Mixtures of HMO and bifidobacterium infantis

Abstract

The present invention provides a composition for use in the prevention and / or treatment of a viral infection in an individual wherein the composition comprises a mixture of Bifidobacterium longum subsp. Infantis and HMO, the HMO mixture consists of 2 '-fucosyllactose (2FL), difucosyllactose (DFL), lactose-N-tetraose (LNT), 6'-sialyllactose (6SL), 3 '-sialyllactose (3SL), and lactose-N-neotetraose (LNnT), and optionally, 3-fucosyllactose (3FL). The present invention also provides compositions for use in preventing and / or reducing the risk of allergen sensitization, preventing and / or reducing the risk of developing respiratory disorders in an individual, and / or preventing and / or reducing the risk of developing asthma in an individual.

Description

Technical Field

[0001] This invention relates to a composition comprising *Bifidobacterium longum subsp. infantis* and a mixture of HMOs, the HMO mixture consisting of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), and lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosylated lactose (3FL). The invention also relates to the use of this composition in human health, for example, for i) prevention and / or treatment of viral infections in individuals, ii) prevention and / or reduction of the risk of allergen sensitization, iii) prevention and / or reduction of the risk of respiratory illnesses in individuals, and / or iv) prevention and / or reduction of the risk of asthma. Background Technology

[0002] Viral infection occurs when a virus replicates within a host cell, thus utilizing the host's resources to promote its own proliferation. Viral infection can also interfere with the host's normal functions and can lead to more serious infection-related disorders, including long-term changes in the immune system (such as inflammatory responses) and subsequent allergic or inflammatory diseases.

[0003] Viral respiratory infections, such as respiratory syncytial virus (RSV), affect nearly 90% of children under two years of age (Karpinnen et al., Clin Microbiol Infect, 2016; 22; 208.e1-e6) and frequently lead to bronchiolitis, an inflammatory bronchial response in infants and young children (Pickles et al., J Pathol, 2015; 235; 266-276). Specifically, severe RSV-induced bronchiolitis is a leading cause of infant morbidity and mortality worldwide (Nair et al., Lancet, 2010; 375; 9725; 2545-1555). Respiratory viruses primarily infect airway epithelial cells. Higher viral loads are associated with increased severity of bronchiolitis, and conversely, rapid reductions in viral load in infants are associated with faster disease resolution (Pickles et al., J Pathol, 2015; 235; 266-276). Well-documented in the literature, during RSV infection, infected and necrotic epithelial cells lead to airway obstruction and inflammation (Pickles et al., J Pathol, 2015; 235; 266-276), thus epithelial cell shedding is a characteristic feature of viral bronchiolitis and is associated with disease severity (Johnson et al., Mod Pathol, 2007; 20; 108-119). Plasma-like dendritic cells (pDCs) are known to play a defensive role in the lesions during RSV infection, and adaptive immune responses, including CD4+ and CD8+ T cells, are important in clearing the virus from the respiratory tract (Openshaw et al., Annu Rev Immunol, 2017; 35; 501-532).

[0004] If the immune defense response to such viral respiratory infections is dysregulated, inflammatory granulocytes, such as neutrophils, along with CD4+ and CD8+ T cell responses, can also contribute to the immunopathological features following respiratory viral infection (Newton et al., Semin Immunopathol, 2016; 38; 471-482). Furthermore, such uncontrolled inflammatory responses can lead to pathological airway smooth muscle remodeling, a hallmark feature of asthma reportedly beginning early in life (O'Reilly et al., JACI, 2013; 131; 1024-1032), and play a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD; Yan F et al., J Transl Med, 2018; 16; 262-270). Therefore, severe viral airway infection early in life represents an independent risk factor for subsequent respiratory diseases such as allergic airway diseases (e.g., asthma; Feldman et al., Am J Respir Crit Care Med, 2015; 191; 34-44) and later chronic obstructive pulmonary disease (Savran O et al., Int J Chron Obstruct Pulmon Dis. 2018; 13: 683-693).

[0005] Breastfeeding is a recognized factor that reduces the severity of respiratory viral infections in infants, either directly through milk bioactive substances (such as human milk oligosaccharides) or indirectly through microbiome-mediated immune benefits.

[0006] In recent years, human milk oligosaccharides (HMOs) have become a topic of great interest due to their role in numerous biological processes occurring in human organisms. Mammalian milk contains at least 130 of these complex oligosaccharides (Urashima et al., Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122-831-1).

[0007] Infancy, especially the first few weeks, the first 3 months, the first 6 months, or the first 12 months, is a crucial period for establishing a balanced gut microbiota. The regulation of the gut microbiota during infancy and early childhood is known to have a significant, predictable impact on future health. For example, the gut microbiome can influence the development of a robust immune system, normal growth, and even the development of obesity later in life. The mode of delivery is also known to affect an infant's initial gut microbiota: infants delivered by cesarean section have been shown to have different gut microbiota compared to those delivered vaginally.

[0008] Bifidobacterium longum subsp. infantis (B. infantis) has been shown to dominate the gut microbiota of breastfed infants and benefit the host by accelerating the maturation of immune responses, balancing the immune system to suppress inflammation, improving intestinal barrier function, and increasing short-chain fatty acid (SCFA) production. Decreased abundance of Bifidobacterium species in infants and young children is associated with chronic diseases, including asthma and obesity, as well as reduced vaccine responses. Researchers hypothesize that loss of Bifidobacterium species in the infant gut in developed populations is associated with an increased incidence of allergic and autoimmune diseases.

[0009] Probiotics, particularly those from the genera *Lactobacillus* and *Bifidobacterium*, are known to support protection against respiratory infections. The role of probiotics in viral respiratory infections was reviewed by Lehtoranta et al. (Lehtoranta et al., *Eur J Clin Microbiol Infect Dis, 2014; 33; 1289-1302*).

[0010] Increasing the abundance of Bifidobacterium species, or particularly Bifidobacterium infantis, in the gut microbiota ecosystem through exogenous application can be difficult, especially for infants fed infant formula. Administering probiotics via nutritional compositions, such as infant formula, is generally intended to promote their implantation into the gut microbiome, thereby triggering the desired benefits associated with said probiotics. The vast diversity of the gut microbiome makes probiotic implantation challenging.

[0011] Due to the loss of Bifidobacterium species in the infant gut and low breastfeeding rates, there is a need to provide infants with both HMOs and bacteria that utilize HMOs (such as Bifidobacterium longum infantis subsp.) to support a healthy microbiome for long-term health.

[0012] Furthermore, the means of preventing or treating viral infections are limited. The number of effective antiviral drugs is limited, such as those used to treat HIV and influenza, and the primary method for controlling viral diseases is vaccination, which aims to prevent outbreaks by building immunity against the virus or a family of viruses.

[0013] Therefore, there is a need for compositions that prevent and / or treat viral infections, more specifically, compositions that prevent or treat respiratory viral infections. Summary of the Invention

[0014] The inventors have surprisingly discovered that early-life synbiotic intervention (a mixture of infantile bifidobacteria and human milk oligosaccharides) provides defense against virus-induced bronchiolitis and promotes immune benefits that persist into adulthood (e.g., as assessed by reducing susceptibility to pollution-induced allergic airway inflammation). Specifically, the inventors have demonstrated that synbiotic intervention leads to rapid resolution of virus-induced lung inflammation and appropriate lung tissue remodeling following virus clearance.

[0015] These findings confirm the use of synbiotics in the protection and treatment of viral infections, particularly viral bronchiolitis, early in life, and reveal the functional benefits of synbiotics in establishing an effective antiviral immune response associated with faster disease resolution. Given that early-life viral respiratory infections represent a major independent risk factor for subsequent asthma, recurrent wheezing, and chronic obstructive pulmonary disease, dietary synbiotic supplementation may also prevent long-term complications associated with early-life viral respiratory infections.

[0016] Therefore, in a first aspect, the present invention provides a composition for use in the prevention and / or treatment of viral infections in an individual, wherein the composition comprises a mixture of *Bifidobacterium longum* subsp. infantis and HMO oligosaccharides, the HMO oligosaccharide mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), and lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosylated lactose (3FL).

[0017] In another aspect, the present invention provides compositions for use in i) preventing and / or reducing the risk of allergen sensitization in an individual's respiratory tract and / or ii) preventing and / or reducing the risk of an individual developing respiratory symptoms, wherein the composition comprises a mixture of *Bifidobacterium longum* subsp. infantis and HMOs, the HMO mixture consisting of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), and lactose-N-neotetrasaccharide (LNnT) and optionally 3-fucosylated lactose (3FL), and wherein the respiratory symptoms are chronic inflammatory diseases of the respiratory tract or allergic respiratory diseases.

[0018] In some embodiments, the composition is used to prevent and / or reduce the risk of allergen sensitization in an individual's respiratory tract.

[0019] In some embodiments, the composition is used to prevent and / or reduce an individual's risk of developing respiratory illnesses.

[0020] In another aspect, the present invention provides a composition for use in preventing and / or reducing the risk of an individual developing asthma, wherein the composition comprises a mixture of Bifidobacterium longum subsp. infantis and HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), and lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosylated lactose (3FL).

[0021] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lactose-N-neotetrasaccharide (LNnT).

[0022] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lactose-N-neotetrasaccharide (LNnT).

[0023] In some implementations, Bifidobacterium longum infantis subspecies is Bifidobacterium longum infantis subspecies LMG 11588.

[0024] In some implementations, Bifidobacterium longum infantis is a strain that has at least 99.9% average nucleotide identity (ANI) with Bifidobacterium longum infantis LMG 11588.

[0025] In some implementations, the HMO mixture is essentially composed of the following substances:

[0026] i. 34% to 85% by weight, preferably 40% to 71% by weight of 2FL;

[0027] ii. 10% to 40% by weight, preferably 12% to 26% by weight of LNT;

[0028] iii. 4% to 14% by weight, preferably 5% to 10% by weight of DFL;

[0029] iv. 9% to 31% by weight, preferably 10% to 28% by weight of a combination of 6SL and 3SL, and

[0030] v. 6% to 30% by weight, preferably 7% to 22% by weight of LNnT.

[0031] In some implementations, the HMO mixture is essentially composed of the following substances:

[0032] i. 20% to 60% by weight, preferably 22% to 55% by weight of 2FL;

[0033] ii. 4% to 30% by weight, preferably 6% to 20% by weight of LNT;

[0034] iii. 1% to 12% by weight, preferably 2% to 8% by weight of DFL;

[0035] iv. 7% to 23% by weight, preferably 8% to 22% by weight of a combination of 6SL and 3SL;

[0036] v. 10% to 50% by weight, preferably 13% to 46% by weight of 3FL, and

[0037] vi. 3% to 25% by weight, preferably 5% to 20% by weight of LNnT.

[0038] In some embodiments, the composition is in the form of a nutritional composition.

[0039] In some implementations, the nutritional composition is selected from infant formula, stage 1 infant formula, stage 2 infant formula, baby food, infant cereal composition, growing milk, fortifiers such as human milk fortifiers or supplements.

[0040] In some implementations, the individual is an infant, toddler, or child. Appropriately, the individual is an infant. Appropriately, the individual is a toddler. Appropriately, the individual is a child.

[0041] In another aspect, the present invention provides the use of the compositions according to the invention in the preparation of a medicament for the prevention and / or treatment of viral infections in an individual.

[0042] In another aspect, the present invention provides the use of the compositions according to the invention in the preparation of a medicament for i) preventing and / or reducing the risk of sensitization to allergens in an individual's respiratory tract and / or ii) preventing and / or reducing the risk of an individual developing respiratory symptoms, wherein the respiratory symptoms are chronic inflammatory diseases of the respiratory tract or allergic respiratory diseases.

[0043] In another aspect, the present invention provides the use of the compositions according to the invention in the preparation of a medicament for the prevention and / or reduction of an individual's risk of developing asthma.

[0044] In another aspect, the present invention provides a method for preventing and / or treating viral infections in an individual, the method comprising administering a composition according to the present invention to the individual.

[0045] In another aspect, the present invention provides i) methods for preventing and / or reducing the risk of allergen sensitization in an individual's respiratory tract and / or ii) methods for preventing and / or reducing the risk of an individual developing respiratory symptoms, the method comprising administering to an individual a composition according to the present invention, wherein the respiratory symptoms are chronic inflammatory diseases of the respiratory tract or allergic respiratory diseases.

[0046] In another aspect, the present invention provides a method for preventing and / or reducing the risk of an individual developing asthma, the method comprising administering a composition according to the present invention to the individual.

[0047] In another aspect, the present invention provides a composition comprising a mixture of Bifidobacterium longum subsp. infantis and HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), and lactose-N-neotetrasaccharide (LNnT).

[0048] In another aspect, the present invention provides a composition comprising a mixture of Bifidobacterium longum subsp. infantis and HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), 3-fucosylated lactose (3FL), and lactose-N-neotetrasaccharide (LNnT). Attached Figure Description

[0049] Figure 1 Schematic diagram of a model of allergic airway inflammation exacerbated by viral airway infection and pollution in early life.

[0050] Figure 2 Early life nutritional intervention using synbiotics (Bifidobacterium infantis + 6HMO) can reduce virus-induced lung lesions.

[0051] Figure 3 and Figure 4 Early life nutritional intervention using synbiotics (Bifidobacterium infantis + 6HMO) can promote immune benefits that last into adulthood.

