way of limitation. 15 Fig.1 illustrates the study design. The participation period comprised 3 phases: i) pre-treatment phase (8 weeks); ii) treatment phase during which the participant receives Bio-K+ pea-based, raspberry-flavoured probiotics drink daily for 14 weeks; iii) wash-out phase (8 weeks). Questionnaires were completed at 5 time- points (T-8pre, T0, T7, T14 and T22); Neuropsychology assessment and blood tests 20 were performed at T0 and T14, EEG was performed at T0, T14 and T22. During the week preceding the T0 and T14 visits, the participant wore an actigraph on the wrist 24 hours a day. Fig. 2 illustrates the Autism Treatment Evaluation Checklist ATEC scores of participants at each study time-point. The ATEC questionnaire was completed at 25 each visit by parents. Gray circles indicate the ATEC score of one participant. Fig.3 illustrates the evolution of the ATEC and its subscale scores during the study. Mean (A) ATEC score and (B) subcale score were compared across the 5 time- points using repeated measure ANOVA in N=17 participants who completed ATEC questionnaire at all time-points. Post-hoc analysis was conducted for pair-wise - 9 - P2771PC comparison between time-points using Bonferroni adjustment for multiple testing. Data represent means with standard error at each time-point. ATEC: Autism Treatment Evaluation Checklist.aP<0.05 compared to T8pre;bP<0.05 compared to T0;cP<0.05 compared to T7;dP<0.05 compared to T22. 5 Fig. 4 illustrates the severity of autism of participants during the study. Classification of autism severity was assessed for each participant before (T0), at the end (T14) and after (T22) treatment. Autism severity was classified in 3 categories according to the ATEC scores: mild (score <49), moderate (score 50- 79) and severe (score ≥ 80) according to Mahapatra et al. in Longitudinal 10 epidemiological study of autism subgroups using Autism Treatment Evaluation Checklist (ATEC) score. Journal of Autism and Developmental Disorders, 2020. 50: p.1497-1508. Fig. 5 illustrates the evolution of the Gastrointestinal Severity Index (GSI) score during the study. Mean GSI score was compared across the 5 time-points using 15 Friedman test. Post-hoc analysis was conducted using Wilcoxon test for pairwise comparison using Bonferroni adjustment for multiple testing data represents mean with standard error of the 17 participants at each time-point.aP<0.05 compared to T8pre;bP<0.05 compared to T0;cP<0.05 compared to T22. Fig.6 illustrates the ATEC and GSI scores during the study. Mean ± standard error 20 (error bars) of baseline-corrected data of ATEC and GSI scores during (T7 and T14) and after supplementation (T22) are represented as percentage of the T0 score for: A) all participants (N=17) who completed GSI and ATEC questionnaires at each time-points; B) participants with severe GI problem (N=13); and C) participants without severe GI problem (N=9). GI problems were classified as severe if GSI 25 score ≥4. Fig. 7 illustrates the severity of gastrointestinal (GI) symptoms during the study. Proportion (%) of participants with severe and non-severe GI problems was assessed at each time-point of the study. GI problems were classified severe if GSI score ≥4. The difference in proportions across the 5 time-points was compared - 10 - P2771PC using Cochran Q test. Post-hoc analysis was performed using McNemar test for pairwise comparison between time-points with false discovery rate correction for multiple testing. GSI: Gastrointestinal Severity Index.aP<0.01 compared to T22. Fig. 8 illustrates the proportion of participants with and without constipation at 5 study time-points. Proportion (%) of participants with constipation was assessed at each time-point of the study based on the first question of GSI questionnaire (See Fig. 12). Constipation was identified in participants having 3-4 stools / week (mild / moderate constipation) and 0-2 stools / week (severe constipation) based on the first question in GSI questionnaire. Participants reporting >5 stools per week 10 were considered not constipated. Fig.9 illustrates changes of Children Sleep Habits Questionnaire (CSHQ) scores of the subjects during study. Fig.9(A) illustrates each individual subjects CSHQ total score and Fig.9(B) was compared across the 5 time-points using repeated measure ANOVA. Post-hoc analysis was conducted for pair-wise comparison between time- 15 points using Bonferroni adjustment for multiple testing. Data represent means with standard error at each time-point.aP<0.05 compared to T8pre.bP<0.05 compared to T0. Fig. 10 illustrates CSHQ subscales as a function of time-point. CSHQ subscales were compared using ANOVA with repeated measures. Post-hoc analysis was 20 performed using McNemar test for pairwise comparison between time-point with false discovery rate correction for multiple testing. Subscale 1: Bedtime Resistance, subscale 2: Sleep Onset Delay, subscale 3: Sleep Duration, subscale 4: Sleep Anxiety, subscale 5: Night Wakings, subscale 6: Parasomnias, subscale 7: Sleep Disordered Breathing, subscale 8: Daytime Sleepiness.aP<0.05 compared to T0; 25bP<0.05 compared to T8-pre. Fig.11 illustrates the changes p-cresol concentration in plasma during study. Mean value was compared between T0 and T14 using paired t-test. Data are presented as group mean for each time point with standard deviation as error bars and symbols represents one participant.ap<0.005 compared to T0. - 11 - P2771PC Fig. 12 illustrates the changes of the Lactobacillus spp. strains and abondance of specific bacterial genera during study. Mean value was compared between times points using paired t-test or mixed-effects model for (A) Lactobacillus spp. (Bio- K+) Cq value (qPCR), and (B) relative abondance of Clostridioides genera and (C) 5 relative abondance of Blautia genera. Data are presented as group mean for each time point with standard deviation as error bars and symbols represents one participant.ap<0.05 pairwise post-hoc comparison with T0 and T22 (L. rhamosus and L. paracasei);bp<0.05 pairwise post-hoc comparison with T0 and T22 (all strains);cp<0.05 compared to T0 (Clostridioides and Blautia). 10 Fig. 13 illustrates an Autism Treatment Evaluation Checklist (ATEC) which was used to calculate the ATEC scores of participants during the study. Fig.14 illustrates a Gastrointestinal Severity Index Questionnaire (GSI) which was used to quantify the severity of GI symptoms of participants during the study. Fig.15 illustrates a Children’s Sleep Habit Questionnaire (CSHQ) which was used 15 to assess sleep quality and sleep disorder of participants during the study. DEFINITIONS Unless otherwise defined, all terms of art, notations and other scientific terminology used herein areintended to have the meanings commonly understood by those 20 persons skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference; thus, the inclusion of such definitions herein should not be construed to represent a substantial difference over what is generally understood in the art. The terms “approximately” and “about” herein refers to the range of the 25 experimental error, which may occur in a measurement. The terms “comprising”, “having”, “including” and “containing” are to be construed open-ended terms (i.e. meaning “including, but not limited to”) and are to be considered as providing support also for terms as “consist essentially of”, “consisting essentially of”, “consist of” or “consisting of”. - 12 - P2771PC The terms “consist essentially of”, “consisting essentially of” are to be construed as semi-closed terms, meaning that no other ingredients which materially affects the basic and novel characteristics of the invention are included (optional excipients may thus included). 5 The terms “consists of”, “consisting of” are to be construed as closed terms. The term “impaired communication” hererin refers to difficulties in developing language skills and understanding what others are saying, challenges with verbal and nonverbal communication skills, including difficulties with: speech and language development, 10 initiating or maintaining conversations, understanding tone of voice, nuances, and idioms, • Interpreting social cues and nonverbal signals, • Developing and using gestures and facial expressions. These communication impairments can vary in severity and impact daily social 15 interactions and relationships for subjects with ASD. The term “impaired sociability” herein refers: 1- to challenges in developing and maintaining meaningful relationships (verbal and nonverbal skills, social interactions, and understanding social cues). 