WO2025190897A1

WO2025190897A1 - Human milk oligosaccharides for lowering the risk of an infection in the gastrointestinal tract

Info

Publication number: WO2025190897A1
Application number: PCT/EP2025/056529
Authority: WO
Inventors: Mari Lilith LUND; Anja Wellejus; Erik JUNCKER BOLL; Stina JENSEN; Sara HESSING; Natalia JIMENEZ
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2024-03-11
Filing date: 2025-03-11
Publication date: 2025-09-18
Anticipated expiration: 2026-09-11
Also published as: CN119867152A

Abstract

The disclosure relates to a composition for use in reducing the risk of an infection in the gastrointestinal tract caused by a pathogenic E. coli in a human subject, said composition comprising at least one human milk oligosaccharide, wherein the human milk oligosaccharide is 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and / or lacto-N-tetraose.

Description

HUMAN MILK OLIGOSACCHARIDES FOR LOWERING THE RISK OF AN INFECTION IN THE GASTROINTESTINAL TRACT

FIELD

The present disclosure relates, in part, to the use of human milk oligosaccharides for reducing the risk of infections in the gastrointestinal tract, particularly diarrheal diseases, caused by Escherichia co// bacteria in a human subject. Further disclosed are combinations of human milk oligosaccharides with specific probiotics. Further disclosed herein are methods, uses, processes, and the like.

BACKGROUND

Human milk oligosaccharides (HMOs) are non-digestible carbohydrates found in human milk. Their importance to infant nutrition is underscored by their position as the third most abundant solid component of human milk, behind lactose and lipids. HMOs can be structurally categorized as (a) fucosylated HMOs such as 2’- and 3-fucosyllactose (2’-FL and 3-FL), (b) neutral non- fucosylated HMOs such as lacto-N-tetraose (LNT) and (c) sialylated HMOs such as 3’- and 6’ sialyllactose (3’-SL and 6’-SL).

Table 1 : Structures of HMOs used herein

HMOs in human milk vary widely based on various influences such as genetics, lactation, and geographic location. While most HMO concentrations decrease over the course of lactation, at least two, 3’-SL and 3-FL, may increase. Different HMOs may work together in complementary ways to support the growth and development of infants.

It has been suggested that HMOs can lower the risk of gut microbiome imbalance due to harmful bacteria (Weichert, Stefan, et al. Nutrition researchi 0 (2013): 831-838). Further, it has been suggested that HMOs can selectively stimulate growth and metabolic activity of helpful bifidobacteria in support of overall gut health (Bode, Lars. Nutrition reviews, 2009, Vol 67 suppl. 2,183.191). Upon infection with a pathogen, the immune system responds with a complex mechanism to eliminate the pathogen and at the same time to avoid damage to the host due to an excessive immune response. The secretion of pro-inflammatory cytokines such as IFN-y and TNF-a plays a crucial role in enhancing the immune response against infections, including those caused by Escherichia coli (Akdis M. et al., 2016 Journal of Allergy and Clinical Immunology, 984-1010; Sanchez-Villamil et al., 2016 Frontiers in Cellular and Infection Microbiology, 6(OCT), p. 219189). For a balanced immune response, the secretion of anti-inflammatory cytokines such as IL-10 and IL-1 receptor antagonist (IL-1 RA) during bacterial infections is relevant for modulating the immune response and preventing excessive inflammation (Raphael et al., 2016 Cytokine, 74(1), pp. 5-17). Therefore, supporting a balanced immune response may be relevant to reducing the risk of an infection with a pathogenic E.coli.

An intact intestinal barrier plays an important role for effective protection against infections of the gastrointestinal tract. The intestinal barrier consists of different elements. One element is a single layer of epithelial cells, which form a physical barrier that plays a crucial role in separating luminal microorganisms from the host immune system and the circulatory system. Tight junctions between the epithelial cells regulate the exchange of substances via the barrier (Bischoff S. C. et al.; BMC Gastroenterology, 2014, 14, 189). The tight junctions can be disrupted by bacterial pathogens and their virulence factors, thereby leading to increased permeability of the epithelial layer (Kbnig et al., Clinical and Translational Gastroenterology, 2016, 7 (10)). Increased permeability and ensuing systemic inflammation are associated with a plethora of health issues, including autoimmune diseases (e.g. inflammatory bowel diseases, arthritis, allergies), metabolic diseases (e.g. obesity), cancer, and neurological disfunctions (Pilla, R., Suchodolski, J.S.; Veterinary Clinics of North America: Small Animal Practice, 51 , 3, 605-621 and Current Opinion in Clinical Nutrition and Metabolic Care 25(3); 178-185). Moreover, it may lead to systemic spread of pathogenic or opportunistic pathogenic bacteria, such as pathogenic E.coli.

E. coli is among the first colonizers and a common inhabitant of the infant gut microbiome. It persists due to e.g., the expression of fimbriae that enables it to attach to colonic epithelial cells (Nowrouzian, F. et al., 2003, Pediatric Research, 54(1), 8-14). It comprises a wide variety of strains ranging from commensals to severe pathogens depending on the presence of virulence factors (Evans DJ Jr. et al. in: Baron S, Medical Microbiology, 4th edition, Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 25).

Enterohemorrhagic E. coli (EHEC) is a pathotype of E. coli associated with foodborne outbreaks worldwide. Clinical manifestations of EHEC infection range from mild diarrhea to severe hemorrhagic colitis and hemolytic uremic syndrome. Infants and children are the main affected patients (Gomes TAT et al, 2016; 47; 3-30). EHEC requires binding to the intestinal epithelium by virtue of adhesins to establish infection (McWilliams BD, Torres AG, Microbiol Spectr. 2014; 2(3)). Thus, reduction of this binding may lower the risk of colonization and ensuing diarrheal disease caused by EHEC and other E. coli pathotypes.

Collectively, E. coli is the leading cause of infantile diarrhea, which is the second most common cause of death among children under the age of five worldwide (Johansson, E. W. et al., 2009, in The United Nations Children’s Fund (UNICEF)ZWorld Health Organization (WHO), Vol. 44, 11 , 1- 68). Thus, reducing the colonization of E. coli in the gastrointestinal tract may lower the risk of diarrheal diseases caused by E. coli.

Strategies are needed that may lower the risk of diarrheal diseases caused by pathogenic E. coli and in particular by the EHEC pathotype.