[0052] Figure 5 Early life nutritional intervention using synbiotics (Bifidobacterium infantis + 5HMO) can promote antiviral immune responses and reduce virus-induced lung inflammation during the peak of infection.

[0053] Figure 6 : The combination of 7-HMO and Bifidobacterium infantis establishes a synergistic effect of Bifidobacterium breve in fecal fermentation cultures. Detailed Implementation

[0054] definition

[0055] As used herein, the following terms have the following meanings.

[0056] The term "individual" refers to an infant, toddler, child, small-for-gestational-age (SGA) infant, or premature infant.

[0057] The term "infant" refers to a child under 12 months of age.

[0058] The term "preschooler" refers to children aged between one and three years old, also known as toddlers.

[0059] The term "child" refers to a child aged between three and twelve years old. Preferably, the term "child" refers to a child aged between three and six years old.

[0060] "Premature infant" or "premature person" refers to an infant or toddler born before full term. Generally, it refers to an infant or toddler born before 36 weeks of gestation.

[0061] The term "small for gestational age" or "SGA" refers to an infant or toddler whose size is smaller than the normal standard for birth at gestational age (most commonly defined as a weight below the 10th percentile for gestational age). In some implementations, SGA may be associated with intrauterine growth restriction (IUGR), a condition in which a fetus fails to reach its potential size.

[0062] The term "low birth weight" should be understood as a birth weight of less than 2500g.

[0063] The term "nutritional composition" refers to a composition that provides nutrients to an individual. This nutritional composition is typically ingested orally or intravenously. It may contain lipid or fat sources, carbohydrate sources, and / or protein sources. In one specific embodiment, the nutritional composition is a ready-to-drink composition, such as a ready-to-drink formula food.

[0064] In one specific embodiment, the nutritional composition of the present invention is a "synthetic nutritional composition". The term "synthetic nutritional composition" means a mixture obtained by chemical and / or biological means, the chemical properties of which may be the same as those of mixtures naturally present in mammalian milk (i.e., the synthetic nutritional composition is not breast milk).

[0065] As used herein, the term "infant formula" refers to a food intended specifically for the nutrition of infants during the first few months of life, and which itself meets the diverse nutritional needs of such individuals (in accordance with Article 2(c) of European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 concerning infant formula and follow-up formula). It also refers to nutritional compositions intended for use in infants, and as defined in the Codex Alimentarius Commission (Codex STAN 72-1981) and for infant specialties (including foods for specific medical purposes).

[0066] The term "infant formula" encompasses both "starter infant formula" and "follow-up formula" or "follow-on formula".

[0067] Stage 2 infant formula or follow-up formula is given starting from the 6th month and includes growing milk. Infant formula constitutes the main liquid element in the gradually diversifying diet of this group.

[0068] The term "GUM" refers to a milk-based beverage that is typically fortified with vitamins and minerals and is intended for use by toddlers or children.

[0069] The term "infant food" refers to food specifically designed to provide nutrition for infants or young children during the first year of their lives.

[0070] The term "infant cereal composition" refers to food designed specifically to provide nutrition for infants or young children during the first year of life.

[0071] The term "fortifier" refers to a liquid or solid nutritional composition suitable for mixing with breast milk or infant formula.

[0072] The term "weaning period" refers to the period during which breast milk is gradually replaced by other foods in the diet of infants or young children.

[0073] "Breast milk" should be understood as the mother's milk or colostrum.

[0074] "Oligosaccharides" are carbohydrate polymers containing small amounts (usually three to ten parts) of common sugars (monosaccharides).

[0075] The term "HMO" refers to human milk oligosaccharides. These carbohydrates are resistant to enzymatic hydrolysis by digestive enzymes (e.g., pancreas and / or brush border), indicating that their potential functions are not directly related to their calorific value. It has been specifically noted in the art that these carbohydrates play a crucial role in early infant and toddler development, such as the maturation of the immune system. Many different types of HMOs have been found in human milk. Each individual oligosaccharide is based on a variety of combinations of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine with these molecules, resulting in a large number of diverse oligosaccharides in human milk; more than 130 such structures have been identified to date. Almost all oligosaccharides have a lactose molecule at the reducing end, and the non-reducing end is occupied by sialic acid and / or fucose (if present). HMOs can be acidic (e.g., oligosaccharides containing charged sialic acid) or neutral (e.g., fucoidylated oligosaccharides). Some examples of HMOs are fucoidylated oligosaccharides, N-acetylated oligosaccharides, and / or sialylated oligosaccharides.

[0076] "Fucosylated oligosaccharides" are oligosaccharides containing fucose residues. These oligosaccharides are neutral. Some examples are LNFP-I (lactose-N-fucopentose I), 2'-FL (2'-fucosyllactose), and 3-FL (3-fucosyllactose).

[0077] The terms “fucosylated oligosaccharides containing α-1,2'-fucosylation epitopes” and “2-fucosylated oligosaccharides” encompass fucosylated oligosaccharides with a certain degree of formal homology, as they contain α-1,2'-fucosylation epitopes and therefore a certain degree of functional homology can be expected.

[0078] The term "N-acetylated oligosaccharide" encompasses both "N-acetyl-aminolactoside" and "oligosaccharide containing N-acetyl-aminolactoside." This oligosaccharide is a neutral oligosaccharide containing N-acetyl-aminolactoside residues. Suitable examples are LNT (lactose-N-tetrasaccharide), para-lactose-N-neohexose (para-LNnH), LNnT (lactose-N-neohexose), and any combination thereof. Other examples are lactose-N-hexasaccharide, lactose-N-neohexose, para-lactose-N-hexasaccharide, para-lactose-N-neohexose, lactose-N-octasaccharide, lactose-N-neohexose, isol-lactose-N-octasaccharide, para-lactose-N-octasaccharide, and lactose-N-decansaccharide.

[0079] "Sialinated oligosaccharides" are oligosaccharides containing charged sialic acid, that is, oligosaccharides with sialic acid residues. These oligosaccharides are acidic. Some examples are 3'-SL (3'-sialyl-lactose) and 6'-SL (6'-sialyl-lactose). The terms "sialylated oligosaccharide" and "sialyl-lactose (SL)" are used interchangeably. The trisaccharide sialyl-lactose consists of lactose at the reducing end and one sialic acid residue at the non-reducing end, which, via α-2,3 binding or α-2,6 binding, produce 3'-SL and 6'-SL, respectively.

[0080] "HMO precursors" are key compounds that intervene in the production of HMOs, such as sialic acid and / or fucose.

[0081] As used herein, the term "GOS" refers to "galacto-oligosaccharides." As used herein, galacto-oligosaccharides (GOS) typically consist of a β-linked galactose moiety and a reduced-terminal galactose or glucose. Such GOS contain β-(1... 2) β-(1 3) β-(1 4) or β-(1) 6) The linked galactose moiety may have a degree of polymerization (DP) of 3 to 8 galactose units. Therefore, the term GOS preferably refers to an oligosaccharide containing at least three galactose units, more preferably an oligosaccharide containing at least four galactose units, and preferably has a degree of polymerization (DP) of 3 to 8 galactose units.

[0082] The nutritional compositions of the present invention may be in solid form (e.g., powder) or liquid form. The amounts of various ingredients (e.g., oligosaccharides) may be expressed in g / 100g of composition on a dry weight basis when the composition is in solid form (e.g., powder), or in g / L of composition concentration when the composition refers to a liquid form (the latter also covers liquid compositions that can be obtained by reconstituted powder in a liquid (such as milk, water), such as reconstituted infant formula or stage 2 infant formula or growing milk or infant cereal products or any other formulation designed to provide nutrition for infants).

[0083] The terms “infant / toddler fed only with human breast milk,” “infant or toddler fed only with breast milk,” “infant or toddler fed exclusively with breast milk,” and “breastfed infant / toddler” are used interchangeably. They refer to infants or toddlers fed with most (i.e., at least 90%, or at least 95%, or at least 99%) or all (100%) of the nutrients and / or energy derived from human breast milk.

[0084] The term "standard nutritional composition" refers to standard synthetic nutritional compositions, such as infant formula, follow-up milk, or growing milk that are already available on the market.

[0085] The terms “microbes,” “microbial community,” and “microbial group” are used interchangeably.

[0086] The terms "intestinal microbiota", "intestinal microbiota", "intestinal microbiota" and "gut microbiota" can be used interchangeably.

[0087] The term "prevention" refers to avoiding the occurrence of a physical condition, illness, or its consequences, and / or reducing its incidence (i.e., decreasing its frequency). Prevention also includes delaying or preventing the onset of symptoms of a disease, disorder, or condition. Prevention may be absolute (preventing the disease from occurring), or it may be effective only for some individuals or for a limited time.

[0088] The term "treatment" means reducing the duration and / or severity of a physical condition, ailment, or its consequences (e.g., the reduction or elimination of symptoms of the condition). Treatment also encompasses reducing, alleviating, or eliminating one or more symptoms associated with the treated disease, disorder, or ailment, and / or slowing, reducing, or halting the progression of the treated disease, disorder, or ailment.

[0089] Prevention and / or treatment of physical conditions, illnesses, or their consequences may occur during treatment (i.e., during the administration of the compositions of the present invention, either immediately after the start of administration or some time after the start of administration, such as days or weeks after the start). However, it may also cover subsequent prevention and / or treatment. The term "subsequent" covers effects after the intervention or treatment has ended. "Subsequent" effects may last from one week to several months or even several years, such as two to four weeks, two to six weeks, two to eight weeks, one to six months, or two to twelve months. Suitablely, after the termination of the intervention or treatment, "subsequent" effects may last from twelve months to twelve years, such as two to ten years or four to five years. Suitablely, "subsequent" effects may persist until the individual is at least five years old, such as at least ten years old, at least twenty years old, or at least thirty years old.

[0090] The term "prebiotic" refers to non-digestible carbohydrates that exert a beneficial effect on the host by selectively stimulating the growth and / or activity of healthy bacteria, such as bifidobacteria in the human colon (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995; 125:1401-12).

[0091] The term "probiotics" refers to microbial cell preparations or microbial cell components that have beneficial effects on the health or well-being of a host. (Salminen S, Ouwehand A, Benno Y. et al., "Probiotics: how should they be defined" Trends Food Sci. Technol. 1999:10 107-10). The microbial cells are generally bacteria or yeast.

[0092] The term “synbiotic” can refer to a component containing both probiotics and prebiotics, or a live microorganism and a substrate selectively utilized by a co-administered live microorganism (see, for example, Swanson, KS et al., 2020. Nature Reviews Gastroenterology & Hepatology, 17(11), pp.687-701).

[0093] The term "cfu" should be understood as colony-forming unit.

[0094] Unless otherwise specified, all percentages are by weight.

[0095] Unless otherwise specified, all weights expressed in g / 100g of composition are dry weights.

[0096] The term "SCFA" refers to short-chain fatty acids.

[0097] The statement "increased SCFA production" means that individuals fed the nutritional composition according to the invention have higher levels of systemic and / or colonic SCFAs compared to those fed the standard composition. SCFA production can be measured using techniques known to those skilled in the art, such as gas-liquid chromatography.

[0098] In this article, the term "gastrointestinal tract" includes the mouth, pharynx, esophagus, stomach, small intestine, large intestine, rectum, and anus. The term "intestine" includes the small intestine, large intestine, and rectum.

[0099] In this article, the term "respiratory tract" refers to the passage formed by the nose, nasal cavity, pharynx, larynx, trachea, bronchi and lungs through which air passes during respiration.

[0100] “Average nucleotide identity (ANI)” is a measure of nucleotide-level genomic similarity between coding regions of two genomes. Average nucleotide identity can be assessed as described here: Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. Oct 2017; 110(10):1281-1286. In an embodiment of the invention, the strain *Bifidobacterium longum* subsp. infant LMG 11588 (also known as ATCC 17930) represents a reference genome to be compared with the microbial genome. Examples of microbial genomes having at least 99.9% ANI with *Bifidobacterium longum* subsp. infant LMG 11588 can be found on PATRIC (https: / / www.patricbrc.org), genome ID 1678.111. In one embodiment of the invention, the *Bifidobacterium longum* subsp. infant strain does not possess potentially transferable antibiotic resistance.

[0101] The ANI (Aspect-Number Indication) of genes shared between two strains is a robust means of comparing the genetic affinity between strains. An ANI value of at least approximately 96% indicates that the strains belong to the same species (Konstantinidis and Tiedje, 2005, ProcNatl Acad Sci USA, 102(7):2567-72; and Goris et al., 2007, Int Syst EvolMicrobiol.57(Pt 1):81-91), while an ANI value of at least approximately 99% indicates that the bacterial genome belongs to the same strain. The ANI between two bacterial genomes is calculated by pairwise comparisons of all sequences common to any two strains and can be determined, for example, using any of the many publicly available ANI tools, including but not limited to OrthoANI with usearch (Yoon et al., 2017, Antonie van Leeuwenhoek 110:1281-1286); ANI calculator, JSpecies (Richter and Rossello-Mora, 2009, Proc Natl Acad Sci USA 106:19126-19131); and JSpeciesWS (Richter et al., 2016, Bioinformatics 32:929-931). Other methods for determining the ANI of two genomes are known in the art (Konstantinidis, KT and Tiedje, 2005, JM, Proc.Natl.Acad.Sci.USA, 102: 2567-2572; and Varghese et al., 2015, NucleicAcids Research, 43(14):6761-6771).