2- to challenges with social interactions and relationships, including 20 difficulties with: • Initiating or maintaining social interactions • Understanding social cues, norms, and expectations • Developing and maintaining friendships • Understanding and interpreting others' emotions and perspectives 25 • Engaging in reciprocal conversations and sharing interests with others These social impairments can impact daily life and relationships, and may manifest differently in subjects with ASD. The term “impaired sensory awareness” herein refers: 1- to the difficulties in processing sensory information perceived by the brain - 13 - P2771PC 2- to difficulties with processing and integrating sensory information from the environment, including: • Hypersensitivity or hyposensitivity to sights, sounds, smells, tastes, or textures 5 • Difficulty filtering out background noise or distractions • Sensory seeking or avoidance behaviors • Over- or under-reactivity to sensory stimuli • Challenges with sensory integration and regulation These sensory impairments can impact daily life, behavior, and emotional 10 regulation in subjects with ASD. The term “impaired cognitive awareness and behaviors” in ASD herein refers: 1- to difficulties with sensory perception, cognitive processing, learning, memory and behaviors 2- to challenges with cognitive processes, including: 15 • Executive function deficits (e.g., planning, organization, time management) • Difficulty with flexibility and adapting to changes • Challenges with problem-solving, abstract thinking, and generalization • Restricted or repetitive patterns of behavior, interests, or activities • Difficulty with self-regulation of emotions and behaviors 20 These cognitive impairments can impact daily functioning, learning, and social interactions in subjects with ASD. The term ATEC questionare herein refers to an Autism Treatment Evaluation Checklist (ATEC) questionnaire (see Fig. 13). The (ATEC) questionnaire is a standardised questionnaire developed by the Autism Research Institute, of 4182 25 Adams Avenue, San Diego, CA 92116 and is used to to measure treatment effects on ASD behavioural symptoms such as language, sociability, sensory or cognitive awareness and attitude for children with ASD [1]. It includes 77 items summed as a total score (0-179) that are divided in 4 subsections: i. Speech / Language Communication (14 items; score range from 0 to 28); ii. Sociability (20 items; score - 14 - P2771PC range from 0 to 40); iii. Sensory / Cognitive Awareness (18 items; score range from 0 to 36); and iv. Health / Physical / Behavior (25 items; score range from 0 to 75) [1] The term ATEC score herein refers to a summed score (0 to 179) of the answers provided to the 77 items in the ATEC questionare. 5 The term Lacticaseibacillus paracasei used herein refers to a bacterial strain of Lacticaseibacillus paracasei that was previously named Lactobacillus casei before the signifficent bacterial taxonomy reclassification of 2020 under which numerous Lactobacillus species were reclassified into new genera. Herein, the terms Lacticaseibacillus paracasei and Lactobacillus casei can be used interchangeably. 10 The term Lacticaseibacillus rhamnosus used herein refers to a bacterial strain of Lacticaseibacillus rhamnosus that was previously named Lactobacillus rhamnosus before the signifficent bacterial taxonomy reclassification of 2020 under which numerous Lactobacillus species were reclassified into new genera. Herein, the terms Lacticaseibacillus rhamnosus and Lactobacillus rhamnosus can be used 15 interchangeably. The term Clostridioides used herein refers to a bacterium that was previously named Clostridium before a bacterial taxonomy reclassification in 2016. Herein, the terms Clostridioides and Clostridium can be used interchangeably. 20 EXAMPLES The following examples are not intended to limit the scope of protection of the present invention. While the invention has been described with the administering step of the nutritionally acceptable format as being done by administering the composition 25 comprising live L. acidophilus, live L. paracasei, and live L. rhamnosus, in a drinkable solution, it will be appreciated by a person skilled in the art that any suitable nutritionally acceptable format of the composition comprising live L. acidophilus, live L. paracasei, and live L. rhamnosus may be provided to administer the composition. In particular, alternative nutritionally acceptable 30 formats include, capsules, tablets, lozenges, gummies, and powders. It will be - 15 - P2771PC appreciated by a person skilled in the art that regardless of the nutritionally acceptable format in which the composition is provided, the resulting effect on the treatment of ASD and on the treatment of the symptoms associated with ASD would be substantially similar as the results of the drinkable solution disclosed in the above detailed embodiment of the invention. While the invention has been described in detail in connection with a cohort of child subjects aged between 4 and 11 years old, it will be appreciated by a person skilled in the art that results disclosed in the detailed embodiment of the invention, would be substantially similar in any type of suitable subject diagnosed with ASD, for example if the subject was an adult subject aged 18 years old and above, or alternatively, in an adolescent subject aged between 12 and 17 years old. Materials and methods Trial design The study was a 30-week, open label, non-randomized, feasibility trial. The study protocol was approved by the Ethics Review Board (ERB) of the CHU Sainte- Justine (#2021-3412) and performed in agreement with ethical principles and good clinical practices. As this study is the first to investigate the effects of a specific probiotic drinkable (Bio-K+) on children with a diagnosis of ASD, the sample size was not calculated, and withdrawn participants and dropouts were not replaced. The participation period comprised 5 assessments time-points over 3 phases (Fig.1). In the first phase, preliminary data were collected 8 weeks before initiating the probiotic supplementation (T-8pre) via personal or virtual meetings with parents to validate that symptoms to be evaluated remained stable before starting treatment. The second phase consisted in a 14-week treatment period with three assessment time-points. The baseline (T0) and the 14 weeks (T14) assessment include completion of questionnaires and a visit at the University Hospital centre Ste- Justine (UHCSJ) to complete testing. The assessment scheduled after 7 weeks (T7) of supplementation was performed by a telephone follow-up to complete the questionnaire planned by the protocol. The third phase consisted of an 8-week - 16 - P2771PC wash-out period to evaluate the persistence of symptoms after probiotic cessation. This assessment (T22) also includes a hospital visit to collect final study data evaluation and questionnaire. Participants 5 Participants were recruited from September 2021 to December 2021 by response to an advertisement posted on the UHCSJ hospital website, Facebook, and Twitter pages, and from April 2022 to June 2022 through consultation of medical charts of children diagnosed with ASD followed at the UHCSJ Integrated Centre for Child Neurodevelopment (CIRENE). 