SUMMARY

The present disclosure provides compositions, uses, methods and the like for reducing the risk of an infection in the gastrointestinal tract, as for example a diarrheal disease, in a human subject. In particular, the present disclosure relates to a composition comprising at least one human milk oligosaccharide (HMO), wherein the human milk oligosaccharide is selected from the group of 3- fucosyllactose (3-FL), 3'-sialyllactose (3’-SL), 6'-sialyllactose (6’-SL) and lacto-N-tetraose (LNT) for lowering the risk of infections in the gastrointestinal tract, in particular diarrheal diseases, in a human subject, in particular in non-adult subjects.

While not wishing to be bound by theory, it is believed that the HMOs inhibit the binding of pathogenic E. coli to the intestinal mucosal barrier and supports intestinal gut barrier integrity. The binding of pathogenic E.coli to epithelial cells is one step in the mechanism of infection. Thus, by inhibiting this binding, it is believed that the risk of infections with pathogenic E. coli is reduced. Further, it is believed that HMOs may enhance the immune response, which is believed to be beneficial for the clearance of pathogens such E.coli.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the reduction of adhesion of a strain of enterohemorrhagic E. coli (EHEC) serotype 0157 to monolayers of human colon cancer cells (Caco-2) in the presence of 3-FL, 3’SL, 6’SL and LNT at a dosage of 10 mg/ml each. Data are expressed as means + standard deviation (n = 3 independent experiments). Statistical significance was determined by One-way ANOVA comparison to the EHEC control group followed by Dunnett’s multiple comparisons test.

Figure 2 shows the results of transepithelial electrical resistance (TEER) measurements of Caco- 2 cell monolayers stimulated with L. rhamnosus, LGG®, 3-FL, combination of the two, or media alone measured for 10 hours in triplicates. (A) shows the TEER percent change relative to the baseline (y-axis) versus the time on the x-axis. (B) shows the TEER area under the curve (AUC) for the 4 conditions for 10 hours. Data are mean + SD (n=3). **** indicate p values < 0.0001 tested using One-way ANOVA with multiple comparisons.

Figure 3 Secretion of anti-inflammatory cytokines IL-10 (A) or IL-1 RA (B) from human PBMCs coincubated with 3-FL, LGG® or the synbiotic combination and challenged with LPS from E. coli for 20 hours.

Figure 4 Secretion of IFN-y (A) and TNF-a (B) from human PBMCs co-incubated with 3-FL, LGG® or the synbiotic combination and challenged with LPS from E. coli for 20 hours. Significant differences are expressed as follows: *= P<0.05, **= P<0.01 , ***= P<0.005 and ****= P<0.001 .

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by persons skilled in the art. Although any methods and materials equivalent or similar to those described herein can be used in the practice of the present disclosure, typical methods and materials are described. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range including the two end values, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

As used herein, the term “and/or” is intended to mean the combined (“and”) and the exclusive (“or”) use, i.e. “A and/or B” is intended to mean “A alone, or B alone, or A and B together”.

As used herein the terms "effective amount", "effective concentration", or "effective dosage" are defined as the amount, concentration, or dosage of a material sufficient to improve the overall health of the subject and confer benefits similar to the ones demonstrated in the examples. The actual effective dosage in absolute numbers depends on factors including the state of health of the subject in question, and other ingredients present. The "effective amount", "effective concentration", or "effective dosage" of the material may be determined by routine assays known to those skilled in the art.

As used herein the term "isolated" means that the bacterial strains described herein are in a form or environment which does not occur in nature, i.e. the strain is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.

A bacterial “strain” as used herein refers to a bacterium which remains genetically unchanged when grown or multiplied and that originates from a single isolate or pure culture. Probiotics are classified by their genus (e.g. Bifidobacterium), species and subspecies (e.g. animalis subs p. lactis), and strains (e.g. DSM 15954 and/or BB-12®). FAO/WHO has stated that probiotic effects are strain specific and that most probiotic characteristics of a particular strain cannot therefore be extrapolated to other strains of the same species.

As used herein, the term “probiotic” refers to a culture of live or freeze-dried microorganisms, dead microorganisms, fragments of microorganisms and extracts or supernatants of microorganisms which, when applied to man or animal, beneficially affects the host (Hill et al. (2014) Expert Consensus Document, The International Scientific Association for Probiotics and Prebiotics. Consensus statement on the scope and appropriate use of the term probiotic).

The term "human milk oligosaccharide" or "HMO", as used herein, unless otherwise specified, refers generally to a number of complex carbohydrates found in human breast milk that can be in acidic or neutral form, and to precursors thereof. Exemplary non-limiting human milk oligosaccharides include 3'- sialyllactose, 6'-sialyllactose, 3-fucosyllactose, 2'-fucosyllactose, and lacto-N-tetraose.

The terms "treat" or "treating" should not be taken to imply that an individual is treated until total recovery. Accordingly, these terms broadly include amelioration and/or prevention of the onset of the symptoms or severity of a particular condition.

The term “lowering or reducing the risk of an infection” include prevention of the onset of the symptoms or reduction of the severity of the symptoms. The risk of an infection is reduced or lowered compared to the situation in absence of the claimed composition.

The term "shelf stable" as used herein, unless otherwise specified, refers to a nutritional product that remains commercially stable after being packaged and then stored at 18-24°C for at least 3 months, including from about 6 months to about 24 months, and also including from about 12 months to about 18 months. The terms "nutritional formulation" or "nutritional composition" as used herein, are used interchangeably and, unless otherwise specified, refer to nutritional liquids, nutritional powders, nutritional supplements, and any other nutritional food product as known in the art. The nutritional powders may be reconstituted to form a nutritional liquid, all of which comprise one or more of fat, protein and carbohydrate and are suitable for oral consumption by a human.

The term "nutritional powder" as used herein, unless otherwise specified, refers to nutritional products in flowable or scoopable form that can be reconstituted with water or another aqueous liquid prior to consumption and includes both spray-dried and dry-mixed dry-blended powders.

The term "newborn" as used herein, unless otherwise specified, refers to a person from birth up to four weeks of age. The term “infant” as used herein, unless otherwise specified, refers to a person 12 months or younger. The term "preterm" as used herein, refers to a baby born prior to 36 weeks of gestation. The term "toddler" as used herein, unless otherwise specified, refers to a person greater than one year of age up to three years of age. The term "child" as used herein, unless otherwise specified, refers to a person greater than three years of age up to twelve years of age.