[0102] Furthermore, in the context of this invention, the terms "comprising" or "including" do not exclude other possible elements. The compositions of this invention (including the various embodiments described herein) may comprise, consist of, or be substantially composed of the following elements: the essential elements and necessary limitations of the invention described herein, and any additional or optional ingredients, components, or limitations described herein or as required.

[0103] Any references to prior art documents in this specification should not be construed as an admission that such prior art is well-known or part of common knowledge in the field.

[0104] The invention will now be described in more detail. It should be noted that the various aspects, features, embodiments, and implementations described in this application may be compatible and / or combined in any combination thereof.

[0105] Therapeutic uses

[0106] Viral infection

[0107] Viral respiratory infections, such as respiratory syncytial virus (RSV), affect nearly 90% of children under two years of age (Karpinnen et al., Clin Microbiol Infect, 2016; 22; 208.e1-e6). These viral respiratory infections in infants and young children often result in bronchiolitis, an inflammatory bronchial response in infants and young children (Pickles et al., J Pathol, 2015; 235; 266-276). Severe RSV-induced bronchiolitis is a leading cause of infant morbidity and mortality worldwide (Nair et al., Lancet, 2010; 375; 9725; 2545-1555).

[0108] Using a mouse pneumonia virus (PVM) model infected with human RSV, the inventors surprisingly discovered that early-life synbiotic intervention (a combination of Bifidobacterium infantis and human milk oligosaccharides) provided protection against virus-induced bronchiolitis. These findings confirm the use of synbiotics in the early-life protection and treatment of viral infections, particularly viral bronchiolitis, and reveal the functional benefits of synbiotics in establishing an effective antiviral immune response associated with faster disease resolution.

[0109] Therefore, the compositions of the present invention are particularly effective for use in the treatment and / or prevention of viral infections in individuals.

[0110] Therefore, in a first aspect, the present invention provides a composition for use in the prevention and / or treatment of viral infections in an individual, wherein the composition comprises a mixture of *Bifidobacterium longum* subsp. infantis and HMO, the HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0111] In another aspect, the present invention provides the use of the compositions according to the invention in the preparation of a medicament for the prevention and / or treatment of viral infections in an individual.

[0112] In another aspect, the present invention provides a method for preventing and / or treating viral infections in an individual, the method comprising administering a composition according to the present invention to the individual.

[0113] Therefore, the compositions of the present invention are also effective in reducing the risk of an individual contracting a viral infection and / or in alleviating symptoms associated with a viral infection.

[0114] Viral infections can be viral gastrointestinal infections or viral respiratory infections. Viral gastrointestinal infections can be viral intestinal infections or viral gastric infections.

[0115] In a preferred embodiment, the viral infection is a viral respiratory infection. The viral respiratory infection can be a viral infection in the upper or lower respiratory tract. In a typical embodiment of the invention, the viral respiratory infection is caused by respiratory syncytial virus (RSV).

[0116] Illnesses associated with viral infections are typically the common cold, influenza (flu), bronchitis, bronchiolitis, pneumonia, sore throat (pharyngitis), sinusitis, non-allergic rhinitis, severe acute respiratory syndrome (SARS), viral croup, otitis media, meningitis, or diarrhea. Generally, when the viral infection is located in the respiratory tract, illnesses associated with respiratory infections include the common cold, influenza (flu), bronchitis, bronchiolitis, pneumonia, sore throat (pharyngitis), sinusitis, non-allergic rhinitis, severe acute respiratory syndrome (SARS), viral croup, or otitis media. Most commonly, illnesses associated with viral respiratory infections are the common cold, influenza (flu), bronchitis, bronchiolitis, or pneumonia.

[0117] Therefore, in a preferred embodiment of the invention, the composition of the invention is used to treat and / or prevent diseases associated with viral respiratory infections, selected from the common cold, influenza (flu), bronchitis, bronchiolitis, and pneumonia. In a more preferred embodiment, the disease associated with the respiratory infection is selected from bronchiolitis and pneumonia, particularly RSV-induced bronchiolitis and / or pneumonia, i.e., bronchiolitis and / or pneumonia caused by RSV. In an even more preferred embodiment, the disease associated with the respiratory infection is bronchiolitis, particularly RSV-induced bronchiolitis.

[0118] The symptoms most commonly associated with viral infections and that can be alleviated by the compositions of the present invention are lung irritation, lung congestion, excessive mucus production, fever, cough, wheezing, shortness of breath, abdominal cramps, diarrhea, or vomiting.

[0119] The above-mentioned infections can be caused by a variety of different viruses, including respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza viruses such as influenza A (IVA) and / or influenza B (IVB), rhinovirus (RV), adenovirus (ADV), metapneumovirus (MPV), bocavirus (BoV), coronavirus (CoV), myxovirus, herpesvirus, enterovirus (EV), parachovirus (PeV), or combinations thereof.

[0120] The compositions of the present invention are particularly effective in treating, preventing, reducing the risk of infection with RSV-induced viral infections, and / or alleviating their symptoms. Therefore, the compositions of the present invention are particularly preferred for use in treating, preventing, reducing the risk of infection with RSV-induced bronchiolitis or RSV-induced pneumonia, and / or alleviating their symptoms.

[0121] The compositions of the present invention can be used to treat and / or prevent viral infections, particularly respiratory infections in people of any age. Therefore, individuals to be treated with the compositions of the present invention may be selected from those aged 0 to <1 year (infants), 1 to <3 years (toddlers), and 3 to <6 years (children), including those aged 3 to <5 years (preschool children).

[0122] Sustained immune benefits

[0123] Viral infections can also disrupt normal host function and lead to more serious infection-related disorders, including immunopathological manifestations following respiratory viral infections (Newton et al., Semin Immunopathol, 2016; 38; 471-482), such as long-term alterations in the immune system (e.g., inflammatory responses) and subsequent allergic or inflammatory diseases. For example, uncontrolled inflammatory responses following respiratory viral infections can lead to pathological airway smooth muscle remodeling, a hallmark feature of asthma reported to begin early in life (O'Reilly et al., JACI, 2013; 131; 1024-1032), and plays a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD; Yan F et al., J Transl Med, 2018; 16; 262-270). Therefore, severe viral airway infection early in life represents a major independent risk factor for subsequent respiratory diseases such as allergic airway diseases (e.g., asthma; Feldman et al., Am J Respir Crit Care Med, 2015; 191; 34-44) and later chronic obstructive pulmonary disease (Savran O et al., Int J Chron Obstruct Pulmon Dis. 2018;13: 683-693).

[0124] Therefore, the compositions of the present invention are particularly effective in promoting sustained immune benefits in individuals.

[0125] Therefore, in another aspect, the present invention provides a composition for use in promoting long-term immune benefits in an individual, wherein the composition comprises a mixture of Bifidobacterium longum subsp. infantis and HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0126] In another aspect, the present invention provides the use of the compositions according to the invention in the preparation of a medicament for promoting long-term immune benefits in an individual.

[0127] In another aspect, the present invention provides a method for promoting long-term immune benefits in an individual, the method comprising administering to the individual a composition according to the present invention.

[0128] The benefits of promoting long-term immunity include:

[0129] i. Promote long-term respiratory health;

[0130] ii. To prevent and / or reduce the risk of sensitization to allergens; and / or

[0131] iii. To prevent and / or reduce the risk of developing respiratory illnesses later in life.

[0132] Where appropriate, promoting long-term immune benefits means promoting long-term respiratory health. Where appropriate, promoting long-term immune benefits means preventing and / or reducing the risk of allergen sensitization. Where appropriate, promoting long-term immune benefits means preventing and / or reducing the risk of developing respiratory illnesses later in life.

[0133] As used herein, the phrase “long-term” encompasses effects following the termination of intervention or treatment. “Long-term” effects can range from one week to several years after the termination of intervention or treatment, for example, 2 to 4 weeks, 2 to 6 weeks, 2 to 8 weeks, 1 to 6 months, 2 to 12 months, 12 months to 12 years, such as 2 to 10 years, or 4 to 5 years. Therefore, “long-term” effects can exist when an individual reaches 3 years of age or older, preferably 3 to 12 years, more preferably 3 to 10 years, even more preferably 3 to 8 years, most preferably 3 to 6 years, and particularly 3 to 5 years or 3 to 4 years. Appropriately, long-term benefits persist until the individual is at least 5 years old, such as at least 10 years, at least 20 years, or at least 30 years old.

[0134] Suitablely, the compositions of the present invention can prevent complications associated with respiratory viral infections. Suitablely, this effect can be long-term prevention of complications associated with respiratory viral infections. Such complications can be those related to the immune system, such as inflammatory responses, and include immunopathological manifestations following respiratory viral infections, such as long-term alterations to the immune system (e.g., long-term alterations to inflammatory responses) and pathological airway smooth muscle remodeling. These complications may further predispose an individual to subsequent allergic or inflammatory diseases, such as allergic respiratory diseases or chronic inflammatory diseases of the respiratory tract.

[0135] Allergic reactions and respiratory symptoms

[0136] Given that early-life viral respiratory infections represent a major independent risk factor for subsequent asthma, recurrent wheezing, and chronic obstructive pulmonary disease (Savran et al., Int J Chron Obstruct, 2015; 191; 34-44; and Feldman et al., 2015 Am J Respir Crit Care Med, 191; 34-44), the compositions of the present invention are also effective in preventing and / or reducing the risk of developing respiratory conditions such as chronic inflammatory diseases of the respiratory tract and allergic airway diseases.

[0137] Since viral infections, particularly RSV infections, are associated with the subsequent development of allergic airway diseases, such as later-onset asthma (Feldman et al. 2015 Am J Respir Crit Care Med, 191; 34-44), the compositions of the present invention are also effective in preventing and / or reducing the risk of allergen sensitization and / or the development of allergic respiratory diseases in an individual's respiratory tract.

[0138] Besides *Bifidobacterium infantis*, *Bifidobacterium breve* is predominant in the feces of breastfed newborns, and there is substantial evidence of its immunomodulatory capabilities (Bozzi Cionci, N. et al., Nutrients, 2018, 10(11)). This evidence includes beneficial modulation of the pulmonary immune response, including reduced airway hyperresponsiveness, activation of type 2 T helper cells, eosinophilia, and production of allergen-specific antibodies (Hougee, S. et al., Int Arch Allergy Immunol, 2010, 151(2): 107-17).

[0139] The inventors have demonstrated a surprising synergistic effect of combining HMO with Bifidobacterium infantis in establishing Bifidobacterium breve in fecal fermentation cultures, and thus linked this synergistic effect to pulmonary immunity, specifically i) preventing and / or treating viral infections in individuals, ii) promoting long-term immune benefits in individuals, iii) preventing and / or reducing the risk of sensitization to allergens in the respiratory tract, iv) preventing and / or reducing the risk of developing respiratory symptoms in individuals, and / or v) preventing and / or reducing the risk of developing asthma.

[0140] Therefore, in another aspect, the present invention provides compositions for use in i) preventing and / or reducing the risk of an individual’s allergen sensitization and / or ii) preventing and / or reducing the risk of an individual’s respiratory illness, wherein the composition comprises a mixture of Bifidobacterium longum subsp. infantis and HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0141] In another aspect, the present invention provides a composition for use in preventing and / or reducing the risk of an individual developing asthma, wherein the composition comprises a mixture of Bifidobacterium longum subsp. infantis and HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0142] In another aspect, the present invention provides the use of the compositions according to the invention in the preparation of a medicament for i) preventing and / or reducing the risk of an individual’s sensitization to an allergen and / or ii) preventing and / or reducing the risk of an individual developing respiratory symptoms.

[0143] In another aspect, the present invention provides the use of the compositions according to the invention in the preparation of a medicament for the prevention and / or reduction of an individual's risk of developing asthma.

[0144] In another aspect, the present invention provides i) methods for preventing and / or reducing the risk of an individual’s allergen sensitization and / or ii) methods for preventing and / or reducing the risk of an individual’s respiratory illness, the method comprising administering to an individual a composition according to the present invention.

[0145] In another aspect, the present invention provides a method for preventing and / or reducing the risk of an individual developing asthma, the method comprising administering a composition according to the present invention to the individual.

[0146] Respiratory disorders include chronic inflammatory diseases of the respiratory tract and allergic respiratory diseases.

[0147] Chronic inflammatory diseases of the respiratory tract include chronic obstructive pulmonary disease (COPD) and asthma, including allergic asthma and non-allergic asthma.

[0148] COPD is a term used for a group of lung diseases, including chronic bronchitis, emphysema, and chronic obstructive airway disease. People with COPD experience difficulty breathing, primarily due to narrowing of their airways.

[0149] Asthma is a chronic respiratory condition characterized by inflammation and bronchospasm, leading to difficulty breathing. It is often associated with allergic reactions or other forms of hypersensitivity. Inflammation and narrowing of the small airways in the lungs can cause asthma symptoms, which can be any combination of cough, wheezing, shortness of breath, and chest tightness. Asthma typically develops in childhood, particularly during the preschool years (3 to 5 years old).

[0150] Allergic respiratory diseases include recurrent wheezing and asthma, including allergic asthma.

[0151] To i) prevent and / or reduce the risk of an individual's allergen sensitization and / or ii) prevent and / or reduce the risk of an individual developing respiratory illnesses, the compositions of the present invention are preferably applied to individuals aged 0 to <3 years, preferably 0 to 2 years, more preferably 0 to <1 year, such as 0 to 6 months. When an individual is 3 years or older, preferably 3 to 12 years, more preferably 3 to 10 years, even more preferably 3 to 8 years, most preferably 3 to 6 years, and particularly 3 to 5 years or 3 to 4 years, this further prevents or reduces the risk of developing respiratory illnesses.