10 To be eligible to enter the study, the identified children had to meet the following inclusion criteria: (1) medical diagnosis of ASD; (2) age between 4–11 years old; (3) normal intelligence as documented by a neuropsychology evaluation at diagnosis; (4) acceptance and ability of the child to consume the pea-based specific drinkable probiotic for the duration of the study. Exclusion criteria were: (1) 15 intellectual disability as documented by neuropsychological assessment; (2) autism in the context of a genetic syndrome such as Fragile X or Tuberous sclerosis Complex; (3) chronic disease such as cancer or diabetes; genetic disorder such as Down Syndrome 21 or 14; (4) immune system disorder; (5) intolerance or allergy to the pea-based specific drinkable probiotic; (6) having taken probiotics during the 20 previous 3 months and; (7) having taken antibiotics in the previous month. All parents provided informed consent. Study Outcomes Recruitment (% patients approached / participants recruited) and retention rate (% participants retained / participants recruited) and completion rate (% 25 completed / expected) of product consumption, study data [sample collection (blood and stool), EEG test, each questionnaire and neuropsychological evaluation] and actigraphy assessment were calculated at the end of the study. The completion rate of product consumption was based on participant capacity to consume the specific pea-based drinkable probiotics daily for the entire 14-week period (i.e. completed) - 17 - P2771PC and expressed as a percentage of total participants included in the study (i.e. expected). The completion rate of study data represents the sum of each completed study data for all participants over the course of the study (i.e. completed), whereas expected data is based on the total number of measures planned during the study 5 according to available participants (i.e. those still included in the study at the time of data collection). The completion rate of actigraphy assessment was computed as the number of participants who completed the Actiwatch assessment by the total number of planned assessments for the available participants. Actigraphy assessment was considered completed if the participant had worn the actigraph for 10 24h for 7 consecutive days. Therefore, if the participant did not comply with wearing the actigraph for the entire 7-day period the data was considered not completed. Bio-K+ Probiotic The probiotic supplement consisted in the pea-based, raspberry-flavored drinkable 15 product fermented by and containing a minimum of 50 x 109CFU of a combination of three strains, L. acidophilus CL1285, L. paracasei LBC80R, L. rhamnosus CLR2 at expiry date. The supplement comprised of live L. acidophilus, live L. paracasei, and live L. rhamnosus preferably in the ranges of 1-5% L. acidophilus, 75-85% L. paracasei and 5-10% L. rhamnosus of the colony forming units (CFU) 20 of the composition. The study product was manufactured and supplied by Kerry (Canada) inc. (Laval, Quebec, Canada). For the duration of the supplementation period, children had to consume one bottle per day (98 g or 98 mL) all at once or over the course of the day. Children were allowed to mix the content of the specific drinkable probiotic bottle with any cold beverage. 25 Testing and data collection Blood tests Blood samples were collected after a 3 to 4 hour fast (9 mL total) at visits T0 and T14. Prior and with the consent of the parent, an anaesthetic cream (Maxilene, 4% of lidocaine, Canada) was applied topically to the child’s arm. - 18 - P2771PC Electroencephalography (EEG) EEG tests were performed at T-8pre, T0 and T22. During the EEG procedure, the child had to wear the 128-channel Electrical Geodesics Incorporated system (Eugene, OR, USA). The duration of each EEG test was 45 min including helmet 5 installation. The EEG protocol included 4 tasks: resting state, face processing using event-related brain potentials (ERP), visual steady-state and auditory steady-state. Continuous EEG resting state was recorded until a total of three minutes of clean signal was obtained (i.e. without movement, muscular or other artefacts). Participants watched a video of an abstract moving shape (rather than a fixation 10 cross) to increase compliance and reduce movement during resting state recording. The face ERP task lasted 6 minutes during which upright and inverted faces and houses were presented and responses to these stimuli were recorded. The visual steady-state task presented visual stimulation blocks at frequencies of 6 Hz, 10 Hz or 12 Hz and for 2 minutes. This task was repeated 4 times, for a total duration of 8 15 minutes. The auditory steady-state task was performed during the viewing of a movie without the soundtrack. Auditory stimulation blocks of 6 Hz and 40 Hz were sustained for 6 minutes. Actigraphy To monitor sleep-wake cycles and possible changes in sleep patterns, children were 20 asked to wear an actigraph for 7 days, 24 hours a day, before the T0 and T14 visits. This wearable device Actiwatch (Actiwatch 64, Mini Mitter Co, Philips Respironics) tracks movements during sleep and awake periods and collects data over an extended period of time, providing an objective measurement for the evaluation of sleep disorders. 25 Assessment of autistic and gastrointestinal symptoms and of sleep At the 5 time-points, 6 standardized questionnaires were used for the assessment of autism symptoms, social communication, GI symptoms and sleep. The Autism Treatment Evaluation Checklist (ATEC) questionnaire (see Fig. 13) is a standardised questionnaire developed by the Autism Research Institute, of 4182 - 19 - P2771PC Adams Avenue, San Diego, CA 92116 and is used to to measure treatment effects on ASD behavioural symptoms such as language, sociability, sensory or cognitive awareness and attitude for children with ASD [1]. It includes 77 items summed as a total score (0-179) that are divided in 4 subsections: i. Speech / Language 5 Communication (14 items; score range from 0 to 28); ii. Sociability (20 items; score range from 0 to 40); iii. Sensory / Cognitive Awareness (18 items; score range from 0 to 36); and iv. Health / Physical / Behavior (25 items; score range from 0 to 75) [1]. A higher total ATEC score indicates greater difficulties or severity and participants were classified as mild (score 20-49), moderate (score 50-79) and severe (score > 10 80) as proposed by Mahapatra et al.2020 [9]. A reduction in ATEC score overtime represent an improvement. The Behavior Rating Inventory of Executive Function (BRIEF) questionnaire is designed to assess executive functions in home and school environments of children with learning and attention disorders, developmental disabilities, depression and other developmental and neurological disorders (Score: 15 0-100) [2]. The Social Communication Questionnaire (SCQ) is used to measure social communication and existing symptoms of ASD (score: 0-19) [3]. The GI Severity Index (GSI) questionnaire (see Fig. 14) quantifies the severity of GI symptoms by attributing a score (0-2) related to 9 components (constipation, diarrhea, stool consistency, stool smell, flatulence, abdominal pain, unexplained 20 daytime irritability, nighttime awakening, abdominal tenderness). The total score is 0-15, as illustrated in Schneider, C.K., et al., Oral human immunoglobulin for children with autism and gastrointestinal dysfunction: a prospective, open-label study. Journal of autism and developmental disorders, 2006.36: p.1053-1064 with higher values corresponding to greater severity. Participant was considered with 25 severe GI disorders when GSI questionnaire score was ≥4. Constipation was identified in participants having 3-4 stools / week (mild / moderate constipation) and 0-2 stools / week (severe constipation) based on the first question of the GSI. Participants reporting ^5 stools per week were considered not constipated. For sleep evaluation, 2 questionnaires were used: 1) The standardize questionnaire on the - 20 - P2771PC Children’s Sleep Habit Questionnaire (CSHQ) (see Fig. 15) and usual sleep schedule. The CSHQ (total score: 33-99) assesses sleep complaints using 8 subscales: (1) Bedtime resistance, (2) Time to sleep, (3) Sleep duration, (4) Anxiety at sleep onset, (5) Nocturnal awakenings, (6) Sleep behaviors, (7) Breathing and (8) 5 Sleepiness. A CSHQ total score of 41 or more means that symptoms are clinically significant and may reflect a sleep disorder as illustrated in Owens, J.A., A. Spirito, and M. McGuinn, The Children's Sleep Habits Questionnaire (CSHQ): psychometric properties of a survey instrument for school-aged children. Sleep- New York-, 2000.23(8): p.1043-1052. 10 Stool samples collection and analysis Stool samples were collected at the 5 time-points (T8-pre, T0, T7, T14 and T22) by the parent at home 1 to 3 days before each time-point. Parents were instructed to freeze the stool sample (-20°C) as soon as it was collected and until the delivery at the research laboratory. Detailed instructions were provided on self-collection, 15 packaging and handling of samples and delivery. Upon receipt, samples were stored at -80°C. After DNA extraction, the composition and diversity of the intestinal microbiota were determined by sequencing the 16SV4 region. Molecular markers of inflammation were measured in stool samples: calprotectin, lactoferrin and the cytokine IL-17A by ELISA, and finally stercobilin and TMA by non-targeted 20 LC / MS-MS. Fecal SCFAs (acetic, propionic, butyric, isobutyric, valeric and isovaleric) were quantified by GC-FID after liquid-liquid extraction. Evaluation of dietary intake Participants’ dietary intakes were collected prior to each stool sample collection to determine if there were significant changes in diet over the course of the trial. 