MIXTURE OF HMOS AND THE TRANSITION MICROORGANISMS BIFIDOBACTERIUM LONGUM

Abstract

The invention relates to a preparation comprising the transitional microorganism Bifidobacterium longum and a mixture of human milk oligosaccharides (HMOs) consisting of 2'-fucosyllactose (2'-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT). The invention also relates to the use of the preparation for the prevention, reduction of risk and / or treatment of infection in subjects. The invention also proposes a preparation for use in promoting long-term immune benefits in subjects, preventing and / or reducing the risk of allergen sensitization, preventing and / or reducing the risk of developing respiratory conditions in subjects, and / or preventing and / or reducing the risk of developing asthma in subjects.

Description

[0001] MIXTURE OF HMOS AND BIFIDOBACTERIUM LONGUM TRANSITIONAL MICROORGANISM

[0002] Field of the Invention

[0003] The present invention is related to a synbiotic, in particular a combination of a Bifidobacterium longum transitional microorganism and a mixture of human milk oligosaccharides consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT). The HMO mixture promotes the growth and / or survival of the Bifidobacterium longum transitional microorganism. The invention also relates to the synbiotic for use in preventing, reducing the risk of and / or treating an infection in a subject. The invention also provides the synbiotic for use in promoting a long-term immune benefit in a subject, preventing and / or reducing the risk of allergen sensitisation, preventing and / or reducing the risk of developing a respiratory condition in a subject, and / or preventing and / or reducing the risk of developing asthma in a subject.

[0004] Background of the Invention

[0005] A viral infection occurs when the virus proliferates inside the host’s cells and hence utilises the host's resources to promote its own multiplication. Viral infections can also interfere with the normal functioning of the host and may lead to more severe infection-related disorders, including long-term alterations in the immune system (such as inflammatory responses) and subsequent allergic or inflammatory diseases later in life.

[0006] Viral respiratory infections, such as respiratory syncytial virus (RSV), affect nearly 90% of children by the age of two (Karpinnen et al, Clin Microbiol Infect, 2016;22;208.e1-e6) and often lead to bronchiolitis, an inflammatory bronchial reaction in infants and young children (Pickles et al, J Pathol, 2015;235;266-276). In particular, severe RSV-induced bronchiolitis is a major cause of morbidity and mortality in infants globally (Nair et al, Lancet, 2010;375;9725;2545- 1555). Respiratory viruses primarily infect the airway epithelium. Higher viral loads have been associated with increased bronchiolitis severity and conversely, rapid viral load reduction in infants was associated with faster disease resolution (Pickles et al, J Pathol, 2015;235;266- 276). It is well documented that infected and necrotic epithelial cells contribute to the airway obstruction and inflammation during RSV infection (Pickles et al, J Pathol, 2015;235;266-276) and as such epithelial cell sloughing is a feature of viral bronchiolitis and associated with disease severity (Johnson et al, Mod Pathol, 2007;20;108-119). Plasmacytoid dendritic cells (pDC) are known to be protective against pathology during RSV infection, and adaptive immune responses including CD4+ and CD8+ T cells are important in viral elimination from the respiratory tract (Openshaw et al, Annu Rev Immunol, 2017;35;501-532).

[0007] 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 lead to immunopathology following respiratory viral infection (Newton et al, Semin Immunopathol, 2016;38;471-482). Moreover, such uncontrolled inflammatory responses can also lead to pathological airway smooth muscle remodelling, a hallmark feature of asthma reported to commence in early life (O’Reilly et al, JACI, 2013; 131 ; 1024-1032) as well as playing a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD; Yan F et al, J Transl Med, 2018;16;262-270). Thus, severe viral airway infections in early life represent an independent risk factor for subsequent development of respiratory diseases such as allergic airway disease (e.g. asthma; Feldman et al, Am J Respir Grit Care Med, 2015;191 ;34-44) and chronic obstructive pulmonary disease (Savran O et al. Int J Chron Obstruct Pulmon Dis. 2018; 13: 683-693) in later life.

[0008] Interactions between the immune system and the microbiome play a crucial role in human health. These interactions start in the prenatal period and are critical for the maturation of the immune system in new-borns and infants. Several factors influence the composition of the infant’s microbiota and subsequently the development of the immune system. They include maternal infection, antibiotic treatment, environmental exposure, mode of delivery, breastfeeding, and food introduction.