[0152] Since viral infections, particularly RSV infections, are often associated with bacterial co-infections (Thorburn et al., Thorax, 2006; 61(7); 611-615) or secondary infections (Sande et al., Nature Communications, 2019; 10; 2218), including antibiotic use, the compositions of the present invention are also effective in preventing or reducing the risk of bacterial co-infections and / or secondary bacterial infections associated with respiratory viral infections in mammals, particularly humans. Pathogens commonly involved in co-infections or secondary infections include Staphylococcus aureus, Streptococcus pneumoniae, and / or Haemophilus influenzae.

[0153] Composition

[0154] The inventors have surprisingly discovered that the combination of *Bifidobacterium longum* subsp. infantis with a mixture of HMOs consisting of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), as well as optionally 3-fucosylated lactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT), provides protection against virus-induced bronchiolitis in early life and promotes immune benefits that persist into adulthood (e.g., assessed by reducing susceptibility to contamination-exacerbated allergic airway inflammation).

[0155] As described above, the composition used according to the present invention comprises a mixture of Bifidobacterium longum subsp. infantis and HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0156] Surprisingly, a specific combination of *Bifidobacterium longum* subsp. infantis with a mixture of HMOs consisting of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosylated lactose (3FL) is effective in protecting against virus-induced bronchiolitis and promoting immune benefits that persist into adulthood.

[0157] Therefore, in another aspect, the present invention provides a composition comprising a mixture of Bifidobacterium longum subsp. infantis and HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosylated lactose (3FL).

[0158] In another aspect, the present invention provides a combination comprising or consisting of a mixture of *Bifidobacterium longum* subsp. *inf.* and HMOs, the HMO mixture comprising 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosylated lactose (3FL). Preferably, the combination comprises a mixture of *Bifidobacterium longum* subsp. *inf.* and HMOs, the HMO mixture comprising 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosylated lactose (3FL).

[0159] The combinations or compositions of the present invention may also contain Bifidobacterium animalis subsp. lactis.

[0160] Therefore, in another aspect, the present invention provides a composition comprising Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, and an HMO mixture comprising 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosylated lactose (3FL).

[0161] In another aspect, the present invention provides a combination comprising or consisting of a mixture of or composed of *Bifidobacterium longum* subsp. *infantica*, *Bifidobacterium animalis* subsp. *lactobacterium*, and HMOs, the HMO mixture comprising 2'-fucosyllactose (2FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosyllactose (3FL). Preferably, the combination consists of a mixture of Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactic acid, and HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosylated lactose (3FL).

[0162] Bifidobacterium longum infant subspecies

[0163] Bifidobacterium longum is a type of Bifidobacterium found in the human gastrointestinal tract. In 2002, three previously distinct species of Bifidobacterium (Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium suis) were merged with biotype Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium suis into a single species named Bifidobacterium longum (Sakata, S. et al., 2002. International journal of systematic and evolutionary microbiology, 52(6), pp. 1945-1951).

[0164] Any suitable strain of *Bifidobacterium longum* subsp. infantis can be used in this invention. Such strains are well known to those skilled in the art. Suitable strains include *Bifidobacterium longum* subsp. infantis LMG 11588 (also known as *Bifidobacterium longum* subsp. infantis NCC3039 or *Bifidobacterium longum* subsp. infantis ATCC 17930) and *Bifidobacterium longum* subsp. infantis ATCC 15697 (also known as *Bifidobacterium longum* subsp. infantis NCC 3078).

[0165] Bifidobacterium longum subsp. infantis can be a strain known to those skilled in the art that has at least 99% (appropriately, at least 99.9%) of the ANI of Bifidobacterium longum subsp. infantis.

[0166] Suitably, *Bifidobacterium longum* subsp. *infantitidis* and *Bifidobacterium longum* subsp. *infantitidis* LMG 11588 (also known as *Bifidobacterium longum* subsp. *infantitidis* NCC3039 or *Bifidobacterium longum* subsp. *infantitidis* ATCC 17930) have an ANI of at least 99% (suitably, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%). Preferably, *Bifidobacterium longum* subsp. *infantitidis* and *Bifidobacterium longum* subsp. *infantitidis* LMG 11588 have an ANI of at least 99.9%.

[0167] Examples of microbial genomes with at least 99.9% ANI of *Bifidobacterium longum* subsp. *infant* 11588 can be found on PATRIC (https: / / www.patricbrc.org), genome ID 1678.111. Therefore, appropriately, *Bifidobacterium longum* subsp. *infant* with PATRIC genome ID 1678.111 can be used in this invention.

[0168] Bifidobacterium longum infantis subspecies LMG 11588 was sold by the Belgian Microbial Collection Center (BCCM) under the LMG accession number LMG 11588.

[0169] Bifidobacterium longum infantis ATCC 15697 was sold by the American Type Culture Collection (ATCC) under accession number ATCC 15697.

[0170] Based on dry weight, the composition or combination according to the present invention may contain 10 g of the composition or combination per g of the composition or combination. 3 cfu to 10 12 CFU of Bifidobacterium longum infantis subsp., preferably 10 7 cfu with 10 12 Between CFU, such as 10 8 cfu with 10 10 Bifidobacterium longum infantis subsp. *CFU*. Appropriately, Bifidobacterium longum infantis subsp. *CFU* is at least about 10 6 CFU / day, at least approximately 10 7 CFU / day or at least approximately 10 8 Administer CFU / day to the individual. Appropriately, *Bifidobacterium longum* subsp. infantis is administered at approximately 10... 12 CFU / day or less, approximately 10 11 CFU / day or less or about 10 10 CFU / day or less is administered to the individual.

[0171] In one implementation, *Bifidobacterium longum* subsp. infantis is live.

[0172] Bifidobacterium animalis subsp. lactis

[0173] Any suitable strain of *Bifidobacterium animalis* subsp. lactis (Bifidobacterium lactis) can be used in this invention. Such strains are well known to those skilled in the art. Suitable strains include *Bifidobacterium animalis* subsp. lactis CNCM I-3446.

[0174] Bifidobacterium animalis subsp. lactis can be a strain that has at least 99% (appropriately, at least 99.9%) of the ANI of a strain of Bifidobacterium animalis subsp. lactis known to the technician.

[0175] Suitably, both *Bifidobacterium animalis* subsp. *lactamase* and *Bifidobacterium animalis* subsp. *lactamase* CNCM I-3446 have an ANI of at least 99% (suitably, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%). Preferably, both *Bifidobacterium animalis* subsp. *lactamase* and *Bifidobacterium animalis* subsp. *lactamase* CNCM I-3446 have an ANI of at least 99.9%.

[0176] Bifidobacterium lactis CNCM I-3446 was deposited by NESTEC SA (NESTEC SA, AVENUE NESTLE 55, CH-1800 VEVEY) on June 7, 2005, at the National Center for Culture Collection of Microorganisms (CNCM) of the Pasteur Institute in France (INSTITUTPASTEUR, 25 RUE DU DOCTEUR ROUX, F-75724 PARIS CEDEX 15, FRANCE) under the Budapest Treaty, with accession number CNCM I-3446.

[0177] Based on dry weight, the composition or combination according to the present invention may contain 10 g of the composition or combination per g of the composition or combination. 3 cfu to 10 12 CFU of Bifidobacterium animalis subsp. lactis, preferably 10 7 cfu with 10 12 Between CFU, such as 10 8 cfu with 10 10 Between CFU and *Bifidobacterium animalis* subsp. *lactobacter*. Appropriately, *Bifidobacterium animalis* subsp. *lactobacter* is at least about 10 6 CFU / day, at least approximately 10 7 CFU / day or at least approximately 10 8 Administer CFU / day to the individual. Appropriately, *Bifidobacterium animalis* subsp. *lactobacter* is administered at approximately 10... 12 CFU / day or less, approximately 10 11 CFU / day or less or about 10 10 CFU / day or less is administered to the individual.

[0178] Based on dry weight, the composition or combination according to the present invention may contain 10 g of the composition or combination per g of the composition or combination. 3 cfu to 10 12 CFU of Bifidobacteria (e.g., a combination of Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis), preferably 10 7 cfu with 10 12 Between CFU, such as 10 8 cfu with 10 10 Bifidobacteria between CFU (e.g., a combination of *Bifidobacterium longum* subsp. *infant* and *Bifidobacterium animalis* subsp. *lactobacter*). Appropriately, the Bifidobacteria (e.g., a combination of *Bifidobacterium longum* subsp. *infant* and *Bifidobacterium animalis* subsp. *lactobacter*) are at least about 10 6 CFU / day, at least approximately 10 7 CFU / day or at least approximately 10 8 Administer CFU / day to the individual. Appropriately, Bifidobacteria (e.g., a combination of Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis) are administered at approximately 10...12 CFU / day or less, approximately 10 11 CFU / day or less, or about 10 10 CFU / day or less is administered to the individual.

[0179] In one implementation, *Bifidobacterium animalis* subsp. *lactamase* is live.

[0180] HMO mixture

[0181] The compositions or combinations of the present invention comprise HMO mixtures.

[0182] In some embodiments, the HMO mixture consists of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL).

[0183] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL) and 3-fucosyllactose (3FL).

[0184] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lactose-N-neotetrasaccharide (LNnT).

[0185] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lactose-N-neotetrasaccharide (LNnT).

[0186] In one embodiment, the HMO mixture contains 16 wt% to 85 wt% of 2FL. Suitably, the HMO mixture may contain 31 wt% to 82 wt%, preferably 41 wt% to 70 wt% of 2FL. Suitably, the HMO mixture may contain 16 wt% to 69 wt%, preferably 22 wt% to 59 wt% of 2FL. Suitably, the HMO mixture may contain 34 wt% to 85 wt%, preferably 40 wt% to 71 wt% of 2FL. Suitably, the HMO mixture may contain 20 wt% to 60 wt%, preferably 22 wt% to 55 wt% of 2FL.

[0187] In one embodiment, the HMO mixture contains 4% to 40% by weight of LNT. Suitably, the HMO mixture may contain 10% to 27% by weight, preferably 14% to 23% by weight of LNT. Suitably, the HMO mixture may contain 9% to 24% by weight, preferably 12% to 21% by weight of LNT. Suitably, the HMO mixture may contain 10% to 40% by weight, preferably 12% to 26% by weight of LNT. Suitably, the HMO mixture may contain 4% to 30% by weight, preferably 6% to 20% by weight of LNT.

[0188] In one embodiment, the HMO mixture contains 1% to 14% by weight of DFL. Suitably, the HMO mixture may contain 4% to 11% by weight, preferably 6% to 10% by weight of DFL. Suitably, the HMO mixture may contain 2% to 10% by weight, preferably 3% to 8% by weight of DFL. Suitably, the HMO mixture may contain 4% to 14% by weight, preferably 5% to 10% by weight of DFL. Suitably, the HMO mixture may contain 1% to 12% by weight, preferably 2% to 8% by weight of DFL.

[0189] In one embodiment, the HMO mixture comprises 6SL and 3SL in an amount combined from 7 wt% to 34 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL in an amount combined from 9 wt% to 34 wt%, preferably from 11 wt% to 29 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL in an amount combined from 8 wt% to 26 wt%, preferably from 11 wt% to 22 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL in an amount combined from 9 wt% to 31 wt%, preferably from 10 wt% to 28 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL in an amount combined from 7 wt% to 23 wt%, preferably from 8 wt% to 22 wt%.

[0190] In one embodiment, the HMO mixture contains 10% to 50% by weight of 3FL. Suitably, the HMO mixture may contain 18% to 50% by weight, preferably 11% to 43% by weight of 3FL. Suitably, the HMO mixture may contain 10% to 50% by weight, preferably 13% to 46% by weight of 3FL.

[0191] In one embodiment, the HMO mixture contains 6% to 30% by weight of LNnT. Suitably, the HMO mixture may contain 6% to 30% by weight, preferably 7% to 22% by weight of LNnT. Suitably, the HMO mixture may contain 3% to 25% by weight, preferably 5% to 20% by weight of LNnT.

[0192] In some implementations, the HMO mixture is essentially composed of the following substances:

[0193] i. 31% to 82% by weight of 2FL;

[0194] ii. 10% to 27% by weight of LNT;

[0195] iii. 4% to 11% by weight of DFL; and

[0196] iv. Combinations of 6SL and 3SL ranging from 9% to 34% by weight.

[0197] In some preferred embodiments, the HMO mixture is essentially composed of the following substances:

[0198] i. 41% to 70% by weight of 2FL;

[0199] ii. 14% to 23% by weight of LNT;

[0200] iii. 6% to 10% by weight of DFL; and

[0201] iv. Combinations of 6SL and 3SL ranging from 11% to 29% by weight.

[0202] In some implementations, the HMO mixture is essentially composed of the following substances:

[0203] i. 16% to 69% by weight of 2FL;

[0204] ii. 9% to 24% LNT by weight;

[0205] iii. 2% to 10% by weight of DFL;

[0206] iv. Combinations of 6SL and 3SL ranging from 8% to 26% by weight; and

[0207] v. 18% to 50% by weight of 3FL.