The term “formula” as used herein, unless otherwise specified, refers to liquid and solid human milk replacements or substitutes that are suitable for consumption by a human.

The term "human milk fortifier" as used herein, unless otherwise specified, refers to liquid and solid nutritional products suitable for mixing with breast milk or formula for consumption by a preterm or term infant.

The terms "susceptible" and "at risk" as used herein, unless otherwise specified, mean having little resistance to a certain condition or disease, including being genetically predisposed, having a family history of, and/or having symptoms of the condition or disease. The terms "modulating" or "modulation" or "modulate" as used herein, unless otherwise specified, refer to the targeted movement of a selected characteristic.

The term “enhanced immune response” relates to an increase of the level of pro-inflammatory cytokines TNF-alpha and/or IFN-gamma compared to the level of the respective cytokines observed without the composition according to the invention.

The term “balanced immune response” relates to a stable level of secreted anti-inflammatory cytokines IL-10 and IL-1 RA compared to the level of the respective cytokines observed without the composition according to the invention. The term purity used in this application refers to chemical purity, thus the degree to which a substance is undiluted or unmixed with extraneous material. Hence, the chemical purity is an indicator of the relationship between the at least one HMO and by-products/impurities.

Chemical purity is expressed as a percentage (%) and is calculated using the following formula: Percent purity= 100x (mass of desired compound in sample)/(total mass of sample)

The purity can be determined by any suitable method known to the person skilled in the art. One suitable method is HPLC (high-performance liquid chromatography). In the obtained chromatogram, the ratio of the area underneath the peak(s) representing the amount of HMO(s) to the sum of areas underneath the peaks representing the HMO(s) and all other compounds than said HMO(s) in the chromatogram is calculated.

All percentages, parts and ratios as used herein, are by weight of the total composition, unless otherwise specified. All such weights, as they pertain to listed ingredients, are based on the active level and, therefore, do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified.

The term “sequence identity of [a certain] %” in the context of two or more nucleotide sequences refers to a relationship between the sequences of two polynucleotides, as determined by sequence comparison (alignment). As used herein, “sequence identity” is determined across the entire length of a sequence. “Sequence identity” means that the two or more sequences have nucleotides in common in the given percentage when compared and aligned. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model, algorithms, or computer program.

Percent sequence identity of nucleotide sequences can be readily calculated by any of the methods known to one of ordinary skill in the art. In preferred embodiments, the “percent identity” of two sequences (e.g., polynucleotide or amino acid sequences) is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST® and XBLAST® programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. Where gaps exist between two sequences, Gapped BLAST ® can be utilized, for example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST® programs, the default parameters of the respective programs (e.g., XBLAST® and NBLAST®) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.

Another local alignment technique which may be used, for example, is based on the Smith- Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) J. Mol. Biol. 147:195-197). A general global alignment technique which may be used, for example, is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) J. Mol. Biol. 48:443-453), which is based on dynamic programming.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

Numerical ranges as used herein are intended to include every number and subset of numbers within that range, whether specifically disclosed or not.

The present invention has been described with reference to various embodiments, aspects, examples, or the like. It is not intended that these elements be read in isolation from one another. Thus, the present disclosure provides for the combination of two or more of the embodiments, aspects, examples, or the like.

All embodiments described herein are intended to be within the scope of the invention disclosed. These and other embodiments of the present invention will become readily apparent to those skilled in the art from the following detailed description of the preferred embodiments having reference to the whole description, the invention not being limited to any particular preferred embodiment(s) disclosed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The present disclosure provides a composition comprising at least one human milk oligosaccharide (HMO), selected from 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose. While not wishing to be bound by theory, it is believed that the composition comprising at least one of these HMOs reduces the risk for a human subject of getting an infection in the gastrointestinal tract caused by a pathogenic E. coll. This is supported by the findings in in vitro experiments that the binding of a pathogenic E. coli to epithelial cells is inhibited in the presence of these HMOs.

To evaluate the influence of the HMOs on the binding of pathogenic E. coli to the intestinal epithelium, a strain of enterohemorrhagic E. coli (EHEC) of pathotype 0157 was pre-incubated or not with the HMOs at different dosages. The pre-incubated E. coli were added to monolayers of human colon adenocarcinoma Caco-2 cells. After a defined incubation time, the number of EHEC adhering to the intestinal cell monolayers was determined. It was found that the HMOs structure-dependently reduced binding of EHEC to the cell monolayers (Figure 1). While not wishing to be bound by theory, it is believed that the reduction of EHEC binding to intestinal epithelial cells, may lower the risk of an infection.

The influence of substances on the intestinal barrier were investigated in another in vitro experiment. Methods for exploring modeling of intestinal barrier function in vitro exist and for this purpose the Caco-2 cell line is routinely used. The intestinal permeability function is not static but can be deliberately modulated by exposure to specific stimuli and insults and followingly can be assessed by measuring the transepithelial electrical resistance (TEER) across the cell monolayers. An increase in TEER following stimulation compared to unstimulated cell monolayers indicate strengthened gut barrier integrity. Conversely, a decrease in TEER relative to that of unstimulated cells is indicative of increased intestinal permeability which - among other undesirable consequences, including systemic inflammation - may increase the risk of systemic dissemination and disease severity caused by pathogenic bacteria.

The cytokine profile induced by pre-treatment of human PBMCs with compositions according to the invention was investigated in response to a challenge with E.coli LPS. This allows to study the immune response against a pathogen, which is crucial for estimating the effectiveness of pathogen clearance during an infection with the pathogen. It is believed that an enhanced and at the same time balanced immune response will be important for reducing the risk and I or severity of an infection in the gastrointestinal tract. An enhancement of the anti-pathogenic immune response is coupled to an increase of the level of pro-inflammatory cytokines, such as TNF-alpha and IFN-gamma.

In certain embodiments, the composition comprising at least one HMO of 3-FL, 3’SL, 6’SL and I or LNT is used to lower the risk of diarrheal diseases. Infections of the gastrointestinal tract often come with diarrhea as a prominent symptom. While not wishing to be bound by theory, it is believed that the application of a composition comprising at least one HMO of 3-FL, 3’SL, 6’SL and I or LNT may lower the risk of diarrheal diseases.

In certain embodiments, the composition comprising at least one HMO of 3-FL, 3’SL, 6’SL and I or LNT is used to reduce the risk of an infection in the gastrointestinal tract by inhibiting the adhesion of E. co// to intestinal epithelial cells. It is known that one step in the mechanism of infection is the attachment to the epithelial cells.