[0009] Breastfeeding is a recognized factor that reduces severity of respiratory viral infection in infants either directly through milk bioactives (e.g. human milk oligosaccharides) or indirectly through microbiome mediated immune benefits.

[0010] Human milk oligosaccharides (HMOs) have become the subject of much interest in recent years due to their roles in numerous biological processes occurring in the human organism. 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).

[0011] Infancy, especially the first weeks, 3 months, 6 months or 12 months of life is a critical period for the establishment of a balanced gut microbiota. It is known that the modulation of the gut microbiota during infancy can prospectively have a great influence on future health status. For example the gut flora can have influence on the development of a strong immune system, normal growth and even on the development of obesity later in life. The gut microbiota and its evolution during the development of the infant is, however, a fine balance between the presence and prevalence (amount) of many populations of gut bacteria. Some gut bacteria are classified as "generally positive" while other ones are "generally negative" (or pathogenic) as to their effect on the overall health of the infant.

[0012] It is known that probiotics, in particular from the Lactobacillus and Bifidobacterium genus, support protection against respiratory tract infections. The role of probiotics in viral respiratory tract infections was reviewed by Lehtoranta and co-workers (Lehtoranta et al, Eur J Clin Microbiol Infect Dis, 2014;33; 1289-1302).

[0013] The weaning period has been described as a non-redundant window for immune imprinting (Cahenzli et al., Cell Host Microbe, 2013, 14(5), 559-70; Olszak et al., Science, 2012, 336(6080): 489-93; Nabhani et al., Immunity, 2019, 50(5), 1276-1288). Healthy immune imprinting promotes appropriate immune responses against environmental challenges, including infections.

[0014] Due to the loss of Bifidobacterium species in the infant gut and low breast-feeding rates, there is a need to provide infants with both HMOs and HMO-utilizing bacteria such as a Bifidobacterium longum transitional microorganism and / or B. longum subsp. infantis to support a healthy microbiome for long-term health.

[0015] Additionally, there are limited means to prevent or treat viral infections. There are limited numbers of effective antiviral drugs, for example drugs used to treat HIV and influenza, and the primary method to control viral disease is vaccination which is intended to prevent outbreaks by building immunity to a virus or a family of viruses.

[0016] There remains a need to develop new strategies for preventing and / or reducing the risk of an infection in an infant or young child, and more specifically to prevent and / or reduce the risk of viral infections (such as viral infections of the respiratory tract).

[0017] Summary of the Invention

[0018] The present inventors have determined that Bifidobacterium longum subsp microorganisms (B. longum transitional) of a clade that is present in the gut microbiome of the transitional feeding period of mammals, particularly humans, may have beneficial effects on preventing and / or reducing the risk of developing infection. For example, the inventors have shown that the B. longum transitional microorganisms are capable of modulating levels of protective cytokines (e.g. IL-6) and / or short-chain fatty acids (SCFAs); and modulating gut barrier permeability, for example following an insult or exacerbation to gut barrier permeability.

[0019] The present inventors have also surprisingly found that synbiotic intervention (a Bifidobacterium longum transitional microorganism in combination with a mix of human milk oligosaccharides) in early life provides beneficial effects on preventing and / or reducing the risk of developing infection.

[0020] Furthermore, the present inventors have surprisingly found that synbiotic intervention (B. infantis in combination with a mix of human milk oligosaccharides) in early life provides protection from virus-induced bronchiolitis and promotes a sustained immune benefit into adulthood (as assessed by reduced susceptibility to pollution-enhanced allergic airway inflammation). Specifically, the present inventors have shown that synbiotic interventions resulted in a rapid resolution of virus-induced lung inflammation and appropriate lung tissue remodeling upon clearance of the virus.

[0021] These findings support the use of the synbiotic in providing protection against and treatment of viral infections, in particular viral bronchiolitis, in early life and uncover functional benefits of the synbiotics to mount effective anti-viral immune responses associated with faster disease resolution. Given that viral respiratory tract infections in early life represent a major independent risk factor for subsequent asthma, recurrent wheeze and chronic obstructive pulmonary disease, dietary supplementation with the synbiotics may also prevent long-term complications associated with viral respiratory tract infections in early life.

[0022] Thus, in a first aspect the present invention provides a composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'- fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0023] In a further aspect the invention provides a combination of a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0024] In a further aspect, the invention provides a composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in preventing, reducing the risk of and / or treating an infection in a subject. Suitably, the infection is a viral infection.