[0208] In some preferred embodiments, the HMO mixture is essentially composed of the following substances:

[0209] i. 22% to 59% by weight of 2FL;

[0210] ii. 12% to 21% by weight of LNT;

[0211] iii. 3% to 8% by weight of DFL;

[0212] iv. Combinations of 6SL and 3SL ranging from 11% to 22% by weight; and

[0213] v. 11% by weight to 43% by weight of 3FL.

[0214] In some implementations, the HMO mixture is essentially composed of the following substances:

[0215] i. 34% to 85% by weight of 2FL;

[0216] ii. 10% to 40% by weight of LNT;

[0217] iii. 4% to 14% by weight of DFL;

[0218] iv. Combinations of 6SL and 3SL ranging from 9% to 31% by weight; and

[0219] v. 6% to 30% by weight of LNnT.

[0220] In some preferred embodiments, the HMO mixture is essentially composed of the following substances:

[0221] i. 40% to 71% by weight of 2FL;

[0222] ii. 12% to 26% by weight of LNT;

[0223] iii. 5% to 10% by weight of DFL; and

[0224] iv. Combinations of 6SL and 3SL ranging from 10% to 28% by weight; and

[0225] v.7% to 22% LNnT by weight.

[0226] In some implementations, the HMO mixture is essentially composed of the following substances:

[0227] i. 20% to 60% by weight of 2FL;

[0228] ii. 4% to 30% by weight of LNT;

[0229] iii. 1% to 12% by weight of DFL;

[0230] iv. Combinations of 6SL and 3SL ranging from 7% to 23% by weight;

[0231] v. 10% to 50% by weight of 3FL; and

[0232] vi. 3% to 25% by weight of LNnT.

[0233] In some preferred embodiments, the HMO mixture is essentially composed of the following substances:

[0234] i. 22% to 55% by weight of 2FL;

[0235] ii. 6% to 20% by weight of LNT;

[0236] iii. 2% to 8% by weight of DFL;

[0237] iv. Combinations of 6SL and 3SL ranging from 8% to 22% by weight;

[0238] v. 13% to 46% by weight of 3FL; and

[0239] vi. 5% to 20% by weight of LNnT.

[0240] When the composition or combination is in liquid form, the total HMO concentration is typically in the range of 0.5 g / L to 10 g / L, preferably in the range of 1 g / L to 7.5 g / L. Specific examples of total HMO concentration levels when the composition or combination is in liquid form include 1 g / L to 5 g / L, 1 g / L to 4 g / L, 2 g / L to 5 g / L, 1 g / L to 3 g / L, or 2 g / L to 4 g / L.

[0241] When the composition or combination is in solid form, the total HMO concentration is typically in the range of 0.35 wt% to 7 wt% (g total HMO / 100g dry composition), preferably in the range of 0.35 wt% to 5 wt%. Specific examples of total HMO concentration levels when the composition or combination is in dry form include 0.5 wt% to 3.5 wt% (g total HMO / 100g dry composition), 0.5 wt% to 2.5 wt%, 1 wt% to 3.5 wt%, 0.5 wt% to 2 wt%, or 1 wt% to 2.5 wt%.

[0242] individual

[0243] In some implementations, the individual is a mammal, such as a human.

[0244] In some embodiments, the compositions according to the invention are used in infants, toddlers, or children.

[0245] In one implementation, the individual is an infant. In another implementation, the individual is a toddler. In yet another implementation, the individual is a child.

[0246] The compositions according to the invention are used in infants or young children. This nutritional composition is particularly suitable for infants under 6 months of age.

[0247] Generally, formula-fed infants have an underdeveloped immune system compared to adults and are more susceptible to viral infections than breastfed infants, with the immune system becoming less developed the younger the infant. Therefore, the composition is particularly useful for preterm infants and / or low or very low birth weight infants, as these infants are even more vulnerable to injury and viral infections. In another embodiment of particular interest, the composition is used for infants delivered by cesarean section. Cesarean-born infants are born in a hospital environment with more pathogens, and antibodies against these pathogens transferred from the mother to the infant are not effective against them. Furthermore, antibiotic administration is a recommended medical practice for cesarean delivery to prevent infection. Such interventions can severely disrupt the (mother's or child's) microbiome, and early-life antibiotic treatment is associated with an increased risk of developing immune-mediated disorders later in life. Cesarean-born infants have delayed and less desirable colonization of the large intestine and are therefore also more susceptible to infection.

[0248] The infant, toddler, or child may be full-term or premature. In one specific embodiment, the nutritional composition of the present invention is used in premature infants, toddlers, or children. Premature infants may have an increased risk of developing poor nutrient utilization, lean body mass syndrome, visceral fat accumulation, and metabolic diseases later in life. Therefore, in one embodiment, the nutritional composition of the present invention is used in premature infants.

[0249] In one implementation, the individual is for infants or young children who are born small for gestational age or have low birth weight.

[0250] Low birth weight infants or toddlers may or may not be premature, and similarly, small for gestational age infants or toddlers may or may not be premature.

[0251] The nutritional composition of the present invention can also be used for infants or young children born by cesarean section or vaginal delivery.

[0252] All infants and young children can benefit from this invention because they are all prone to, or may be prone to, an unbalanced gut microbiota at some age.

[0253] In some advantageous embodiments of the invention, the nutritional composition is used for infants or young children with a fragile or unbalanced microbiome or microbiome dysbiosis, such as premature infants, infants born by cesarean section, infants born small for gestational age or with low birth weight, hospitalized infants / young children, infants / young children who have received or have received antibiotic treatment, and / or infants / young children who have or have had intestinal infections and / or intestinal inflammation.

[0254] Indeed, it is foreseeable that the compositions of the present invention may be even more beneficial for infants whose gut microbiota may be impaired at birth or for weak infants / young children (such as premature infants and / or infants born by cesarean section). Furthermore, it is foreseeable that the compositions of the present invention may be even more beneficial, particularly for infants / young children who exhibit intestinal disturbances (such as diarrhea, infection, or colic) especially after birth, for example, during the first four weeks after birth.

[0255] In embodiments of the invention, the compositions of the invention are intended for infants who are premature, born by cesarean section, born small for gestational age, or have low birth weight, or exhibit an unbalanced or abnormal gut microbiota, or have had or have had intestinal infections and / or intestinal inflammation, particularly when the infant is 0 to 6 months of age. Without being bound by theory, it is believed that younger infants benefit even more from the compositions of the invention, particularly when the infant has an unbalanced gut microbiota (or is at risk of having an unbalanced gut microbiota) and / or has a weakened health condition (as illustrated in the conditions cited above).

[0256] The age and duration of administration (or administration or feeding) of the nutritional composition can be determined as needed.

[0257] In one embodiment, the infant or toddler is 0-36 months old, such as 0-12 months or 0-6 months. It is foreseeable that the compositions of the present invention may be more advantageous for newborns (0 to 4 weeks or 0 to 8 weeks) because their intestines may be more vulnerable.

[0258] The mammal to be treated is preferably a human, but the mammal may also be a non-human mammal, such as a non-human mammal selected from pigs, cattle, horses, dogs, cats, goats, sheep and rabbits.

[0259] In other embodiments, the individual is a young animal, preferably a pet.

[0260] Pets can be mammals such as dogs or cats, or rodents such as mice, rats and guinea pigs, rabbits, etc. In some implementations, pets are small dog breeds.

[0261] The term "juvenile" can refer to an individual who has not yet reached adulthood.

[0262] In some embodiments, the nutritional composition according to the invention may be used before and / or during the weaning period.

[0263] In some embodiments, the nutritional compositions according to the invention are used in individuals at risk and / or in need.

[0264] Individuals at risk and / or in need may be bottle-fed and / or formula-fed.

[0265] In one embodiment, the composition of the present invention is given to an individual as a supplemental composition to breast milk. In some embodiments, the individual is breastfed for at least the first 2 weeks, the first 1 month, 2 months, 4 months, or 6 months. In one embodiment, the nutritional composition of the present invention is given to the individual after this period of breastfeeding, or given to the individual along with breast milk during this period of breastfeeding. In another embodiment, the composition is given to the individual as the sole or primary nutritional composition for at least a period of time (e.g., after the first, second, or fourth month of life), for at least 1 month, 2 months, 4 months, or 6 months. In one embodiment, the nutritional composition of the present invention is a complete nutritional composition (meeting all or most of the individual's nutritional needs). In another embodiment, the nutritional composition of the present invention is a supplement or fortifier intended for use, for example, supplementing human milk or supplementing infant formula or follow-up formula.

[0266] Nutritional composition

[0267] In some embodiments, the compositions of the present invention are in the form of nutritional compositions.

[0268] The nutritional compositions according to the present invention may be, for example, infant formula, stage 1 infant formula, follow-up or stage 2 infant formula, growing milk, baby food, infant cereal compositions, fortifiers (such as human milk fortifiers), supplements, pet food, or pet food supplements. In some specific embodiments, the compositions of the present invention are infant formula, fortifiers, or supplements intended for infants aged 4 months or 6 months. In a preferred embodiment, the nutritional compositions of the present invention are infant formula.

[0269] In some other embodiments, the nutritional composition of the present invention is a fortifier. The fortifier may be a breast milk fortifier (e.g., human milk fortifier) ​​or a formula food fortifier (such as an infant formula fortifier or a follow-up formula / stage 2 infant formula fortifier).

[0270] When a nutritional composition is a supplement, it can be provided in unit doses. In such cases, it is particularly useful to limit the amount of oligosaccharides and probiotics according to the daily dose administered to infants, toddlers, or children.

[0271] When the nutritional composition is a supplement, it may contain an HMO composition as described herein and Bifidobacterium infantis, and may contain no additional nutrients other than the excipients necessary to obtain a stable nutritional composition.

[0272] The nutritional composition of the present invention may be in solid (e.g., powder), liquid, or gel form. In one particular embodiment, the nutritional composition is a supplement, wherein the supplement is in powder form and provided in sachets, preferably with 0.1 g / sachet to 20 g / sachet, for example, 1 g / sachet to 10 g / sachet, or provided in syrup form, preferably with a total solids concentration of 5 g / 100 mL to 75 g / 100 mL (5% to 75% (w / v)). When the supplement is in powder form, it may contain a carrier. However, it is preferred that the supplement does not contain a carrier. When the supplement is in syrup form, the components are preferably dissolved or suspended in water acidified with citrate.

[0273] In one embodiment, the nutritional composition according to the invention is a hypoallergenic composition. In another embodiment, the composition according to the invention is a hypoallergenic nutritional composition.

[0274] Other ingredients

[0275] The compositions or combinations according to the invention may also contain other types of oligosaccharides, polysaccharides, and / or fibers and / or their precursors. Other oligosaccharides and / or fibers and / or their precursors may be selected from the list including: human milk oligosaccharides (HMOs), galactooligosaccharides (GOS), fructooligosaccharides (FOS), xylooligosaccharides (XOS), cellulose oligosaccharides (COS), arabinoxylan, arabinogalactan, xylan, inulin, polydextrose, β-glucan, pectin, any combination thereof, and any derivative thereof (e.g., partially hydrolyzed). Their amounts may be between 0% by weight and 10% by weight of the composition. In one specific embodiment, the nutritional composition may also contain at least one BMO (a bovine milk-derived oligosaccharide).

[0276] Additional HMOs that may be included in the nutritional compositions according to the invention may be selected from lactose-N-fucopentose (e.g., lactose-N-fucopentose I, lactose-N-fucopentose II, lactose-N-fucopentose III, lactose-N-fucopentose V), lactose-N-fucohexasose, lactose-N-difucohexasose I, fucosyllacto-N-hexasose, fucosyllacto-N-neohexose, and difucosyllacto-N-hexasose. Fucose-N-hexasaccharide I, fucose-N-neohexaccharide II, p-lactose-N-neohexaccharide (p-LNnH), lactose-N-hexasaccharide, lactose-N-neohexaccharide, p-lactose-N-hexasaccharide, p-lactose-N-neohexaccharide, lactose-N-octasaccharide, lactose-N-neohexaccharide, isolose-N-octasaccharide, p-lactose-N-octasaccharide, lactose-N-decasaccharide, and any combination thereof.

[0277] In some embodiments, the composition or combination according to the invention comprises at least one additional HMO.

[0278] In other embodiments, the compositions or combinations according to the invention do not contain any additional HMOs. Therefore, the prebiotic oligosaccharide mixture as described herein can be the only HMO in the compositions or combinations of the invention.

[0279] The compositions or combinations of the present invention may further comprise at least one additional probiotic (or probiotic strain), such as at least one additional probiotic strain.

[0280] The most commonly used probiotics are mainly bacteria and yeasts from the following genera: Lactobacillus spp., Lacticaseibacillus spp., Streptococcus spp., Enterococcus spp., Bifidobacterium spp., and Saccharomyces spp.

[0281] In some specific embodiments, the probiotics are probiotic bacterial strains. In some specific embodiments, they are specifically Bifidobacterium and / or Lactobacillus.

[0282] Suitable probiotic strains include *Lactobacillus rhamnosus* ATCC 53103, *Lactobacillus rhamnosus* CGMCC 1.3724, *Lactobacillus paracasei* CNCM I-2116, *Lactobacillus johnsonii* CNCMI-1225 (from Valio Oy, Finland), *Streptococcus salivarius* DSM 13084 (sold by BLIS Technologies Limited, New Zealand under the name KI2), *Bifidobacterium lactis* (specially sold by Christian Hansen, Denmark under the trademark Bb 12), *Bifidobacterium longum* CNCM I-2618 (*Bifidobacterium longum* NCC2705), *Bifidobacterium breve* (sold by Danisco under the trademark Bb-03), and *Bifidobacterium breve* (sold by Morinaga under the trademark M-16V), such as *Procter*. &GambIe Co. sells Bifidobacterium infantis under the trademark Bifantis, and Institut Rosell (Lallemand) sells Bifidobacterium breve under the trademark R0070.