In certain embodiments, the composition comprising at least one HMO of 3-FL, 3’SL, 6’SL and I or LNT is used to reduce the risk of an infection in the gastrointestinal tract by supporting the intestinal barrier function.

In certain embodiments the composition comprises one, or two, or three HMOs selected from 3- FL, 3’SL, 6’SL or LNT.

In certain embodiments, the composition comprises two HMOs selected from 3-FL, 3’SL, 6’SL or

LNT. In certain embodiments, the composition comprises one HMO selected from 3-FL, 3’SL, 6’SL or

LNT.

In certain embodiments, the composition comprises 3’SL and / or 6’SL as the only HMOs. In in vitro experiments, these HMOs exhibit an inhibitory effect on the binding of pathogenic E.coli to epithelial cells.

In certain embodiments, the composition comprises 3-FL and I or LNT as the only HMOs. In in vitro experiments, these HMOs exhibit an inhibitory effect on the binding of pathogenic E.coli to epithelial cells.

In certain embodiments, the composition comprises 3-FL as the only HMO. In in vitro experiments, this HMO has been shown to improve the intestinal barrier function.

In certain embodiments, the present composition comprises an effective amount of a probiotic strain. For example, where the probiotic is present it is preferred, the probiotic has a concentration ranging from 0.05 x 10⁹ CFU/dose to 30 x 10⁹ CFU/dose, preferably from 0.5 x 10⁹ CFU/dose to 25 x 10⁹ CFU/dose. A dose herein refers to the amount taken on one day.

In certain embodiments, the present compositions may comprise at least one probiotic strain, for example, Lactococcus lactis subsp. lactis biovar. Diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus (including but not limited to Lactobacillus acidophilus, Lacticaseibacillus easel subsp. easel, Lacticaseibacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus salivarius), any strain belonging to the genus Bifidobacterium (including but not limited to Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum), or any strain from the genera of Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridia, Coprococcus, Dorea, Eubacterium, Faecalibacterium or Roseburia or the family Coriobacteriaceae, as well as suitable combinations of the foregoing.

The present compositions may comprise at least one strain of a bacterium selected from the group comprising Bifidobacterium animalis subsp. lactis deposited as DSM 15954, Lactobacillus acidophilus deposited as DSM 13241 , Lactobacillus rhamnosus deposited as ATCC 53103, Lacticaseibacillus paracasei subsp. paracasei deposited as ATCC 55544, Lacticaseibacillus paracasei deposited as LMG-17806, Streptococcus thermophilus deposited as DSM 15957, Lactobacillus fermentum deposited as NM02/31074, Lacticaseibacillus paracasei subsp. paracasei deposited as CCTCC M204012 and suitable combinations thereof.

In certain embodiments, the composition comprises the probiotic Lacticaseibacillus rhamnosus GG. It is preferred that the probiotic is LGG® produced and sold by Chr. Hansen A/S. It is preferred that the nucleotide sequence of the Lacticaseibacillus rhamnosus GG has at least 99.00% identity to SEQ ID NO. 1 , preferably at least 99.10%, at least 99.20%, at least 99.30%, at least 99.40%, at least 99.50%, at least 99.60%, at least 99.70%, at least 99.80% identity to SEQ ID NO.1 , more preferably at least 99.90%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98% identity to SEQ ID NO. 1 , even more preferably at least 99.99% identity to SEQ ID NO. 1 or is identical to SEQ ID NO. 1 .

In certain embodiments, the composition comprises the probiotic Lacticaseibacillus rhamnosus GG and the nucleotide sequence of the Lacticaseibacillus rhamnosus GG differs in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides from the nucleotide sequence of SEQ ID NO.1 . Preferably, the nucleotide sequence of the Lacticaseibacillus rhamnosus GG differs in no more than 5000, 4000, 3000, 2750, 2500, 2250, 2000, 1750, 1500, 1250, 1000, 750, 500, 400 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15 nucleotides from the nucleotide sequence of SEQ ID NO.1 .

The nucleotide sequence of SEQ ID NO. 1 is available under reference number GCF_028475085.1 in the ncbi database (htps://www.ncbi.nlm.nih.qov/) as Lacticaseibacillus rhamnosus GG genome assembly ASM2847508v1 . Reference is made to the database in the version accessible on 07 March 2024.

It is known that Lacticaseibacillus rhamnosus GG LGG® can inhibit pathogenic E.coli (Petrova M.l. et al., 2016, PLoS ONE, 11 (8), e0161337) . In vitro experiments show that LGG® also improved intestinal barrier function (Fig. 2).

In certain embodiments, the composition comprises Lacticaseibacillus rhamnosus GG and 3-FL, preferably Lacticaseibacillus rhamnosus GG and 3-FL as the only HMO. In vitro experiments show that LGG® and 3-FL have a beneficial effect on the intestinal barrier function (Fig. 2 and 3). In in vitro experiments, the pretreatment of human PBMCs with the combination of 3-FL and LGG® resulted in a significant boost in the secretion of TNF-a and IFN-y compared to an E. coli LPS challenge.

TNF-a is known for its ability to stimulate immune cell function, enhancing the phagocytic activity of macrophages and neutrophils, which are essential for the clearance of bacterial infections such as E. coli. This cytokine not only promotes the recruitment of immune cells to the site of infection but also enhances their bactericidal functions (Trevejo et al., 2001 Proceedings of the National Academy of Sciences of the United States of America, 98(21), pp. 12162-12167; Jeong et al., 2019 J. Microbiol. Biotechnol., 29(8), pp. 1248-1254, Jeong et al., 2020 J. Microbiol. Biotechnol., 30(9), pp. 1395-1403). Similarly, IFN-y enhances bactericidal activity of macrophages and has been shown to enhance the expression of major histocompatibility complex (MHC) molecules, which are crucial for further promoting adaptive immune responses (Wu et al., 2019 Immunology, 158(4), pp. 304-313). Therefore, the boost of these cytokines induced by LGG® and 3-FL might reduce the risk of developing E. coll infections.