[0025] In a further aspect, the invention provides a composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in promoting a long-term immune benefit in a subject.

[0026] In a further aspect, the invention provides a composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0027] In some embodiments, the composition is for use in preventing and / or reducing the risk of allergen sensitisation in a subject.

[0028] In some embodiments, the composition is for use in preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0029] In a further aspect, the invention provides a composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in preventing and / or reducing the risk of developing asthma in a subject.

[0030] The invention further provides a prebiotic for use in preventing and / or reducing the risk of an infection in a subject by promoting the growth and / or survival of a Bifidobacterium longum transitional microorganism in the gut of the infant or young child, wherein the prebiotic is a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0031] The invention also provides a combination of a Bifidobacterium longum transitional microorganism and a HMO mixture for use in preventing, reducing the risk of, and / or treating an infection in a subject; wherein the HMO mixture consists of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT). Suitably, the infection is a viral infection.

[0032] In a further aspect, the invention provides a combination of a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in promoting a long-term immune benefit in a subject.

[0033] In a further aspect, the invention provides combination of a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0034] In some embodiments, the combination is for use in preventing and / or reducing the risk of allergen sensitisation in a subject.

[0035] In some embodiments, the combination is for use in preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0036] In a further aspect, the invention provides a combination of a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in preventing and / or reducing the risk of developing asthma in a subject.

[0037] In some embodiments, the composition or combination further comprises a Bifidobacterium longum subsp. infantis and / or Bifidobacterium lactis microorganism.

[0038] In some embodiments, the Bifidobacterium longum subsp. infantis is Bifidobacterium longum subsp. infantis LMG 11588.

[0039] In some embodiments, the Bifidobacterium longum subsp. infantis is a strain having an Average Nucleotide Identity (AN I) of at least 99.9% to Bifidobacterium longum subsp. infantis LMG 11588.

[0040] In some embodiments, the Bifidobacterium lactis is Bifidobacterium lactis CNCM 1-3446.

[0041] In some embodiments, the Bifidobacterium lactis is a strain having an Average Nucleotide Identity (ANI) of at least 99.9% ANI to Bifidobacterium lactis CNCM 1-3446.

[0042] In some embodiments, the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, and 3SL.

[0043] In some embodiments, the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL, and 3-FL. In some embodiments, the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL, and LNnT.

[0044] In some embodiments, the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL, 3-FL, and LNnT.

[0045] In some embodiments, the HMO mixture consists essentially of: i. 31 wt% to 82 wt% of 2’-FL, preferably 41wt% to 70 wt%; ii. 10 wt% to 27 wt% of LNT, preferably 14 wt% to 23 wt%; iii. 4 wt% to 11 wt% of DFL, preferably 6 wt% to 10 wt%; and iv. 9 wt% to 34 wt% of 6SL and 3SL combined, preferably 11 wt% to 29 wt%.

[0046] In some embodiments, the HMO mixture consists essentially of: i. 16 wt% to 69 wt% of 2’-FL, preferably 22 wt% to 59 wt%; ii. 9 wt% to 24 wt% of LNT, preferably 12 wt% to 21 wt%; iii. 2 wt% to 10 wt% of DFL, preferably 3 wt% to 8 wt%; iv. 8 wt% to 26 wt% of 6SL and 3SL combined, preferably 11 wt% to 22 wt%; and v. 8 wt% to 50 wt% of 3-FL, preferably 11 wt% to 43 wt%

[0047] In some embodiments, the HMO mixture consists essentially of: i. 34 wt% to 85 wt% of 2’-FL, preferably 40 wt% to 71 wt%; ii. 10 wt% to 40 wt% of LNT, preferably 12 wt% to 26 wt%; iii. 4 wt% to 14 wt% of DFL, preferably 5 wt% to 10 wt%; iv. 9 wt% to 31 wt% of 6SL and 3SL combined, preferably 10 wt% to 28 wt% and; v. 6 wt% to 30 wt% of LNnT, preferably 7 wt% to 22 wt%.