[0283] On a dry weight basis, the composition or combination according to the invention may contain at least one (additional) probiotic strain of 10e3 cfu to 10e12 cfu per g of composition or combination, more preferably a probiotic strain of between 10e7 cfu and 10e12 cfu (such as between 10e8 cfu and 10e10 cfu).

[0284] In one embodiment, the probiotics are live. In another embodiment, the probiotics are non-replicating or inactivated. In some other embodiments, both live and inactivated probiotics may be present simultaneously. Probiotic components and metabolites may also be added.

[0285] The nutritional compositions according to the invention typically contain a protein source. The amount of protein can be from 1.6 g / 100 kcal to 3 g / 100 kcal. In some embodiments, particularly when the composition is intended for preterm infants, the amount of protein can be between 2.4 g / 100 kcal and 4 g / 100 kcal or more than 3.6 g / 100 kcal. In some other embodiments, the amount of protein can be less than 2.0 g / 100 kcal, for example, from 1.8 g / 100 kcal to 2 g / 100 kcal, or less than 1.8 g / 100 kcal.

[0286] Protein sources based on whey, casein, or mixtures thereof, or soy-based protein sources, may be used. Regarding the whey protein of interest, the protein source may be based on acidic whey or sweet whey, or mixtures thereof, and may contain any desired proportions of α-lactalbumin and β-lactoglobulin.

[0287] In some advantageous implementations, the protein source is whey-based (i.e., more than 50% of the protein comes from whey protein, such as 60% or 70%).

[0288] The protein can be whole or hydrolyzed, or a mixture of whole and hydrolyzed proteins. The term "whole" means that the major components of the protein are intact, i.e., the molecular structure is not altered, for example, at least 80% of the protein is unchanged, such as at least 85% of the protein is unchanged, preferably at least 90% of the protein is unchanged, even more preferably at least 95% of the protein is unchanged, such as at least 98% of the protein is unchanged. In one specific embodiment, 100% of the protein is unchanged.

[0289] The term "hydrolyzed" means, in the context of this invention, that a protein has been hydrolyzed or broken down into its constituent amino acids. The protein may be completely or partially hydrolyzed. For example, providing partially hydrolyzed protein (with a degree of hydrolysis between 2% and 20%) may be desirable for infants or young children considered at risk of bovine milk allergies. If a hydrolyzed protein is required, the hydrolysis process can be carried out as needed and as is known in the art. For example, whey protein hydrolysates can be prepared by enzymatic hydrolysis of whey fractions in one or more steps. If the whey fraction used as a raw material is substantially lactose-free, it has been found that the protein undergoes far less lysine blockage during the hydrolysis process. This allows the degree of lysine blockage to be reduced from about 15% by weight of total lysine to less than about 10% by weight of lysine; for example, about 7% by weight of lysine, which significantly improves the nutritional quality of the protein source.

[0290] In one embodiment of the invention, at least 70% of the protein is hydrolyzed, preferably at least 80%, such as at least 85%, even more preferably at least 90%, such as at least 95%, and particularly at least 98%. In one specific embodiment, 100% of the protein is hydrolyzed.

[0291] In one specific embodiment, the protein in the nutritional composition is hydrolyzed, fully hydrolyzed, or partially hydrolyzed. The degree of hydrolysis (DH) of the protein may be between 8 and 40, or between 20 and 60, or between 20 and 80, or greater than 10, 20, 40, 60, 80, or 90.

[0292] Alternatively, the protein component can be replaced with a mixture or synthetic amino acids, for example, for premature or low birth weight infants.

[0293] In one embodiment, the nutritional composition or growing milk according to the invention is a hypoallergenic composition. In another embodiment, the composition according to the invention is a hypoallergenic nutritional composition or growing milk.

[0294] The nutritional compositions according to the invention typically contain a carbohydrate source. This is particularly preferred when the nutritional compositions of the invention are for infant formula. In this case, any carbohydrate source commonly found in infant formula can be used, such as lactose, sucrose, saccharin, maltodextrin, starch, and mixtures thereof, but one of the preferred carbohydrate sources is lactose.

[0295] The nutritional compositions according to the invention typically contain a lipid source. This is particularly relevant when the nutritional compositions of the invention are for infant formula. In this case, the lipid source can be any lipid or fat suitable for use in infant formula. Some suitable fat sources include palm oil, structured triglyceride oil, high-oleic sunflower oil and high-oleic safflower oil, and medium-chain triglyceride oil. Essential fatty acids linoleic acid and α-linolenic acid, as well as small amounts of oils containing large amounts of pre-formed arachidonic acid and docosahexaenoic acid, such as fish oil or microbial oil, may also be added. The ratio of n-6 fatty acids to n-3 fatty acids in the fat source can be from about 5:1 to about 15:1, for example from about 8:1 to about 10:1.

[0296] The nutritional compositions of the present invention may also contain all vitamins and minerals considered essential for a daily diet and required in significant amounts. Minimum requirements for certain vitamins and minerals have been determined. Examples of minerals, vitamins, and other nutrients optionally present in the compositions of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are typically added in salt form. The presence and amounts of specific minerals and other vitamins will vary depending on the target population.

[0297] If necessary, the nutritional compositions of the present invention may contain emulsifiers and stabilizers, such as soybean, lecithin, mono- and di-citrate citrate, etc.

[0298] The nutritional composition of the present invention may also contain other substances that may have beneficial effects, such as nucleotides, nucleosides, etc.

[0299] The nutritional composition of the present invention may further contain carotenoids. In some specific embodiments of the present invention, the nutritional composition of the present invention does not contain any carotenoids.

[0300] Manufacturing of nutritional compositions

[0301] The nutritional compositions according to the invention can be prepared in any suitable manner. The compositions will now be described by way of example.

[0302] For example, formulated foods (such as infant formula) can be prepared by blending protein sources, carbohydrate sources, and fat sources together in appropriate proportions. If used, an emulsifier may be added at this stage. Vitamins and minerals may be added at this stage, but they are usually added later to avoid thermal degradation. Before blending, any lipophilic vitamins, emulsifiers, etc., can be dissolved in the fat source. Water (preferably water subjected to reverse osmosis) can then be mixed in to form a liquid mixture. The water temperature is suitably between about 50°C and about 80°C to aid in the dispersion of the components. Commercially available liquefying agents can be used to form the liquid mixture.

[0303] Oligosaccharides can be added at this stage, especially when the final product is in liquid form. If the final product is in powder form, these components can also be added at this stage as needed.

[0304] Then, the liquid mixture is homogenized in, for example, in two stages.

[0305] The liquid mixture can then be heat-treated to reduce the bacterial load, for example by rapidly heating the liquid mixture to a temperature between about 80°C and about 150°C for a duration between about 5 seconds and about 5 minutes. This can be done by steam injection, autoclaving, or a heat exchanger (e.g., a plate heat exchanger).

[0306] The liquid mixture is then cooled, for example, to between approximately 60°C and approximately 85°C by rapid cooling. It is then homogenized again, for example, in two stages, between approximately 10 MPa and approximately 30 MPa in the first stage and between approximately 2 MPa and approximately 10 MPa in the second stage. The homogenized mixture can then be further cooled to add any heat-sensitive components, such as vitamins and minerals. The pH and solids content of the homogenized mixture can then be conveniently adjusted.

[0307] If the final product is a powder, the homogenized mixture is transferred to a suitable drying apparatus, such as a spray dryer or freeze dryer, and converted into a powder. The moisture content of the powder should be less than about 5% by weight. Oligosaccharides may also be added at this stage, or alternatively, by dry mixing them with probiotic strains in the form of crystalline syrup, or by blending them with probiotic strains, followed by spray drying or freeze drying of the mixture.

[0308] If a liquid composition is preferred, the homogenized mixture can be sterilized and then filled into a suitable container under aseptic conditions, or it can be filled into a container first and then distilled.

[0309] In another embodiment, the composition of the present invention may be a supplement. The supplement may be in the form of, for example, tablets, capsules, lozenges, or liquids. The supplement may also contain protective hydrophilic colloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulation agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, flavor masking agents, weighting agents, gelling agents, and gelling agents. The supplement may also contain conventional pharmaceutical additives and adjuvants, excipients, and diluents, including but not limited to: water, gelatin of any source, plant gums, lignin sulfonates, talc, sugars, starches, gum arabic, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, fillers, etc.

[0310] In addition, supplements may contain organic or inorganic carrier materials suitable for oral or parenteral administration, as well as vitamins, trace minerals, and other micronutrients recommended by government agencies such as the USRDA.

[0311] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure. Numerical ranges include the numbers defining the range. Unless otherwise specified, any nucleic acid sequences are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right, respectively, from amino to carboxyl.

[0312] Where a range of values ​​is provided, it should be understood that, unless the context explicitly indicates otherwise, each intermediate value between the upper and lower limits of the range is also specifically disclosed, accurate to one-tenth of the lower limit unit. Each smaller range between any specified value or intermediate value in the specified range and any other specified value or intermediate value in the specified range is covered within this disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range in which any limit is included, neither limit is included, or both limits are included is also covered within this disclosure, subject to any specifically excluded limit in the specified range. Where the range includes one or both limits, the range excluding any one or both of those included limits is also included in this disclosure.

[0313] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the context clearly specifies otherwise.

[0314] As used herein, the terms “comprising” and “consisting of” are synonymous with “including” or “containing”, and are inclusive or open-ended, and do not exclude additional unlisted members, elements, or method steps. The terms “comprising” and “consisting of” also include the term “composed of”.

[0315] Unless otherwise specified, the present invention will be practiced using conventional chemical, biochemical, molecular biological, microbiological, and immunological techniques, all of which are within the capabilities of a person skilled in the art. Such techniques are described in the literature. See, for example, Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, FM, et al. (1995 and periodically supplemented), Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons; Roe, B., Crabtree, J., and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JM, and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ, (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM, and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. These general texts are incorporated herein by reference.

[0316] The publications discussed herein are provided only for their disclosure prior to the filing date of this patent application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended herein.

[0317] The present invention will now be further described by way of examples, which are intended to help those skilled in the art to implement the invention and are not intended to limit the scope of the invention in any way.

[0318] Example

[0319] Example 1 - Effects on early life viral airway infections and allergic airway inflammation exacerbated by pollution

[0320] Detailed experimental design

[0321] Figure 1 A schematic diagram of a model of early-life viral airway infection and allergic airway inflammation exacerbated by pollution is shown. The experimental design is described in more detail below.

[0322] Starting three weeks before gestation, WT C57BL / 6 mice were fed a control fiber or low-fiber diet. Throughout the experiment, from day 0 (PND) to day 66 (PND66), the offspring of these mice were fed a diet corresponding to that of their mothers.

[0323] At PND5, young mice fed a low-fiber diet were randomly assigned to different experimental groups (n=14-16 / group). Mice were fed once daily with different combinations of nutrients (6HMO; Bifidobacterium infantis LGM11588; Bifidobacterium infantis LGM11588+6HMO) via intragastric gavage (ig) at a concentration of 50 μl in saline solution. This continued until PND20. The control group was fed only saline solution.

[0324] In PND10, all animals (n=13–16 / group) were intranasally infected with 10 PFU of PVM (PVM, J3666) in a total volume of 40 μl saline solution (20 μl / nostril) to induce bronchiolitis. The virus-containing solution was delivered as droplets into the nostrils using a P20 pipette. Mice anesthetized with isoflurane were kept in a supine position to ensure proper aspiration of the solution.

[0325] Animals (n=7-8 / group) were sacrificed at PND20 (the peak of the immunopathological period of bronchiolitis) to measure virus-induced lung inflammation and related pathology.

[0326] Starting from PND42, animals recovering from PVM infection (n=7-8 / group) were intranasally sensitized once a week (i.e., at PND42, PND49, PND56, PND63) with a combination of cockroach allergen extract (CRE, 1 mg / application) and particulate PM2.5 (10 mg), ending at PND63, to induce allergic airway inflammation exacerbated by pollution.

[0327] Animals were sacrificed at PND20 (N=7-8 / group, peak of immunopathological bronchiolitis) to test the effect of nutritional intervention on promoting protective immunity, and at PND66 (N=7-8 / group) to assess susceptibility to contamination-induced allergic respiratory inflammation following viral airway infection.

[0328] Probiotic dosage

[0329] 10 6 CFU / day.

[0330] HMO dosage

[0331] Based on the latest recommendations for HMOs in infant formula, a concentration of 4 mg / day and a mixture of 6 HMOs (2FL / DFL, LNT, 3SL, 6SL, 3FL) were selected (Phase 1: 1.8-2 g / L / day for the first 6 months).

[0332] The calculations are based on the following: 1-week-old infants consume 500 mL of breast milk per day and 10-day-old mouse pups consume up to 2 mL of milk per day (Source: contemporary topics in laboratory animal science / American Association for Laboratory Animal Science 43(3):50-3).

[0333] The ratios of HMO components are inspired by the composition of breast milk: 2FL / DFL component 53%; LNT component 19%; 6SL component 8%; 3SL component 6.2%; 3FL component 13.8%.