Additionally, maintaining appropriate levels of anti-inflammatory cytokines such as IL-10 and IL- I RA during bacterial infections is crucial for modulating the immune response and preventing self-harm due to excessive inflammation (Sun et al., 2015 Mucosal Immunology doi:10.1038/mi.2015.49; Akdis et al., 2016 Journal of Allergy and Clinical Immunology, pp. 984- 1010). In vitro experiments show that the levels of these cytokines secreted by human PBMCs after the pretreatment with LGG® and 3FL are maintained, supporting a balanced immune response against E. coll and reducing the risk of tissue damage.

In certain embodiments, the composition is a nutritional composition such as a formula or a dietary supplement.

A preferred composition herein is a nutritional composition such as a formula. The nutritional compositions may be in any product form comprising the ingredients described herein, and which is safe and effective for oral administration. The nutritional compositions may be formulated with optional ingredients such as those described herein.

The nutritional compositions of the present disclosure are preferably formulated as dietary product forms, which are defined herein as those embodiments comprising the ingredients of the present disclosure in a product form that then contains at least one of fat, protein, and carbohydrate, and preferably also contains vitamins, minerals, or combinations thereof.

The nutritional compositions may be formulated with sufficient kinds and amounts of nutrients to provide a sole, primary, or supplemental source of nutrition, or to provide a specialized nutritional product for use in individuals afflicted with specific diseases or conditions or with a targeted nutritional benefit as described below. Specific non-limiting examples of product forms suitable for use as HMO-containing compositions as disclosed herein include, for example, liquid and powdered dietary supplements, liquid and powdered human milk fortifiers, liquid, and powdered formula.

Nutritional liquids include both concentrated and ready-to-feed nutritional liquids. These nutritional liquids are most typically formulated as suspensions or emulsions, although other liquid forms are within the scope of the present disclosure. Nutritional emulsions suitable for use may be aqueous emulsions comprising proteins, fats, and carbohydrates. These emulsions are generally flowable or drinkable liquids at from about 1 °C to about 25°C and are typically in the form of oil- in-water, water-in-oil, or complex aqueous emulsions, although such emulsions are most typically in the form of oil-in-water emulsions having a continuous aqueous phase and a discontinuous oil phase.

The nutritional emulsions may be and typically are shelf stable. The nutritional emulsions typically contain up to about 95% by weight of water, including from about 50% to about 95%, also including from about 60% to about 90%, and also including from about 70% to about 85%, of water by weight of the nutritional emulsions. The nutritional emulsions may have a variety of product densities, but most typically have a density greater than about 1 g/mL, including greater than about 1.05 g/mL, including greater than about 1.055 g/mL to about 1.12 g/mL, and also including from about 1.085 g/mL to about 1.10 g/mL. The nutritional emulsions may have a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the emulsions comprise generally at least 660 kcal/liter, about 675 kcal/liter to about 820 kcal/liter, about 680 kcal/liter to about 800 kcal/liter. In some embodiments, the emulsion may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter. In some specific embodiments, the emulsion may have a caloric density of 25, or 50, or 75, or 100 kcal/liter. The nutritional emulsion may have a pH ranging from about 3.5 to about 8, from about 4.5 to about 7.5, including from about 5.5 to about 7.3, including from about 6.2 to about 7.2. Although the serving size for the nutritional emulsion can vary depending upon a number of variables, a typical serving size is generally at least 1 mL, or even at least 2 mL, or even at least 5 mL, or even at least 10 mL, or even at least 25 mL, including ranges from about 1 mL to about 300 mL, including from about 4 mL to about 250 mL, and including from about 10 mL to about 240 mL.

The nutritional solids may be in any solid form but are typically in the form of flowable or substantially flowable particulate compositions, or at least particulate compositions, that may optionally be compressed into tablets. Particularly suitable nutritional solid product forms include spray dried, agglomerated and/or dry-blended powder compositions. The compositions can easily be scooped and measured with a spoon or similar other device and can easily be reconstituted by the intended user with a suitable aqueous liquid, typically water, to form a nutritional composition for immediate oral or enteral use. In this context, "immediate" use generally means within about 48 hours, most typically within about 24 hours, preferably right after reconstitution. The nutritional powders may be reconstituted with water prior to use to a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the powders are reconstituted with water to form compositions comprising generally at least 660 kcal/liter, about 675 kcal/liter to about 820 kcal/liter, about 680 kcal/liter to about 800 kcal/liter. In some embodiments, the reconstituted powder may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter. In some specific embodiments, the reconstituted powder may have a caloric density of 25, or 50, or 75, or 100 kcal/liter.

The present compositions may be useful in newborns, infants, toddlers, or children. The present compositions may be useful in newborns. The present compositions may be useful in infants. E. coli is the leading cause of infantile diarrhea, which indicates that infants may particularly benefit from the present composition.

The present composition may comprise an effective amount of 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and/or lacto-N-tetraose.

The present composition may comprise at least 0.01%, by weight, 3-fucosyllactose; at least 0.01%, by weight, 3'-sialyllactose; at least 0.01%, by weight, 6'-sialyllactose; and I or at least 0.01%, by weight, lacto-N-tetraose.

The present composition may comprise 3-fucosyllactose, 3’sialyllactose, 6’-sialyllactose and /or lacto-N-tetraose in any suitable amount, such as, for example, at least about 0.001 g/dose, at least about 0.01 g/dose, at least about 0.05 g/dose, at least about 0.1 g/dose, at least about 0.5 g/dose. The compositions may, for example in the case of a dietary supplement, comprise less than about 20 g/dose, less than about 10 g/dose, less than about 5 g/dose, less than about 4 g/dose, less than about 3 g/dose, less than about 2 g/dose, less than about 1 .5 g/dose of 3- fucosyllactose, 3’sialyllactose, 6’-sialyllactose and /or lacto-N-tetraose.

The present compositions may comprise individual HMOs in any suitable amount, such as, for example, at least 0.001 mg/mL, including from about 0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about 10 mg/mL, including from about 0.01 mg/mL to about 5 mg/mL (mg of particular HMO per mL of composition).