3- 25 day food records and 24-hour recalls were used to calculate energy and nutrient intakes using a nutrient calculation tool built in-house employing nutrient values from the 2010 Canadian Nutrient File. Information on the intake of vitamin / mineral supplements was also collected. - 21 - P2771PC Neuropsychological evaluation The neuropsychological evaluation used was the Weschler Intelligence Scale for Children - Fifth Edition (WISC-V) for children aged 6 years to 11 years and 11 months or the WISC-IV (Fourth Edition) for children under 6 years old [4-6]. The 5 neuropsychological evaluation was offered in 3 languages according to the preferences of the participants and / or their parents: French, English, or Spanish. Following the completion of these test batteries a score was attributed to participants and intelligence was classified as extremely high (score ≥ 130); very high (score: 120-129); high average (score: 110-119); average (score: 90-109); low 10 average (score: 80-89); very low (score: 70-79); extremely low (score: 50-69). Extraction of bacterial DNA from faecal samples and sequencing of the 16S rRNA gene amplicon Sample preparation was performed as described by Roussel et al. (2018, 2024). DNAs were stored at -20°C until 16S rRNA gene library preparation. Library 15 preparation and sequencing were carried out as described in detail by Roussel et al (2022). Sequencing was performed on samples diluted to a final concentration of 10 pM using the MiSeq 600 cycle V3 reagent kit (Illumina, San Diego, USA) on an Illumina MiSeq system (Illumina, San Diego, USA). Quantitative PCR analysis of L. acidophilus CL1285, L. paracasei LBC80R, L. 20 rhamnosus CLR2 and C. difficile strains The quantitative PCR (qPCR) was performed as described by Masset et al. (2023) using primers specific for L. acidophilus CL1285 (NCFM-F / R), L. paracasei LBC80R (3989H7-F / R) et L. rhamnosus CLR2 (3990q3-F / R) and C. difficile FliC, identified in most flagellated and non-flagellated strains of C. difficile (FliC-F / R). 25 Plasmatic and bacterial p-cresol measurement Plasma and bacterial supernatant were obtained by centrifugation, respectively, of blood sample or bacterial cultures at 3,000 × g for 15 min at +4 °C.200 µl of plasma or supernatant were mixed separately with 50 µl of internal standard (1,4- chlorophenol at 800 ppm and 2,3,5-trichlorophenol at 600 ppm) and 200 µl of ethyl - 22 - P2771PC acetate (EA). The mixtures were centrifuged at 12,000 × g for 15 minutes at +4 °C and the organic phase was removed and stored. A further 200 µl of EA was added to the aqueous phase and centrifuged at 12,000 × g for 15 minutes at +4 °C. The two organic phases for each sample were combined.50 µl of this mixture was added 5 to 50 µl of N,O-bis (triméthylsilyl)trifluoroacétamide (BSTFA) and incubated for 3 hours at 70 °C in a sand oven. The rate of p-cresol production was measured by GC-MS as described by Armbruster et al. (1994) and de Loor (2005). Data analysis Description of participants’ characteristics at recruitment is computed as mean ± 10 standard deviation (SD) for continuous variables (age at recruitment) and as percentage (%) of total participants for categorial variables (sex, sibling, non- verbal, GI problems, sleep problems, ADHD, epilepsy and brain abnormality symptoms). For feasibility data, the descriptive statistics for continuous variables are presented as percentage (%). The mean questionnaire completion rate is the 15 average completion rate of the 8 questionnaires (ATEC, SCQ, BRIEF, GI, questionnaire on usual sleep schedules, CSHQ, 3-day food records and 24 hour food record). Assessment of supplementation effect was conducted using scores derived from the ATEC (total and each of the four subscales), the GSI and the CSHQ questionnaires at each time-point which were presented as mean ± standard error 20 (SE). The change in total and each subscale score for ATEC and CHSQ questionnaires during the study was assessed using a repeated measures analysis of variance (ANOVA) and Bonferroni correction is applied for multiple testing in post-hoc pairwise comparisons. Changes in GSI scores overtime are assessed using Friedman test followed by Wilcoxon test as post-hoc analysis with Bonferroni 25 correction for multiple comparisons. To assess changes in GSI (Gastrointestinal Severity Index) scores over time, we used the Friedman test, which is a non- parametric method suitable for analyzing ordinal data. This test helped to determine if there were statistically significant differences in GSI scores across multiple time points. Once significant differences were identified with the Friedman test, we - 23 - P2771PC conducted pairwise comparisons using the Wilcoxon signed-rank test. This post- hoc analysis allowed the pinpointing of specific time points that had significant changes in GSI scores. To ensure the accuracy of the results and control for the increased risk of Type I errors due to multiple comparisons, the applied the Bonferroni correction was applied. This adjustment helped maintain the overall error rate at a controlled level, ensuring the robustness of the results. To further described the variation of ATEC and GSI during the study, the percentage of change in both scores from T0 was calculated for T7, T14 and T22 using T0 (i.e., baseline- before treatment score) result as 100% for each participant. The rate of reduction of the ATEC score before (T0) and after (T14) treatment is expressed as efficacy index (E) where E = (T0 - T14) / T0 × 100%. Treatment efficacity was then determined for each participant based on calculated E as follow: highly effective (ETE >50%); effective (EE 20-50%); ineffective (IE <20%) and total effectiveness rate (TE) = (ETE + EE) / total number of cases × 100%. The proportions of participants according to ASD severity (mild / moderate / severe) and severe GI problems (yes / no) were presented as a percentage (%) at each time- points. The proportion of participants with severe ASD (“severe” vs. “mild and moderate”) and with severe GI problems (yes vs. no) was compared across all time- points using the Cochran Q test and post-hoc analysis was conducted for pairwise comparison between time-points using McNemar test with false discovery rate correction for multiple testing. EEG data was compared between T0 and T14 using Wilcoxon paired tests. Production of p-cresol was compared between T0 and T14 using paired t-test student. Gut microbial community and amplicon data statistics analysis were performed as described by Roussel et al. (2022) Results Recruitment of participants and cohort description Thirty-eight participants were evaluated for eligibility according to the inclusion / exclusion criteria. Initially, the recruitment announcement was published - 24 - P2771PC on the CHU Sainte-Justine website and social networks (i.e. Facebook and Twitter). A total of 23 parents expressed interest in including their children in the study. Of these, 15 participants were retained, including 14 boys and one girl. The other 7 children were not enrolled for not meeting the inclusion / exclusion criteria for age (N=4: older than 12 years) and difficulty to commute to CHU Sainte-Justine (N=4). To increase the number of girls in the cohort, we recruited via medical charts. Out of 15 identified potential participants, 8 were recruited including 6 girls and 2 boys. The reasons that motivated the refusal of 7 parents to include their child in the study were: lack of financial compensation (N=2), weekday testing / lack of time (N=4) and difficulty to commute (N=1). The recruitment rate through both methods was 60.5%. Of the 23 children enrolled, 65.2% (N=15) were recruited via social networks and 34.8% via medical charts. Demographic characteristics of the recruited participants are described in Table 1. The mean age at recruitment was 6.7 years (range of 4.0 to 11.11 years) with 69.6% of boys (N=16). A total of 5 autistic siblings (21.7%) were included in this study. These children were part of three families, each of which had at least two children with a diagnosis of ASD. Of the 23 participants, 7 were non-verbal (30.4%), one had epilepsy (4.5%), 2 had ADHD (8.7%), one had celiac disease (4.5%) and 2 followed a gluten and casein free diet (8.7%). Severe GI symptoms were found in 65.2% of participants and severe sleep disturbances in 91.3%. According to the neuropsychological assessment, 66.7% of