[0334] In some experiments, based on the latest recommendations for HMOs in infant formula, a concentration of 4 mg / day and a blend of 5 HMOs (2FL, DFL, LNT, 3SL, 6SL) were selected (Phase 1: 1.8-2 g / L / day for the first 6 months). The proportions of the 5 HMO blends used were: 2FL / DFL 65.70%; LNT 23.40%; 6SL 9%; and 3SL 2%.

[0335] low fiber diet

[0336] WT C57BL / 6 mice effectively cleared PVM virus. This mild infection was not associated with changes in lung tissue remodeling, which predispose infants to allergic airway inflammation later in life. To increase susceptibility to PVM infection and associated risk of allergic airway inflammation in adulthood, mother mice and their corresponding offspring were fed a low-fiber diet throughout the experiment (susceptible group; Trompette, Nat. Med, 2014). Animals fed a normal-fiber diet served as protected controls (protected group).

[0337] Statistical analysis

[0338] Nonparametric two-way ANOVA was used, followed by a Bonferroni post-hoc test to determine statistical significance. A result was considered significant when p ≤ 0.05. P≤0.05; P≤0.01; P ≤ 0.001. All statistical analyses were performed using Prism GraphPad software.

[0339] result

[0340] PVM infection model

[0341] Mouse pneumonia virus (PVM) is associated with the human respiratory syncytial virus (RSV) pathogen, affecting >90% of children under two years of age. PVM has been used to study respiratory viral replication and subsequent inflammatory responses as part of the natural host-pathogen relationship. Therefore, PVM infection in mice reproduces many of the clinical and pathological features of the more severe forms of RSV infection in infants—which, if not properly controlled, have lasting consequences for lung function and predispose to anaphylactic airway reactions later in life (Dyer KD, Viruses, 2012). The highly pathogenic PVM strain J3666 was chosen for these studies to align with the human pathophysiology of RSV infection in infants.

[0342] Severe bronchiolitis early in life is associated with excessive mucus secretion and airway epithelial cell shedding. Dead epithelial cells, along with viscous exudate, can form dense plugs in the bronchoalveolar spaces, obstructing respiration. Controlled mucus secretion and epithelial cell death are beneficial defense mechanisms that limit viral transmission. These responses need to be strictly regulated after viral clearance to avoid chronic and pathological tissue remodeling.

[0343] The pathological features of severe viral lower respiratory tract infections include airway epithelial cell (AEC) shedding, excessive mucus secretion, and airway smooth muscle (ASM) remodeling. AEC shedding is characteristic of viral bronchiolitis and is associated with disease severity and viral load.

[0344] AEC shedding was quantified by measuring the length of sloughed airway epithelium and expressing it as a percentage of the basement membrane length of the airways in at least five airways per mouse. Quantitative assessment of mucus secretion and pathological lung tissue remodeling (as another marker of severe bronchiolitis) was performed by quantifying Muc5ac-positive AECs, and ASM remodeling was assessed by area / airway circumference (Lynch JP, JEM 2018).

[0345] Compared with placebo, early life nutritional intervention using synbiotics (Bifidobacterium infantis + 6HMO) significantly reduced infection-mediated airway epithelial cell (AEC) shedding. Figure 2 A) Mucus secretion ( Figure 2 B) and pathological tissue remodeling ( Figure 2 C). Notably, synbiotic intervention showed superior efficacy compared to prebiotic (6HMO) or probiotic (Bifidobacterium infantis) intervention alone in reducing infection-mediated AEC shedding, mucus secretion, and pathological airway remodeling.

[0346] To evaluate whether early life nutritional supplementation with synbiotics, in addition to evoking an effective antiviral immune response, also leads to controlled resolution of lung inflammation after viral clearance, the number of neutrophils and / or eosinophils in the airways of mice infected with PVM was assessed by flow cytometry. Importantly, early life supplementation with synbiotics resulted in controlled resolution of lung inflammation after viral clearance, as assessed by a significant reduction in the number of granulocytes (neutrophils and eosinophils) in the airways in response to PVM infection.

[0347] Early life nutritional intervention using a second synbiotic (Bifidobacterium infantis + 5HMO) resulted in a significant upregulation of the antiviral cytokine IFN-λ 5 days post-infection (peak of viral infection). Figure 5 A), which is related to factors such as the number of lung neutrophils ( Figure 5 B) and the number of eosinophils in the lungs ( Figure 5 C) The significantly reduced quantified granulocyte infiltration into the inflamed lung is associated with intervention with synbiotics (Bifidobacterium infantis + 5HMO) in early life, indicating that intervention can reduce virus-induced severe lung inflammation. These data suggest that early life supplementation with synbiotics can control an effective antiviral immune response and promote rapid resolution of lung inflammation, thus facilitating recovery from respiratory infections.

[0348] A model of allergic airway inflammation exacerbated by pollution

[0349] Severe RSV infection early in life represents a significant risk factor for the later development of allergic airway diseases. Outdoor air pollution is a major health problem worldwide. Exposure to particulate matter (PM) is associated with the exacerbation of several respiratory diseases, including allergic asthma. To simulate this globally relevant condition, animals recovering from severe PVM infection were sensitized with cockroach allergens in the presence of pollutant PM2.5 to induce allergic airway inflammation. This experimental approach allows testing whether early-life (window of opportunity) nutritional interventions not only alter susceptibility to viral bronchiolitis but may also have lasting immune benefits into adulthood.

[0350] Early-life respiratory viral infections represent a potential tipping point in the balance between long-term respiratory health and chronic airway diseases such as airway hypersensitivity. This study shows that early-life synbiotic intervention not only reduces the severity of airway viral infections but also promotes immune benefits that persist into adulthood, as assessed by reducing susceptibility to pollution-exacerbated allergic airway inflammation.

[0351] This is manifested by lung ILC2 as assessed by flow cytometry ( Figure 3 A) and eosinophil count ( Figure 3 Both (B) decreased. The reduction in lung inflammation was also associated with a decrease in mucus score ( Figure 4 A) and organizational restructuring and improvement ( Figure 4 (B) is associated with this. Notably, it is consistent with reduced susceptibility to viral airway infections (see [link]). Figure 2 Synbiotic intervention (Bifidobacterium infantis + 6HMO) showed superior efficacy to prebiotic (6HMO) or probiotic (Bifidobacterium infantis) intervention alone in reducing allergic airway inflammation exacerbated by pollution in adulthood.

[0352] in conclusion

[0353] Using a PVM-induced bronchiolitis and early-life nutritional intervention model in newborn mice, we demonstrated the effectiveness of synbiotic intervention (a combination of Bifidobacterium infantis and human milk oligosaccharides (5HMO or 6HMO)). It can provide fine-grained antiviral inducement Prevention of bronchitis. Importantly, we demonstrated that synbiotic intervention (a combination of Bifidobacterium infantis and human milk oligosaccharides (6HMOs)) provides defense against virus-induced bronchiolitis. Provides excellent efficacy It is superior to 6HMO alone or Bifidobacterium infantis alone. Specifically, synbiotic intervention leads to rapid resolution of virus-induced lung inflammation and appropriate lung tissue remodeling after virus clearance.

[0354] Since the initial host response to respiratory viruses represents a potential tipping point in the balance between long-term respiratory health and chronic respiratory disease, these findings suggest that early-life nutritional interventions using these synbiotics (Bifidobacterium infantis + 6 HMO or Bifidobacterium infantis + 5 HMO) may represent a preventative strategy against severe bronchiolitis in infants and the associated risk of developing allergic airway disease in school age. Indeed, early-life nutritional synbiotic interventions not only reduced the severity of airway viral infections but also promoted immune benefits that persist into adulthood, as assessed by reducing susceptibility to pollution-exacerbated allergic airway inflammation. In particular, these results provide strong evidence that early-life nutritional synbiotic interventions attenuate pathological airway remodeling, which is known to increase the risk of developing chronic inflammatory diseases later in life, such as asthma or COPD.

[0355] Example 2 - Study on in vitro fecal fermentation

[0356] Experimental Design

[0357] This study was conducted using Cryptobiotix SA, employing its proprietary in vitro human gastrointestinal replication model and colonic microbiota incubation, as described in Van den Abbeele et al., Frontiers Microbiol. 2023.

[0358] Two groups were used in the study. These consisted of the following:

[0359] 1. Blends of 7 HMOs: 2FL, 3FL, DFL, LNT, LNnT, 3SL, 6SL

[0360] 2.7 HMOs (2FL, 3FL, DFL, LNT, LNnT, 3SL, 6SL) + Bifidobacterium infantis LMG1188

[0361] Each group consisted of six bioreactors (n=6), each inoculated with the fecal microbiota of six individual donors. Donors were 3-month-old (±3 weeks) infants fed only with HMO-free / probiotic-free formula. Exclusion criteria included antibiotic use within 30 days prior to sample delivery for study purposes and a history of necrotizing enterocolitis (NEC) or intestinal surgery. This resulted in the enrollment of six specific test individuals with a mean age of 3.1 (±0.5) months. Intestinal absorption and colonic incubation conditions were then established (see Van den Abbele et al., Frontiers Microbiol. 2023).

[0362] A blend of seven HMOs was added, resulting in a final concentration of 2.66 g / L. The g / L composition of each HMO is described in the table below.

[0363]

[0364] Bifidobacterium infantis LMG11588 at 5×10 7 CFU / mL concentration measurement.

[0365] Colon incubation was performed for 24 hours, with samples taken at 0 and 24 hours:

[0366] SCFA levels were determined by HPLC.

[0367] Determining the 16S rRNA community profile of the microbiome

[0368] result

[0369] As described by Van den Abbeele et al., 2023 (DOI: 10.3389 / fmicb.2023.1131662), the level of *Bifidobacterium breve* in liquid was quantified after 24 hours of incubation. Under both conditions, the 7-HMO blend was present as the sole carbon source. The 6-donor mean was plotted. The left bar chart represents the *Bifidobacterium breve* level when only the 7-HMO blend was present and when only 5 × 10⁻⁶ HMO was present. 7 The sum of Bifidobacterium breve levels at CFU / mL of Bifidobacterium infantis LMG 11588 (cumulative level). The right-hand bar chart represents the Bifidobacterium breve levels when the same active ingredient is present together (synergistic level). The yield without the active ingredient is subtracted from each individual condition (blank subtraction). When 7-HMO is co-present with Bifidobacterium infantis LMG 11588, their interaction results in higher Bifidobacterium breve levels than in the 7-HMO blend alone or in the presence of 5 × 10⁻⁶ CFU / mL. 7 The expected total level of Bifidobacterium infantis LMG at CFU / mL was 11588. Figure 6 Therefore, the data demonstrate the synergistic effect of the combination of 7-HMO and Bifidobacterium infantis in establishing Bifidobacterium breve in fecal fermentation cultures.

[0370] in conclusion

[0371] Using fecal fermentation studies, we demonstrated the synergistic effect of combining 7-HMO with Bifidobacterium infantis in establishing Bifidobacterium breve.

[0372] Besides *Bifidobacterium infantis*, *Bifidobacterium breve* is predominant in the feces of breastfed newborns, and there is substantial evidence of its immunomodulatory capabilities (Bozzi Cionci, N. et al., Nutrients, 2018, 10(11)). This evidence includes beneficial modulation of the pulmonary immune response, including reduced airway hyperresponsiveness, activation of type 2 T helper cells, eosinophilia, and production of allergen-specific antibodies (Hougee, S. et al., Int Arch Allergy Immunol, 2010, 151(2): 107-17).

[0373] Therefore, the synergistic effect shown here is related to lung immunity.

[0374] All publications mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the invention protected by the claims should not be unduly limited to such specific embodiments. Indeed, various modifications to the described modes of carrying out the invention that will be apparent to those skilled in molecular biology or related fields are intended to fall within the scope of the following claims.

[0375] Various preferred features and embodiments of the invention will now be described with reference to the following numbered paragraphs:

[0376] 1. A composition for the prevention and / or treatment of viral infections in an individual, wherein the composition comprises a mixture of *Bifidobacterium longum* subsp. infantis and HMO, the HMO mixture comprising 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0377] 2. A composition used to promote long-term immune benefits in an individual, wherein the composition comprises a mixture of Bifidobacterium longum subsp. infantis and HMOs, the HMO mixture comprising 2'-fucosyllactose (2FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL) and optionally 3-fucosyllactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0378] 3. A composition used in i) preventing and / or reducing the risk of an individual’s allergen sensitization and / or ii) preventing and / or reducing the risk of an individual’s respiratory illness, wherein the composition comprises a mixture of Bifidobacterium longum subsp. infantis and HMO, said HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0379] 4. A composition used in the prevention and / or reduction of an individual's risk of developing asthma, wherein the composition comprises a mixture of Bifidobacterium longum subsp. infantis and HMO, said HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0380] 5. The composition for the use according to any of the preceding paragraphs, wherein the HMO mixture comprises 2'-fucosyllactose (2FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL) and 3-fucosyllactose (3FL).

[0381] 6. A composition for the purpose according to any one of paragraphs 1 to 4, wherein the HMO mixture comprises 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL) and lactose-N-neotetrasaccharide (LNnT).

[0382] 7. A composition for the purpose according to any one of paragraphs 1 to 4, wherein the HMO mixture comprises 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), 3-fucosylated lactose (3FL), and lactose-N-neotetrasaccharide (LNnT).

[0383] 8. The composition for the use according to any of the preceding paragraphs, wherein the Bifidobacterium longum subsp. infantis is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having at least 99.9% average nucleotide identity (ANI) with Bifidobacterium longum subsp. infantis LMG 11588.