Where the composition is a nutritional powder, the concentration of individual HMOs in the nutritional powder is preferably from about 0.001% to about 5%, including from about 0.01% to about 1% (by weight of the nutritional powder). Where the composition is a ready- to-feed nutritional liquid, the concentration of individual HMOs is preferably from about 0.001% to about 0.50%, including from about 0.001% to about 0.15%), including from about 0.01% to about 0.10%, and further including from about 0.01%) to about 0.03% (by weight of the ready-to-feed nutritional liquid). Where the composition is a concentrated nutritional liquid, the concentration of individual HMOs is preferably from about 0.002% to about 0.6%, including from about 0.002% to about 0.3%, including from about 0.02% to about 0.20% (by weight of the concentrated nutritional liquid). The present composition may be in the form of a powder. Formulating compositions with HMOs can be somewhat problematic. It has been found that a more reproducible and consistent composition can be achieved through controlling the particle size distribution (PSD) of the HMO. While not wishing to be bound by theory, it is believed that having a somewhat narrow PSD improves the flowability of the HMO enabling a more effective mixing with the other ingredients. In addition, it is believed that a PSD within a certain range provides a better solubility profile. Particle size of an HMO may be determined using a standard method, such as using a sieve tower, which separates the powder into the different fractions after a defined time with a predefined amplitude. The sieves used in such a method may be sieves which comply with DIN ISO 3310-1.

It is preferred that the HMOs, in particular 3-FL, used in the present compositions have the following particle size characteristics:

Percent through mesh #230 (63 pm) - less than about 20%, less than about 18%, less than about 16%, less than or equal to about 15%.

Percent through mesh #100 (150 pm) - greater than about 75%, greater than about 70%, greater than about 65%, greater than or equal to about 60%.

Percent through mesh #45 (355 pm) - greater than about 95%, greater than about 92%, greater than or equal to about 90%.

Percent through mesh #20 (850 pm) - 100%.

It is preferred that the human milk oligosaccharides when in form of a powder, have a water activity a_w between 0.10 and 0.3, preferably between 0.10 and 0.25. The water activity may be determined using known hygrometers; preferably the water activity is determined according to ISO 18787:2017. The water activity ensures the microbial stability of the powder and prevents contamination with undesired microorganisms. This is in particular important for compositions comprising probiotic strains.

The present composition preferably comprises HMOs of synthetic origin, such as HMOs produced by microbial fermentation, or alternatively HMOs produced by biocatalysis or chemical synthesis. In particular, microbial fermentation allows production on an industrial scale in high purities, which may be used in nutritional compositions. For the present composition, preferably HMOs are used that have a purity of more than 85%, preferably a purity of more than 90%, more preferably a purity of more than 95%.

The present compositions may comprise probiotics. The compositions may be formulated as combined or as separate compositions of the probiotic strains and the HMO. The present compositions may comprise these probiotics in any suitable form for administration to the subject. In a preferred embodiment, the compositions may comprise the bacteria in dried form, which can be obtained by freeze-drying, spray-drying, lyophilization, or the like.

If the bacteria are freeze-dried, they are generally mixed with a cryoprotectant before they are freeze-dried. The term “a cryoprotectant” is used herein to refer to a substance that is able to improve the survival during freezing and/or drying and to improve the storage stability of bacteria. The cryoprotectant used herein preferably comprises a saccharide and/or a sugar alcohol such as inositol.

The saccharide may be a mono-, di-, oligo- or polysaccharide, or a mixture of at least two saccharides. Useful monosaccharides include, for example, glucose (also known as dextrose), fructose, ribose and galactose and useful disaccharides include, for example, sucrose, trehalose, maltose and lactose. The composition may comprise one or more mono- or disaccharides, such as one, two, or three or even more different saccharides.

The cryoprotectant may comprise a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.

The cryoprotectant may further comprise a peptide, protein, protein hydrolysate or a mixture thereof. Examples of peptides and proteins to be used are casein, pea, whey, albumin, glutamic acid or gelatin, and any isolate or hydrolysate thereof. Other additives, e.g. antioxidants such as sodium ascorbate, sodium citrate, trisodium citrate dihydrate and cysteine hydrochloride may also be present. Skim milk powder and yeast extract may also be ingredients.

The compositions of the present disclosure may optionally include anti-inflammatories such as long-chain polyunsaturated fatty acids (LCPUFAs) and/or antioxidants such as carotenoids. LCPUFAs may be included in the compositions to provide nutritional support and to enhance growth and functional development of the intestinal epithelium and associated immune cell populations. Exemplary LCPUFAs for use in the present compositions include, for example, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), linoleic acid, linolenic acid (alpha linolenic acid) and gamma-linolenic acid derived from oil sources such as plant oils, marine plankton, fungal oils, and fish oils. The present compositions preferably comprise total concentrations of LCPUFA of from about 0.01 mM to about 10 mM and including from about 0.01 mM to about 1 mM. Alternatively, the compositions comprise total concentrations of LCPUFA of from about 0.001 g/L to about 1 g L.

Additionally, antioxidants such as carotenoids, and particularly, combinations of the carotenoids, lutein, lycopene, zeaxanthin and/or beta-carotene may be included in the present compositions. The compositions of the present disclosure may further comprise other optional components that may modify the physical, chemical, aesthetic, or processing characteristics of the composition or to serve as pharmaceutical or additional nutritional components. Non-limiting examples of such optional ingredients include preservatives, emulsifying agents, buffers, pharmaceutical actives, nutrients, colorants, flavors, thickening agents and stabilizers, flowing agents, minerals, emulsifying agents, lubricants, sweetening agents, and the like.

A flowing agent or anti-caking agent may be included in the present compositions to retard clumping or caking of the powder over time and to make a powder embodiment flow easily from its container. Non-limiting examples include tricalcium phosphate, silicates, and combinations thereof. The concentration of the flowing agent or anti-caking agent in the nutritional composition varies depending upon the product form, the other selected ingredients, the desired flow properties, and so forth, but most typically range from about 0.1% to about 4%, including from about 0.5% to about 2%, by weight of the nutritional composition.

The compositions of the present disclosure may be prepared by any known or otherwise effective manufacturing technique for preparing the selected product solid or liquid form. Many such techniques are known for any given product form such as nutritional liquids or powders and can easily be applied by one of ordinary skill in the art to the nutritional compositions described herein.

Those skilled in the art will appreciate that the administration of compositions disclosed herein can be carried out with dose levels and dosing regimens as required depending on the circumstances and on the condition of the subject. Suitable dosage regimes can be determined based on the teaching of the present application. Dosage regimens may be adjusted to provide the optimal support of the subject. It will be appreciated that the exact amounts and rates of administration will depend on a number of factors such as the age, body weight, general health, sex, and dietary requirements of the subject. Based on the teaching herein those skilled in the art can, by routine trial and experimentation, determine suitable dosage regimes on a case-by-case basis.