participants (N=10) had extremely low intelligence, 26.7% (N=4) average and 6.6% (N=1) low intelligence (Table 3). Incidental findings identified during the study: brain abnormalities were detected by EEG in 3 participants (13.6%) including one participant who had a known diagnosis of epilepsy and levels of hs-CRP > 1.0 mg / L were found in 2 children. Feasibility of the study protocol The feasibility thresholds for the study are described in Table 2. The study drop- out rate was 4.25% (target for success <35%), as one participant did not complete the study for logistics considerations. Because this participant dropped out of the study after the T-8pre time-point, he was not taken into consideration in the calculation of success rates of the subsequent visits. All participants (100%, N=22) - 25 - P2771PC accepted to consume the required portion of the probiotic supplement daily for 14 weeks. For 3 participants, the product was mixed with orange juice (N=2) or drinkable yogurt (N=1). Blood punctures were performed on 95.4% of schedules procedures. However, in 4 cases related to 4 different participants, blood was not successfully collected (totally or partially) for technical reasons (i.e. presence of small or deep veins). Since the procedure was accepted by the children and performed nonetheless, these data were considered positive as to their feasibility. Also, 89.2% of stool samples were collected. Of missing samples, two samples were not stored properly and 10 were not provided. The EEG tests were performed in 86.3% of cases. Five participants were unable to complete 9 EEG tests: 2 participants did not perform the test at T0 and T14 (4 tests), one participant was unable to complete it at T14 and T22 (2 tests), one participant was unable to do it at T0 (1 test) and 2 participants were unable to complete it at T22 (2 tests). The actigraph was worn 42 times for the entire duration of the test (7 consecutive days). Two participants worn the actigraph for only 5 out of 7 days. In total, the actigraph acceptance rate was 94.4%. Parents were able to complete 84.4% of all the questionnaires (pre-established threshold of feasibility >80%). The neuropsychological evaluation completion rate was 56.8%. The evaluation could not be performed with 7 non-verbal children and with one child who was stressed at both T0 and T14 visits. Moreover, 3 other participants did not complete the neuropsychological evaluation at T0, but successfully completed it at T14. Results of the impact of the specific probiotic on autistic and GI symptoms Our data indicate that probiotics treatment alleviated the symptoms in children with ASD. After 14 weeks of intervention, there was a reduction in ATEC score for all participants, reflecting an improvement in autistic symptoms (Fig. 2). The mean ATEC total score decreased by 27.1 ± 3.2 points (P<0.001), which represents 43% of T0 score (Fig. 3A). Scores for the four ATEC domains were also lower after supplementation, with the greatest decline observed in the Health / Physical / Behavior sub-score (-14.6 points ± 2.2) (Fig. 3B). The probiotic supplementation appeared to impact the severity of autism, according to ATEC score classification: 6 participants initially classified with moderate autism were, - 26 - P2771PC after treatment, in the mild category, and 3 participants initially classified with severe autism were classified as moderate thereafter (Fig.4). In parallel, there was a decrease in the GSI score, reflecting an improvement in GI symptoms (-2.9 ± 0.61 P<0.01) (Fig.5) which represents a 32.5% reduction of the 5 score measured before supplementation. Interestingly, 8 weeks after stopping probiotic supplementation (T22), ATEC and GSI scores had returned to their initial levels (Fig.s 3A, 5 and 6A). When the cohort was stratified according to the presence of severe GI problems (GSI score ^ 4), the same tendency in the evolution of ATEC and GSI scores was observed in both groups (Fig.6B and 6C). Also, the 10 proportion of participants with severe GI problems was lower at the T14 visit, compared to T22 (11.8% vs.70.6%; P<0.01) (Fig.7). At the start of the study, 15 participants (65.2%) had constipation. After probiotic supplementation (T14), constipation was still present in only 5 children (22.7%) but was deemed less severe (Fig.8). Finally, the total efficiency rate of treatment was 95.2% as 20 participants 15 out of 21 had at least an improvement of the ATEC score of at least 20% of ATEC following supplementation (Table 5). Mode of action Regarding ASD, differences were found in gut microbiota composition of autistic children compared to neurotypical peers by comparing stool samples. Specifically, 20 a higher bacterial colonization of Clostridioides, Desulfovibrio and Bacteroides species at the genus level and lower abundance of Bifidobacterium species were observed in autistic children compared to controls. This shift in bacterial composition led to modifications in fecal levels of the metabolite butyrate (a short- chain fatty acid, SCFA) and glutamate. The free amino acid glutamate and its 25 decarboxylated form, the γ-aminobutyric acid (GABA), act as neurotransmitters in the nervous central system. GABA is produced by several Bacteroides species, and by some Lactobacillus species as well, such as L. rhamnosus and L. acidophilus. Moreover, some species of the Clostridiaceae family ferment tyrosine to produce p-cresol, a metabolite that has been linked to autism. As many metabolites produced 30 by gut bacteria (i.e. GABA, SCFA) influence brain function or neuroinflammation, - 27 - P2771PC the resulting dysbiosis contributes to ASD and its symptoms via modulation of neuroimmune and neuroendocrine signaling. Due to the association between ASD and disruption of the immune system, mainly through increase of certain inflammatory cytokines including IL-17A and TNF-α, the chronic inflammation 5 and neuroinflammation leads to behavioral and cognitive problems and therefore influences the etiological pathway of ASD. Compared to controls, higher levels of calprotectin, a protein found in neutrophils, were found in fecal samples of ASD children, suggesting local inflammatory response of GI tissue. Results of the impact of the specific probiotic on sleep 10 Probiotic supplementation resulted in a significant change in sleep quality (Fig.9). There was a significant reduction in the mean total CSHQ score after supplementation (T7 and T14) compared with the pre-treatment phase (T8-pre and T0), ranging from -4.7 ± 1.1 (P<0.05) to -7.4 ± 1.4 (P<0.005) (Fig.9). In addition, probiotic supplementation promoted a significant variation in the scores of 4 15 subscales: sleep duration (subscale 3), parasomnias (subscale 6), sleep-disordered breathing (subscale 7), daytime sleepiness (subscale 8) (Fig.10). Results of the impact of de specific probiotic on p-cresol synthesis Our data indicates that the probiotic supplementation led to a clear reduction in p- cresol levels in plasma. After 14 weeks of supplementation, there was a significant20 reduction in plasmatic p-cresol levels for all participants (Fig.11). The mean of p- cresol level decrease by 4.2 ± 1.6 µg / ml (P<0.005) which represents 82.7% of T0 level. Results of the impact of de specific probiotic on the abundance of p-cresol- producing bacteria 25 Quantification of L. acidophilus CL1285, L. paracasei LBC80R, and L. rhamnosus CLR2 strains by qPCR confirmed their presence during the supplementation phase and their absence before and after (Fig.12A), supporting compliance with the study product. Also, microbiome analysis revealed a decrease in the relative abundance of the genera Clostridioides and Blautia, known to synthesize p-cresol (Fig. 12B - 28 - P2771PC and C) after probiotic supplementation. Also, qPCR targeting flagellin FliC of C. difficile detected these strains at baseline in fecal samples of 8 out of 23 participants. In all 8 participants, the targeted C. difficile strains were not detectable after 14 weeks of supplementation. 5 Discussion The findings of this study confirm that the consumption of a specific drinkable probiotic (Bio-K+) by children diagnosed with ASD improved the symptoms associated with ASD including GI symptoms and symptoms of sleep disorders. Initially, the recruitment of 30 children with a diagnosis of ASD was planned. 