[0384] 9. The composition for the stated use according to any of the preceding paragraphs, wherein the composition further comprises Bifidobacterium animalis subsp. lactis.

[0385] 10. The composition according to paragraph 9 for the stated use, wherein the Bifidobacterium animalis subsp. lactis is Bifidobacterium animalis subsp. lactis CNCM I-3446 or a strain having at least 99.9% average nucleotide identity (ANI) with Bifidobacterium animalis subsp. lactis CNCM I-3446.

[0386] 11. A composition for the stated purpose according to any one of paragraphs 1 to 5 or 8 to 10, wherein the HMO mixture is substantially composed of the following substances:

[0387] i. 31% to 82% by weight, preferably 41% to 70% by weight of 2FL;

[0388] ii. 10% to 27% by weight, preferably 14% to 23% by weight of LNT;

[0389] iii. 4% to 11% by weight, preferably 6% to 10% by weight of DFL; and

[0390] iv. 9% to 34% by weight, preferably 11% to 29% by weight of a combination of 6SL and 3SL.

[0391] 12. The composition according to any one of paragraphs 1 to 4, 6, or 8 to 10, wherein the HMO mixture is substantially composed of the following substances:

[0392] i. 16% to 69% by weight, preferably 22% to 59% by weight of 2FL;

[0393] ii. 9% to 24% by weight, preferably 12% to 21% by weight of LNT;

[0394] iii. 2% to 10% by weight, preferably 3% to 8% by weight of DFL;

[0395] iv. 8% to 26% by weight, preferably 11% to 22% by weight of 6SL and 3SL; and

[0396] v. 18% to 50% by weight, preferably 11% to 43% by weight of 3FL.

[0397] 13. A composition for the stated purpose according to any one of paragraphs 1 to 5 or 8 to 10, wherein the HMO mixture is substantially composed of the following substances:

[0398] i. 34% to 85% by weight, preferably 40% to 71% by weight of 2FL;

[0399] ii. 10% to 40% by weight, preferably 12% to 26% by weight of LNT;

[0400] iii. 4% to 14% by weight, preferably 5% to 10% by weight of DFL;

[0401] iv. 9% to 31% by weight, preferably 10% to 28% by weight of a combination of 6SL and 3SL, and

[0402] v. 6% to 30% by weight, preferably 7% to 22% by weight of LNnT.

[0403] 14. The composition according to any one of paragraphs 1 to 4, 6, or 8 to 10, wherein the HMO mixture is substantially composed of the following substances:

[0404] i. 20% to 60% by weight, preferably 22% to 55% by weight of 2FL;

[0405] ii. 4% to 30% by weight, preferably 6% to 20% by weight of LNT;

[0406] iii. 1% to 12% by weight, preferably 2% to 8% by weight of DFL;

[0407] iv. 7% to 23% by weight, preferably 8% to 22% by weight of a combination of 6SL and 3SL;

[0408] v. 10% to 50% by weight, preferably 13% to 46% by weight of 3FL, and

[0409] vi. 3% to 25% by weight, preferably 5% to 20% by weight of LNnT.

[0410] 15. A composition for the purpose according to any of the preceding paragraphs, wherein the composition is a nutritional composition selected from infant formula, stage 1 infant formula, follow-up formula or stage 2 infant formula, baby food, infant cereal composition, growing milk, fortifiers such as human milk fortifiers, or supplements.

[0411] 16. The composition for the stated purpose according to any of the preceding paragraphs, wherein the individual is an infant, toddler, or child.

[0412] 17. A composition for the purpose according to any one of paragraphs 1 or 5 to 16, wherein the composition is used in the prevention and / or treatment of viral respiratory infections.

[0413] 18. The composition for the purpose according to any one of paragraphs 1 or 5 to 17, wherein the viral infection causes a disease selected from the common cold, influenza (flu), bronchitis, bronchiolitis and pneumonia.

[0414] 19. The composition according to paragraph 18 for the stated purpose, wherein the disease is bronchiolitis or pneumonia, preferably bronchiolitis.

[0415] 20. A composition for the purpose according to any one of paragraphs 1 or 5 to 18, wherein the composition is used to reduce the risk of contracting the viral infection and / or to alleviate symptoms associated with the viral infection.

[0416] 21. The composition according to paragraph 20 for the stated use, wherein the symptoms associated with the viral infection are selected from lung irritation, lung congestion, excessive mucus production, fever, cough, wheezing, shortness of breath, abdominal cramps, diarrhea, and vomiting.

[0417] 22. The composition for the purpose according to any one of paragraphs 1 or 5 to 21, wherein the viral infection is caused by a virus selected from respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus such as influenza A (IVA) and / or influenza B (IVB), rhinovirus (RV), adenovirus (ADV), metapneumovirus (MPV), bocavirus (BoV), coronavirus (CoV), myxovirus, herpesvirus, enterovirus (EV), and paraenterovirus (PeV), or any combination thereof.

[0418] 23. The composition for the purpose according to any one of paragraphs 1 or 5 to 22, wherein the viral infection is respiratory syncytial virus (RSV) or rhinovirus (RV) infection, preferably RSV infection.

[0419] 24. The composition for the stated use according to any one of paragraphs 2 or 5 to 16, wherein the long-term immune benefits to an individual include:

[0420] i. Promote long-term respiratory health;

[0421] ii. To prevent and / or reduce the risk of sensitization to allergens; and / or

[0422] iii. To prevent and / or reduce the risk of developing respiratory illnesses later in life.

[0423] 25. The composition for the purpose according to any one of paragraphs 3, 5 to 16 or 24, wherein the respiratory condition is a chronic inflammatory disease of the respiratory tract or an allergic respiratory disease.

[0424] 26. The composition according to paragraph 25 for the stated purpose, wherein the chronic inflammatory disease of the respiratory tract is asthma or chronic obstructive pulmonary disease (COPD).

[0425] 27. The composition according to paragraph 25 for the stated purpose, wherein the allergic respiratory disease is recurrent wheezing or asthma, preferably allergic asthma.

[0426] 28. A composition for the purpose according to any one of paragraphs 3, 5 to 16 or 24 to 27, wherein the composition is used in i) preventing and / or reducing the risk of an individual’s sensitization to an allergen and / or ii) preventing and / or reducing the risk of the individual developing respiratory illnesses later in life.

[0427] 29. A composition for the purpose according to any one of paragraphs 4 to 16 or 24 to 27, wherein the composition is used to prevent and / or reduce the risk of the individual developing asthma later in life.

[0428] 30. The composition used for the purposes according to paragraph 28 or 29, wherein the subsequent period is 12 months to 12 years after the termination of the treatment.

[0429] 31. A method, said method being used for

[0430] i) Prevention and / or treatment of viral infections in individuals;

[0431] ii) Promotes long-term immune benefits in individuals;

[0432] iii) Prevent and / or reduce the risk of individual sensitization to allergens;

[0433] iv) Prevent and / or reduce an individual's risk of developing respiratory illnesses; and / or

[0434] v) Prevent individual asthma,

[0435] The method includes administering to the individual a composition as defined in any one of paragraphs 1 to 30.

[0436] 32. A composition comprising Bifidobacterium longum subsp. infantis and a mixture of HMOs, the HMO mixture comprising 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), 3-fucosylated lactose (3FL) and lactose-N-neotetrasaccharide (LNnT).

[0437] 33. The composition according to paragraph 32, wherein the Bifidobacterium longum infant subspecies is Bifidobacterium longum infant subspecies LMG 11588 or a strain having at least 99.9% average nucleotide identity (ANI) with Bifidobacterium longum infant subspecies LMG 11588.

[0438] 34. The composition according to paragraph 32 or paragraph 33, wherein the composition further comprises Bifidobacterium animalis subsp. lactis.

[0439] 35. The composition according to paragraph 34 for the stated use, wherein the Bifidobacterium animalis subsp. lactis is Bifidobacterium animalis subsp. lactis CNCM I-3446 or a strain having at least 99.9% average nucleotide identity (ANI) with Bifidobacterium animalis subsp. lactis CNCM I-3446.

[0440] 36. The composition according to any one of paragraphs 32 to 36, wherein the composition is a nutritional composition selected from infant formula, stage 1 infant formula, follow-up formula or stage 2 infant formula, baby food, infant cereal composition, growing milk, fortifiers such as human milk fortifiers, or supplements.

[0441]

[0442]

[0443]

Claims

1. A composition for the prevention and / or treatment of viral infections in an individual, wherein the composition comprises a mixture of *Bifidobacterium longum* subsp. infantis and HMOs, the HMO mixture comprising 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), and lactose-N-neotetrasaccharide (LNnT), and optionally 3-fucosylated lactose (3FL).

2. A composition used in i) preventing and / or reducing the risk of allergen sensitization in an individual's respiratory tract and / or ii) preventing and / or reducing the risk of an individual developing respiratory symptoms, wherein the composition comprises a mixture of *Bifidobacterium longum* subsp. infantis and HMOs, the HMO mixture comprising 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), and lactose-N-neotetrasaccharide (LNnT) and optionally 3-fucosylated lactose (3FL), and wherein the respiratory symptoms are chronic inflammatory diseases of the respiratory tract or allergic respiratory diseases.

3. A composition used in the prevention and / or reduction of an individual's risk of developing asthma, wherein the composition comprises a mixture of Bifidobacterium longum subsp. infantis and HMOs, said HMO mixture being composed of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), and lactose-N-neotetrasaccharide (LNnT) and optionally 3-fucosylated lactose (3FL).

4. The composition for the use according to any one of claims 1 to 3, wherein the HMO mixture comprises 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL) and lactose-N-neotetrasaccharide (LNnT).

5. The composition for the said use according to any one of claims 1 to 4, wherein the HMO mixture is substantially composed of the following substances: i. 34% to 85% by weight, preferably 40% to 71% by weight of 2FL; ii. 10% to 40% by weight, preferably 12% to 26% by weight of LNT; iii. 4% to 14% by weight, preferably 5% to 10% by weight of DFL; iv. 9% to 31% by weight, preferably 10% to 28% by weight of a combination of 6SL and 3SL, and v. 6% to 30% by weight, preferably 7% to 22% by weight of LNnT.

6. The composition for the use according to any one of claims 1 to 3, wherein the HMO mixture comprises 2'-fucosyllactose (2FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL) and lactose-N-neotetrasaccharide (LNnT).

7. The composition according to any one of claims 1 to 3 or 6, wherein the HMO mixture is substantially composed of the following substances: i. 20% to 60% by weight, preferably 22% to 55% by weight of 2FL; ii. 4% to 30% by weight, preferably 6% to 20% by weight of LNT; iii. 1% to 12% by weight, preferably 2% to 8% by weight of DFL; iv. 7% to 23% by weight, preferably 8% to 22% by weight of a combination of 6SL and 3SL; v. 10% to 50% by weight, preferably 13% to 46% by weight of 3FL, and vi. 3% to 25% by weight, preferably 5% to 20% by weight of LNnT.

8. The composition for the use according to any one of the preceding claims, wherein the *Bifidobacterium longum* subsp. *infantitidis* is *Bifidobacterium longum* subsp. *infantitidis* LMG 11588 or a strain having at least 99.9% average nucleotide identity (ANI) with *Bifidobacterium longum* subsp. *infantitidis* LMG 11588.

9. A composition for the stated use according to any one of the preceding claims, wherein the composition is a nutritional composition selected from infant formula, stage 1 infant formula, follow-up formula or stage 2 infant formula, baby food, infant cereal composition, growing milk, fortifiers such as human milk fortifiers, or supplements.

10. A composition for the stated use according to any one of the preceding claims, wherein the individual is an infant, toddler, or child.

11. The composition for the purpose according to any one of claims 1 to 10, wherein the composition is used to reduce the risk of contracting the viral infection and / or to alleviate symptoms associated with the viral infection.

12. The composition for the purpose according to any one of claims 1 to 11, wherein the viral infection is caused by a virus selected from respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus such as influenza A (IVA) and / or influenza B (IVB), rhinovirus (RV), adenovirus (ADV), metapneumovirus (MPV), bocavirus (BoV), coronavirus (CoV), myxovirus, herpesvirus, enterovirus (EV), and paraenterovirus (PeV), or any combination thereof, preferably wherein the viral infection is caused by RSV.

13. The composition for the use according to any one of claims 2 to 10, wherein the chronic inflammatory disease of the respiratory tract is asthma or chronic obstructive pulmonary disease (COPD).

14. The composition for the purpose according to any one of claims 2 or 4 to 10, wherein the allergic respiratory disease is recurrent wheezing or asthma.

15. A method, said method being used for i) Prevention and / or treatment of viral infections in individuals; ii) Prevent and / or reduce the risk of individual sensitization to allergens; iii) To prevent and / or reduce an individual's risk of developing respiratory illnesses; and / or iv) Prevention of individual asthma, The method includes administering to the individual a composition as defined in any one of claims 1 to 14.

16. A composition comprising Bifidobacterium longum subsp. infantis and a mixture of HMOs, the HMO mixture comprising 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL), 3-fucosylated lactose (3FL) and lactose-N-neotetrasaccharide (LNnT).

17. A composition comprising Bifidobacterium longum subsp. infantis and a mixture of HMOs, the HMO mixture comprising 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), 3'-sialylated lactose (3SL) and lactose-N-neotetrasaccharide (LNnT).

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