EXAMPLES

Example 1

The HMOs 3-FL, LNT, 3’-SL, and 6’-SL, were produced by Chr. Hansen HMO GmbH, Rheinbreitbach, Germany. Stocks of HMO solutions were dissolved in water.

The human epithelial intestinal cancer cell line Caco-2 (ACC 169, DSMZ, passages 5-20) was maintained in Dulbecco’s Modified Eagle Medium (DMEM) GlutaMAX Supplement (Gibco) supplemented with 1% non-essential amino acids (Merck Life Science), 1% penicillin- streptomycin (10,000 U/mL) (Gibco), and 10% heat-inactivated fetal bovine serum (Gibco) at 37°C in a 5% CO2 atmosphere. The cells were seeded at a density of 8x10⁴ cells/well in 24-well culture plates, and the media was replaced every 3-4 days until cells were ready for use in the experiment after 14 days.

A strain of EHEC 0157 (DSM 17076) was grown agitated overnight in Luria-Bertani (LB) broth at 37°C, then washed twice using Hanks balanced salt solution (HBSS, Gibco), and resuspended and ODeoonm-norrnalized to 0.5 in DMEM. The EHEC strain was then diluted 1 :20 in DMEM to approximately 2x10⁶ colony forming units (CFU)/ml and pre-incubated or not with the single HMOs at a dosage of 10mg/ml with agitation at room temperature. After 2hrs of pre-incubation, the cell culture media was removed from the Caco-2 cell monolayers, which were then gently washed twice using HBSS. The EHEC strain suspensions (with and without HMOs) were then added to the Caco-2 cell monolayers, and after 1 hr of incubation, the media containing nonadhering EHEC was removed. The Caco-2 cell monolayers were gently washed trice in HBSS and following 3 washing steps, 0.1% Triton X-100 in HBSS was added to wells to release the adhering EHEC. The number of EHEC adhering to the intestinal cell monolayers was then quantified by serial plating on tryptic soy broth (plates) and counting of CFU) following overnight incubation at 37°C.

Results

The results showed that individual HMOs (tested at 10mg/ml) structure-dependently reduced EHEC binding with statistical significance (p<0.01) achieved for 3FL, 3’SL, 6’SL and LNT and the greatest effect observed for 3’SL and 6’SL (approx. 40% reduction). Data are expressed as means + standard deviation (n = 3 independent experiments). Statistical significance was determined by One-way ANOVA comparison to the EHEC control group followed by Dunnett’s multiple comparisons test.

Example 2

L. rhamnosus, LGG® (SEQ ID NO.1) and 3-FL (3-fucosyllactose) combined introduces higher intestinal barrier tightness as evaluated by transepithelial electrical resistance (TEER) across a Caco-2 cell monolayer in vitro compared with the components tested individually.

Culturing of Caco-2 cells

The human intestinal epithelial Caco-2 cell line (DSMZ ACC 169, Leibniz-lnstitut DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was cultured in complete DMEM medium containing DMEM GlutaMax supplemented with 20% heat inactivated fetal bovine serum, 1 X MEM non-essential amino acids and 1 X Pen-Strep-Amp B at 5% CO2 at 37°C. Caco-2 cell passages 7-25 were used. When the cells were approximately 50% confluent the medium was removed, and the cells were washed twice in Phosphate buffered saline (PBS). The cells were trypsinized by adding 1 mL of trypsin and left for 3 min in the CO2 incubator at 37°C. Approximately 10 mL of medium was added to the trypsinized cells, they were counted and a concentration of 100.000 cells/mL in complete DMEM was prepared. A volume of 500 pL of the cell suspension was used to seed each apical compartment of 12 mm Corning® Transwell® with 0.4 pm pore polyester membrane cell culture inserts 3460, where after 1 .5 mL of supplemented DMEM was added to the basolateral compartment. Cells were cultured on the inserts for 21 days with change of medium twice a week. After 21 days the transwells were moved to the CellZscope (NanoAnalytics, Germany). 1.65 mL and 0.8 mL of antibiotics (Abx) free complete medium was added in the basolateral and apical compartments, respectively. The CellZscope was placed overnight in a CO2 incubator (5%) at 37°C, and TEER was measured every hour using automated data collection. This overnight measurement of TEER before the experimental start allowed for determination of baseline TEER in each well and as a quality control of a stable electrical resistance.

Preparation of 3-fucosyllactose (3-FL) in DMEM medium

A solution of 3-fucosyllactose (3-FL) was prepared in complete DMEM medium without Abx.

Preparation of Lacticaseibacillus rhamnosus, LGG® (SEQ ID NO.1)

The day prior to co-incubation with the Caco-2 cells a frozen stock of Lacticaseibacillus rhamnosus, LGG® (SEQ ID NO.1) was inoculated in MRS. A dilution row was generated by transferring 1 mL of mixed inoculated culture with 9 mL MRS and this was repeated 5 times. The strain was cultured anaerobically overnight at 37°C. On the day of co-incubation bacterial growth was evaluated by measuring optical density at 600 nm (ODeoo) and cultures representing late exponential/early stationary phase were selected. The culture was centrifuged at 6000 x g for 5 min, to collect the bacteria pellet. The supernatant was discarded and 35 mL of 37°C warm PBS was added, and the bacteria were washed and spun down at 6000 x g for 5 min. This washing procedure was repeated twice. Bacterial cells were harvested by spinning at 6000 x g for 5 min and the supernatant was discarded. Bacterial cells were resuspended in 5 mL pre-heated complete DMEM medium without Abx and ODeoo was adjusted.

Stimulation of Caco-2 cells with L. rhamnosus, LGG® (SEQ ID NO.1) and 3-FL alone or in combination

To stimulate the Caco-2 cells, CellZscope measurements were paused, and the CellZscope was removed from the CO2 incubator and apical medium was removed from each transwell. Bacterial solution (final concentration OD 0.5), 3-FL solution (2% final concentration), combinations, or media control (DMEM) was added to the relevant wells (each in triplicate).

The CellZscope was returned to the CO2 incubator and the TEER measurements were resumed and continued overnight. Changes in TEER during bacterial stimulation were calculated relative to the latest value recorded immediately prior to the stimulation (baseline measurement, set to 100%). Area under the curve was calculated after 10 hours for each well.

Results

All tested conditions; LGG® alone, 3-FL alone and the combination of the two had significantly increased Area under the curve (AUC) TEER measurements after 10 hours. Further, the combination of L.rhamnosus, LGG® and 3-FL had significantly higher AUC TEER measurements compared to the components tested individually.