10 However, the interim analysis showed that the study objectives were largely achieved earlier and, following the recommendation of the Data and Safety Monitoring Board, recruitment was ended after the 23rdparticipant, as the continuation of the study was deemed futile. In doing so, we avoided unnecessarily exposing children to procedures and related stress, and we optimized the use of 15 resources. One aspect that was evaluated in this study was the use of validated questionnaires as tools to capture data with the targeted population. When assessing the impact of an intervention on autistic and other clinical symptoms in children with a diagnosis of ASD, different questionnaires can be considered. 20 The composition of the tested cohort minimally reflects the socio-demographic portrait of autism reported in the scientific literature. The proportion of girls vs. boys (30%) in our cohort is representative of the autistic population in Canada. The 2023 CHSCY report documented that 29% of children with a diagnosis of ASD aged 1 to 11 years are girls [7]. 25 GI dysfunctions have been reported in 9-91% of individuals with ASD. This is consistent with the data of this study, which report that 91% of participants have at least one GI problem and of which 65.2% had severe symptoms. Also, 65.2% and 21.7% of participants had constipation and diarrhea, respectively. Constipation is reported to be the primary GI comorbidity in individuals with a diagnosis of ASD, - 29 - P2771PC and chronic constipation is typically the most common disorder, encountered in up to 80% of children with a diagnosis ASD. Alternating constipation and diarrhea is also present in this population. Notably, a beneficial impact of the probiotic supplementation comprising live L. 5 acidophilus, live L. paracasei, and live L. rhamnosus, on the total ATEC score, its 4 sub-categories and on the GSI score was found. For comparison, studies evaluating the impact of probiotics on ATEC and GSI scores are summarized in Table 4. The reduction of the ATEC scores following probiotics supplementation range from 7.5% to 32.4%. Clearly, the 42% reduction observed in the present study 10 using live L. acidophilus, live L. paracasei, and live L. rhamnosus, indicates a superior impact on autistic symptoms over the prior art. GI abnormalities may be a manifestation of an underlying inflammatory process, which is hypothesized to be related to intestinal dysbiosis, or simply reflect sensory hyperreactivity to abdominal signals. The capacity of the specific drinkable 15 probiotic to improve the quality of life of study participants, including dysphoria, health anxiety, food avoidance disorder and social interaction, is mediated by restoration of gut microbiota. The results of the present study illustrate a beneficial effect of the specific drinkable probiotic on ASD and on improvement in symptoms associated with ASD including, GI symptoms, and sleep disorders. In the present 20 study, the improvement or disappearance of constipation is a consequence of the intestinal microbiota normalization following probiotic treatment. In the present study, sleep disorders affected 91.3% of participants. These sleep disorders comprise bedtime resistance, prolonged sleep onset, long or numerous night-time awakenings and early morning awakenings and are associated with 25 “somatic complaints”; “anxious / depressed” state; and “social problems” in children with a diagnosis of ASD. The results of the present study show an improvement in the sleep of children diagnosed with ASD following the probiotic supplementation, as perceived by their parents. It was observed that the total scores and subscale scores did not vary significantly throughout the treatment period, suggesting a - 30 - P2771PC stabilization of its effect on items measured by the CSHQ. However, it was observed that the total score and subscale measured by the CSHQ, returned to pre- supplementation levels after probiotics discontinuation. Accordingly, our pilot data supports mechanistic hypotheses involving the 5 metabolite p-cresol. The 3 lactobacilli L. acidophilus CL1285, L. paracasei LBC80R, and L. rhamnosus CLR2 inhibit the growth of some bacteria producing metabolites that have been linked to autism. Compared to controls, autistic children are more likely to be colonized by genus of bacteria known to synthesize p-cresol, such as Clostridioides. The metabolite p-cresol is a neurotoxin that has been linked 10 to autism in several studies. Given that it has been demonstrated that the 3 lactobacilli strain of Bio-K+ inhibit the growth of Clostridioides difficile, we assume that they also affect the production of their metabolites such as p-cresol. Additionally, one of the metabolites known to be involved in sleep disorders is p- cresol. The improvement in sleep disturbances observed in the participants of this 15 study could be a consequence of the reduction in p-cresol level promoted by the three lactobacilli. Conclusion The results illustrate that probiotic supplementation with a composition comprising live L. acidophilus, live L. paracasei, and live L. rhamnosus, in children with a 20 diagnosis of ASD improve the symptoms of ASD. Advantageously, supplementation improves the Sleep and GI symptoms associated with ASD. The study data shows the efficacy of the composition of the invention in improving behavioural symptoms in children with ASD and in improving GI and sleep disorders associated with ASD. 25 - 31 - P2771PC TABLES Table 1. Participants’ characteristics and demographic data at recruitment Characteristics N=23 Sex, n (%) Male 16 (69.6) Female 7 (30.4) Age at recruitment (years), mean ± SD 6.7 ± 2.2 Autistic siblings 5 (21.7) Non-verbal 7 (30.4) GI problemsa15 (65.2) Diarrhea 5 (21.7) Constipation 15 (65.2) Sleep problemsb21 (91.3) ADHD 4 (17.4) Epilepsy 1 (4.5) Brain abnormality 3 (13.6) Celiac disease 1 (4.5) Under gluten and casein free diet 3 (13.6) 5aParticipant has at least one gastrointestinal problem according to GSI questionnaire.bParticipants are considered to have sleep problems at a CSHQ score ≥ 41. SD: standard deviation. ATEC: Autism Treatment Evaluation Checklist; ADHD: attention-deficit hyperactivity disorder; GI: gastrointestinal. 10 15 - 32 - P2771PC Table 2. Description of feasibility data aOne participant dropped out of the study after the T-8pre time-point, he was not taken into 5consideration in the calculation of success rates of the subsequent visits. The pre-established successthresholds are adapted from [8]. EEG: electroencephalogram; ATEC: Autism Treatment Evaluation Checklist; SCQ: Social Communication Questionnaire; GSI: Gastrointestinal Severity Index; CSHQ: Children’s Sleep Habit Questionnaire; BRIEF: Behavior Rating Inventory of Executive Function. - 33 - P2771PC Table 3. Autistic and gastrointestinal symptoms at study baseline and evaluation of sex differences Symptoms Sex All Girls Boys P-valueaAutistic N=23 N=7 N=16 ATEC score, mean ± SD Total 59.3 ± 29.6 61.0 ± 58.5 ± 0.83 41.2 24.6 i. Speech / language 10.5 ± 9.5 13.9 ± 9.0 ± 9.0 0.28 communication 10.3 ii. Sociability 12.7 ± 8.0 12.6 ± 12.8 ± 6.7 0.57 10.3 iii. Sensory / cognitive awareness 10.4 ± 7.1 11.1 ± 9.6 10.1 ± 6.1 0.91 iv. Health / physical / behavior 25.6 ± 11.3 23.4 ± 26.6 ± 0.55 13.2 10.7 ASD severityb, N (%) 1.00 Mild 10 (43.5) 3 (42.9) 7 (43.8) Moderate 8 (34.8) 2 (28.6) 6 (37.5) Severe 5 (21.7) 2 (28.6) 3 (18.8) Gastrointestinal N=23 N=7 N=16 GSI Score, mean ± SD 4.0 ± 2.7 2.6 ± 2.1 4.6 ± 2.8 0.10 GI problemsc, N (%) 0.19 Yes 12 (52.2) 2 (28.6) 10 (62.5) Neuropsychological N=15 N=2 N=13 Intelligence scaled, N (%) 0.50 Extremely high 0 0 0 Very high 0 0 0 High average 0 0 0 Average 4 (26.7) 2 (100) 2 (15.4) - 34 - P2771PC Low average 1 (6.7) 0 1 (7.6) Very low 0 0 0 Extremely low 10 (66.7) 0 10 (76.9) Data were collected at T-8pre (autistic and gastrointestinal) and T0 time-point (neuropsychological) and compared between participants according to their sex (girls vs. boys).aDifferences between sex were compared using Mann-Whitney U-test (ATEC and GSI score) and Fisher exact test (ASD severity, GI problems and 5 Intelligence scale).bASD severity was classified in 3 categories according to the ATEC scores: mild (score <49), moderate (score 50-79) and severe (score ≥ 80) according Mahapatra et al. [9]. cGI problems were determined in participants based on GSI score. dWISC-V and IV batteries were used to evaluate intelligence: Extremely high (score ≥ 130); very high (score: 120-129); high average (score: 110-119); average (score: 90-109); low average (score: 80-89); very low (score: 1070-79); extremely low (score: 50-69). ASD: autism specter disorder; GI: gastrointestinal, GSI:Gastrointestinal Severity Index.