Example 3

Isolation of Peripheral blood mononuclear cells (PBMCs)

PBMCs isolation was performed in SepMateTM tubes (50 ml) via Ficoll-Plague PlusTM density gradient centrifugation (1200g, 10 min, RT). Five buffy coats were collected from healthy anonymous donors (Righospitalet, Blodbank, Copenhagen, Denmark) on the day of experiment. The isolated PBMC were washed twice in PBMC media consisting of RPMI medium (Sigma Aldrich), 2-mecaptoethanol (final concentration of 50 pM, Sigma Aldrich) and HEPES buffer (final 10 mM, Sigma Aldrich). Subsequently, the cells were counted using a NucleoCounter®-MC200™ and resuspended in the same medium supplemented with heat-inactivated fetal bovine serum (FBS, final 10%, Gibco). The PBMCs suspension was adjusted to 2x10⁶ cells/ml and plated at 96-well plates (1x10⁵ cells/well) for at least 1 h (37°C, under 5%CO2) before a stimulation.

Preparation of Bacteria

LGG® was inoculated from a frozen stock from the Chr. Hansen culture collection and cultured overnight at 37°C in pH 6.5 MRS (de Man, Rogosa and Sharpe) broth (Difco). A 10-fold dilution series was prepared from the overnight culture and incubated overnight under the same conditions. A culture representing late exponential growth phase was selected based on optical density ODeoo measurements. The bacterial culture was centrifuged for 2 min at 6000g, washed twice in Hank’s Balanced Salt Solution (HBSS) and resuspended in antibiotic-free complete DC medium at a stock concentration of 6.7x10⁷intact cells/ml.

Stimulation of PBMCs

PBMCs from 5 healthy donors were stimulated with either LGG®, 3-FL, or left untreated. The final concentration of LGG® was 1x10⁶ IC/well, corresponding to an MOI of 1 :10 (PBMC:LGG®), final concentration of 3-FL was 0.5% and the synbiotic combination was 1x10⁶ LGG®IC/well plus 0.5% 3-FL, all wells were treated with Tetracycline (4pg/ml final concentration, Sigma Aldrich). After 3 hours of incubation all wells were challenged with Lipopolysaccharide (LPS) from Escherichia coli (cat. number L2654, Sigma Aldrich) at a final concentration of 500ng/ml. After a total of 20 hours of incubation the cell supernatants were collected on AcroPrep filtertop plates (PallTMNew York, USA), centrifuged (1500 x g, 5 min, at 4°C) and stored at -80°C prior to cytokine measurement. Cytokine quantification

Levels of Tumor necrosis factor-a (TNF-a), Interleukin-10 (IL-10), Interleukin -1 receptor antagonist (IL-1 RA) and Interferon-y (IFN-y) secreted from PBMCs were quantified using an MSD® Multi-Spot Assay System MESO Scale QuickPlex™ (MSD Maryland, USA) according to the manufacturer’s instructions. Data are expressed in picograms per milliliter (pg/ml).

Statistical analysis

Secreted levels of IFN-y were compared using a Friedman test with a Dunn’s multiple comparison test. For the remaining cytokines (TNF-a, IL-1 RA and IL-10) an RM-one-way ANOVA with Tukey’s multiple comparison test was used. An a level of 0.05 was considered significant.

Results

As shown in Figure 3, co-incubation of PBMCs with 3-FL, LGG® or the synbiotic combination did not result in significant differences on the secretion of anti-inflammatory cytokines IL-10 (Fig.3A) and IL-1 RA (Fig.3B) compared to the secretion induced by the E. coli LPS challenge alone. Furthermore, while 3-FL had no significant effect on IFN-y secretion, LGG® significantly boosted its secretion, and the synbiotic combination showed a tendency to enhance IFN-y levels (Fig.4A). Regarding TNF-a secretion, 3-FL did not induce any significant changes, whereas LGG® significantly increased its levels, and the synbiotic combination led to a higher significant increase (Fig.4B).

Claims

1 . A composition for use in reducing the risk of an infection in the gastrointestinal tract caused by a pathogenic E. coli in a human subject, said composition comprising at least one human milk oligosaccharide, wherein the human milk oligosaccharide is 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and I or lacto-N-tetraose.

2. The composition for use according to claim 1 , wherein the infection in the gastrointestinal tract is a diarrheal disease.

3. The composition for use according to any one of the preceding claims, wherein the risk of an infection in the gastrointestinal tract is reduced through the inhibition of adhesion of E. coli to intestinal epithelial cells.

4. The composition for use according to any one of the preceding claims, wherein the pathogenic E. coli is of the enterohemorrhagic E. coli (EHEC) pathotype, wherein the EHEC preferably has serotype 0157.

5. The composition for use according to any one of the preceding claims, wherein the composition is a nutritional composition.

6. The composition for use according to any one of the preceding claims, wherein the risk of an infection in the gastrointestinal tract is reduced through an enhancement of the immune response, preferably through an increase of the level of pro-inflammatory cytokines.

7. The composition for use of any one of the preceding claims, wherein the composition comprises at least 0.5 x 10⁹ CFU/dose of a probiotic.

8. The composition for use of claim 7, wherein the probiotic is Lacticaseibacillus rhamnosus GG.

9. The composition for use of any one of the preceding claims, wherein the composition comprises an effective amount of 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and I or lacto-N-tetraose.

10. The composition for use of any one of the preceding claims, wherein the composition is a powder.

11 . The composition for use of claim 10, wherein the composition comprises 3-fucosyllactose and the 3-fucosyllactose has a particle size distribution, as measured using sieves complying to DIN ISO 3310-1 , of less than about 20% through mesh #230 (63 pm), greater than about 65% through mesh #100 (150 pm), greater than about 92% through mesh #45 (355 pm), and 100% through mesh #20 (850 pm).

12. The composition for use of any one of claims 10 or 11 , wherein the water activity a_w of the human milk oligosaccharides is between 0,10 and 0,30, preferably between 0,10 and 0,25.

13. The composition for use of claim 7 or 8, wherein the composition comprises the human milk oligosaccharide 3-fucosyllactose, wherein 3-fucosyllactose preferably is the only human milk oligosaccharide.

14. The composition of any one of claims 1 to 13, wherein the subject is a newborn oran infant.

15. The composition of any one of claims 1 to 13, wherein the subject is a toddler or a child.