[0002] - 35 - P2771PC Table 4. Review of the effect of probiotics on ATEC and / or GSI scores - 36 - P2771PC N / A: outcome not measured. CFU: colony forming unit. - 37 - P2771PC Table 5. Efficiency of the specific drinkable probiotic supplementation based on ATEC score 51Efficiency rate was calculated based on the total ATEC score for each participant as follow: (T0 - T14) / T0 × 100%.2Effectiveness level is based on efficiency rate, where >50%: highly effective, 20-50%: effective and <20%: ineffective.3Total effectiveness rate is calculated as: (high effective + effective) / total number of cases × 100%. 10 15

[0003] - 38 - P2771PC References 1. Rimland, B. and M. Edelson, The Autism treatment evaluation checklist (ATEC). San Diego. CA: Autism Research Institute, 1999. 2. Gioia, G.A., et al., Behavior rating inventory of executive function. 1996: Psychological Assessment Resources. 3. Rutter, M., et al., Social communication questionnaire (SCQ). 2003. Los Angeles, CA: Western Psychological Services. 4. Weiss, L.G., et al., WISC-V assessment and interpretation: Scientist- practitioner perspectives.2015: Academic Press. 5. Wechsler, D., WISC-IV: Weschler Intelligence Scale for Children. 2003: Pearson. 6. Kaufman, A.S., et al., Test review: Wechsler intelligence scale for children, (WISC-IV). Journal of psychoeducational assessment, 2006.24(3): p.278- 295. 7. Canada, P.H.A.o., Autism spectrum disorder: Highlights from the 2019 Canadian health survey on children and youth. 2023: https: / / www.canada.ca / content / dam / phac- aspc / documents / services / publications / diseases-conditions / autism- spectrum-disorder-canadian-health-survey-children-youth-2019 / autism- spectrum-disorder-canadian-health-survey-children-youth-2019.pdf. 8. Carmassi, C., et al., Systematic review of sleep disturbances and circadian sleep desynchronization in autism spectrum disorder: toward an integrative model of a self-reinforcing loop. Frontiers in Psychiatry, 2019.10: p.366. 9. Mahapatra, S., et al., Longitudinal epidemiological study of autism subgroups using Autism Treatment Evaluation Checklist (ATEC) score. Journal of Autism and Developmental Disorders, 2020.50: p.1497-1508. 10. Niu, M., et al., Characterization of intestinal microbiota and probiotics treatment in children with autism spectrum disorders in China. Frontiers in neurology, 2019.10: p.1084. 11. Tharawadeephimuk, W., et al. Preliminary study of probiotics and kynurenine pathway in autism spectrum disorder. in 2019 16th International Conference on Electrical Engineering / Electronics, Computer, Telecommunications and Information Technology (ECTI-CON). 2019. IEEE. 12. Wang, Y., et al., Probiotics and fructo-oligosaccharide intervention modulate the microbiota-gut brain axis to improve autism spectrum reducing also the hyper-serotonergic state and the dopamine metabolism disorder. Pharmacological research, 2020.157: p.104784. 13. Shaaban, S.Y., et al., The role of probiotics in children with autism spectrum disorder: A prospective, open-label study. Nutritional neuroscience, 2018. 21(9): p.676-681. 14. EI-Alfy, M., A. Youssef, and R. Sabrey, A Study on Effect of Probiotic Supplementation on Gastrointestinal Symptoms, Cognition and Behavior in - 39 - P2771PC Egyptian Children with Autism Spectrum Disorder. Egypt. J. Paediatr, 2019. 36: p.327-337. 15. Li, Y.-Q., et al., Effect of probiotics combined with applied behavior analysis in the treatment of children with autism spectrum disorder: a prospective randomized controlled trial.2021. 16. West, R., et al., Improvements in gastrointestinal symptoms among children with autism spectrum disorder receiving the Delpro® probiotic and immunomodulator formulation. J. Prob. Health, 2013.1(1): p.1-6. 17. Roussel C, Galia W, Leriche F, Chalancon S, Denis S, et al., Comparison of conventional plating, PMA-qPCR, and flow cytometry for the determination of viable enterotoxigenic Escherichia coli along a gastrointestinal in vitro model. Appl Microbiol Biotechnol. 2018;102(22):9793–9802. doi:10.1007 / s00253-018-9380-z. 18. Roussel, Charlène, Sola, Mathilde, LESSARD-LORD, Jacob, et al. Human gut microbiota and their production of endocannabinoid-like mediators are directly affected by a dietary oil. Gut Microbes, 2024, vol. 16, no 1, p. 2335879. 19. Roussel C, Guebara SAB, Plante PL, Desjardins Y, Marzo VD, Silvestri C. Short-term supplementation with ω-3 polyunsaturated fatty acids modulates primarily mucolytic species from the gut luminal mucin niche in a human fermentation system. Gut Microbes.2022;14(1):2120344. 20. Masset, Zoé, gunaratnam, Sathursha, millette, Mathieu, et al. Transcriptome analysis of the Clostridioides difficile response to a specific lactobacilli probiotic formulation: Explanations for its mechanisms of action. Journal of Applied Microbiology, 2023, vol.134, no 3, p. lxad047 21. Report: Design and validation of primers used for the quantification, by qPCR, of strains L. acidophilus CL1285, L. casei LBC80R, L. rhamnosus CLR2 in feces sample. June 2019. 22. Bezawada, N., et al., Autism Spectrum Disorder and the Gut Microbiota in Children: A Systematic Review. Ann Nutr Metab, 2020.76(1): p.16-29. 23. Basra, M., et al., Exploring the neurotoxic effects of microbial metabolites: A potential link between p-Cresol and autism spectrum disorders? Brain Research, 2024: p.149427. 24. Gao, X.W., et al., Dose-response efficacy of a proprietary probiotic formula of Lactobacillus acidophilus CL1285 and Lactobacillus casei LBC80R for antibiotic-associated diarrhea and Clostridium difficile-associated diarrhea prophylaxis in adult patients. Am J Gastroenterol, 2010. 105(7): p.1636-41. 25. McFarland, L.V., et al., Primary prevention of Clostridium difficile infections with a specific probiotic combining Lactobacillus acidophilus, L. casei, and L. rhamnosus strains: assessing the evidence. J Hosp Infect, 2018.99(4): p.443-452. 26. Ignacio, A., et al., Correlation between body mass index and faecal microbiota from children. Clin Microbiol Infect, 2016.22(3): p.258.e1-8. 27. Tasteyre, A., et al., Phenotypic and genotypic diversity of the flagellin gene (fliC) among Clostridium difficile isolates from different serogroups. - 40 - P2771PC Journal of Clinical Microbiology, 2000.38(9): p.3179-3186. 28. Lv, Z., Huang, L., Song, Y., Lan, Y., Sun, S., et al., (2024). Investigation of causal effects of blood metabolites on insomnia and circadian rhythm sleep wake disorders. Frontiers in Sleep, 3, 1333154. 29. Schneider, C.K., et al., Oral human immunoglobulin for children with autism and gastrointestinal dysfunction: a prospective, open-label study. Journal of autism and developmental disorders, 2006.36: p.1053-1064. 30. Owens, J.A., A. Spirito, and M. McGuinn, The Children's Sleep Habits Questionnaire (CSHQ): psychometric properties of a survey instrument for school-aged children. Sleep-New York-, 2000.23(8): p.1043-1052. 31. Armbruster, David A., Margaret D. Tillman, and Linda M. Hubbs. "Limit of detection (LQD) / limit of quantitation (LOQ): comparison of the empirical and the statistical methods exemplified with GC-MS assays of abused drugs." Clinical chemistry 40.7 (1994): 1233-1238. 32. de Loor, Henriette, et al. "Gas chromatographic–mass spectrometric analysis for measurement of p-cresol and its conjugated metabolites in uremic and normal serum." Clinical chemistry 51.8 (2005): 1535-1538..

[0004] - 41 - P2771PC CLAIMS 1. Composition comprising a combination of live Lactobacillus acidophilus, us rhamnosus, SD) and in the additional food itional protein, , more protein in said posited acillus at the mnosus deposit - 42 - P2771PC 8. Composition for use according to any of the previous claims, wherein said sleep disorders are selected from a group comprising sleep duration, parasomnias, sleep disordered breathing and daytime sleepiness. ii f i f h i li h i i - 43 - P2771PC (CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus, more preferably said composition comprises 0.5-5% of L. acidophilus, 75-99% of L. paracasei and 2-10% of L. rhamnosus of the colony forming units (CFU) of said combination of live L. acidophilus, live 5 L. paracasei and live L. rhamnosus. 13. Composition for use according to claim 12, wherein said composition comprises 1-5% L. acidophilus, 75-85% L. paracasei and 5-10% L. rhamnosus of the colony forming units (CFU) of said combination of live ein the n adult etween and 11 in said mnosus east 14 n more acillus acillus ns in a acillus acillus ly said D. Bird & Bird

Claims

- 44 - P2771PC Abstract The present invention refers to the use of a composition comprising live Lactobacillus acidophilus, live Lacticaseibacillus paracasei and live Lacticaseibacillus rhamnosus in the treatment of Autism spectrum disorder (ASD) 5 and in the treatment of multiple symptoms associated with ASD, selected from impaired communication, impaired sociability, impaired sensory awareness, impaired cognitive awareness and behaviors, gastrointestinal disorders or sleep disorders. The composition of the present invention is a nutritionally acceptable composition 10 comprising the combination of live Lactobacillus acidophilus, live Lacticaseibacillus paracasei and live Lacticaseibacillus rhamnosus selected from a group comprising a drinkable solution, a capsule, a tablet, a lozenge, gummies, powders, sachets, stick packs, , fermented beverages, juices or yogurt.

8. Composition for use according to any of the previous claims, wherein said sleep disorders are selected from a group comprising sleep duration, parasomnias, sleep disordered breathing and daytime sleepiness.

9. Composition for use according to any of the previous claims, wherein said composition is a nutritionally acceptable composition selected from a group comprising a drinkable solution, a capsule, a tablet, a lozenge, gummies, sachets, stick packs or powders, preferably said nutritionally acceptable composition is selected from the group comprising fermented beverages, juices or yogurt, more preferably said composition is a probiotic drinkable solution comprising the combination of live L. acidophilus, live L. paracasei and live L. rhamnosus.

10. Composition for use according to any of the previous claims comprising a combination of live L. acidophilus, live L. paracasei and live L. rhamnosus in the range of at least 10 billion colony forming units (CFU) to 200 billion colony forming units (CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus.

11. Composition for use according to claim 10 comprising least 10 billion colony forming units (CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus, preferably said composition comprises at least 50 billion colony forming units (CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus, more preferably said composition comprises at least 100 or 150 billion colony forming units (CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus.

12. Composition for use according to any of the previous claims, wherein said composition comprises 0.1 to 5% of / .. acidophilus, 65-99% of / .. paracasei and 0.5-10% of L. rhamnosus of the colony forming units (CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus, preferably said composition comprises 0.2-5% of L. acidophilus, 70-99% of L. paracasei and 1-10% of L. rhamnosus of the colony forming units(CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus, more preferably said composition comprises 0.5-5% of L. acidophilus, 75-99% of L. paracasei and 2-10% of L. rhamnosus of the colony forming units (CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus.

13. Composition for use according to claim 12, wherein said composition comprises 1-5% L. acidophilus, 75-85% L. paracasei and 5-10% L. rhamnosus of the colony forming units (CFU) of said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus.

14. Composition for use according to any of the previous claims, wherein the subject diagnosed with ASD is under the age of 18 years old or it is an adult subject over the age of 18 years old, preferably the subject is aged between 4 and 17 years old, more preferably the subject is aged between 4 and 11 years old.

15. Composition for use according to any of the previous claims, wherein said combination of live L. acidophilus, live L. paracasei and live L. rhamnosus is administered at least once a day, preferably for a period of at least 14 weeks, more preferably for a period of at least 20 weeks, even more preferably for a period of at least 30 weeks.

16. Use of the composition comprising a combination of live Lactobacillus acidophilus, live Lacticaseibacillus paracasei and live Lacticaseibacillus rhamnosus for inhibiting the growth of Clostridioides difficile strains in a subject affected by ASD.

17. Use of the composition comprising a combination of live Lactobacillus acidophilus, live Lacticaseibacillus paracasei and live Lacticaseibacillus rhamnosus for inducing a decrease of the level of p-cresol, preferably said level of / ?-cresol is reduced of about 80% in a subject affected by ASD.Bird & Bird

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