[0048] In some embodiments, the HMO mixture consists essentially of: i. 20 wt% to 60 wt% of 2’-FL, preferably 22 wt% to 55 wt%; ii. 4 wt% to 30 wt% of LNT, preferably 6 wt% to 20 wt%; iii. 1 wt% to 12 wt % of DFL, preferably 2 wt% to 8 wt%; iv. 7 wt% to 23 wt% of 6SL and 3SL combined, preferably 8 wt% to 22 wt%; v. 10 wt% to 50 wt% of 3-FL, preferably 13 wt% to 46 wt% and vi. 3 wt% to 25 wt% of LNnT, preferably 5 wt% to 20 wt%. In some embodiments, the Bifidobacterium longum transitional microorganism is: (i) capable of metabolizing the HMOs; (ii) preferentially utilizes 3-FL over 2’-FL; (iii) is capable of metabolizing a glycan substrate selected from the group recited in any of Tables 1 to 3; and / or (iv) encodes one or more CAZymes selected from the group recited in Table 1 , preferably wherein the Bifidobacterium longum transitional microorganism further encodes one or more CAZymes selected from Table 2 and 3.

[0049] In some embodiments, the Bifidobacterium longum transitional microorganism has an Average Nucleotide Identity (ANI) of at least 98% with at least one Bifidobacterium longum strain selected in the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687, CMCC-P0001 (ATCC BAA-2753), and any combination thereof

[0050] In some embodiments, the composition is in the form of a nutritional composition.

[0051] In some embodiments, the nutritional composition is selected from an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement.

[0052] In some embodiments, the subject is an infant, a young child or a child. Preferably, the subject is an infant or a young child.

[0053] In some embodiments, the subject is an infant.

[0054] In some embodiments, the subject is a young child.

[0055] In some embodiments, the subject is a child.

[0056] Brief Description of the Drawings

[0057] Figure 1 - Average Nucleotide Identity (ANI) LIPGMA based phylogenetic tree of strains belonging to the B. longum species. The scale represents the percentage of identity at each branch point.

[0058] Figure 2 - Short chain fatty acids (SCFAs) production (i.e acetate, butyrate and propionate) over 48h of batch fermentation with 3-fucosylactose (3-FL). A. Heatmap shows the z score of the Nuclear magnetic resonance (NMR) peak intensity of 3-FL, TCA cycle, SCFAs intermediates and SCFAs. Three conditions were tested, i.e., fermentation with no supplementation, supplementation with B. longum transitional strain (NCC5004), or supplementation with B. longum subsp. infantis (NCC3089). Each condition was performed in triplicate using one baby fecal inoculum. Samples were collected at the beginning (TO) at 24 h (T24) and at the end of the fermentation (T48). B. Abundance of B. longum transitional strain (NCC5004) and B. longum subsp. infantis (NCC3089) at TO, T24 and T48 of batch fermentation with 3-FL as measured by strain specific qPCR.

[0059] Figure 3 - Short chain fatty acids (SCFAs) production (i.e. acetate, butyrate and propionate) over 48h of batch fermentation with pea fiber rich in arabinan. A. Heatmap shows the z score of the Nuclear magnetic resonance (NMR) peak intensity of TCA cycle, SCFAs intermediates and SCFAs. Three conditions were tested, i.e., fermentation with no supplementation, supplementation with B. longum transitional strain (NCC5002), or supplementation with B. longum subsp. infantis (NCC3089). Each condition was performed in triplicate using one baby fecal inoculum. Samples were collected at the beginning (TO) at 24 h (T24) and at the end of the fermentation (T48). B. Abundance of B. longum transitional strain (NCC5002) and B. longum subsp. infantis (NCC3089) at TO, T24 and T48 of batch fermentation with pea fiber as measured by strain specific qPCR.

[0060] Figure 4 - Interleukin 6 (IL-6) production by monocytes following training with different probiotics and stimulated thereafter with LPS. Bars represent median IL-6 response with dotted line demonstrating the IL-6 level by untrained monocytes

[0061] Figure 5 - Transepithelial electrical resistance (TEER) of Caco-2 monolayers incubated with transitional B. longum NCC5002 (black line), B. lactis NCC2818 (grey line) or vehicle (dotted line) for 24h followed by a challenge with pro-inflammatory cytokines.

[0062] Figure 6 - Measurement of the flux of micromolecules across Caco-2 cell monolayers after incubation with transitional B. longum NCC5002 (black line), B. lactis NCC2818 (grey line) or vehicle (dotted line) followed by a challenge with pro-inflammatory cytokines.

[0063] Figure 7 - Representation of glycoside hydrolases (GH) and polysaccharide lyases (PL) in the genomes of the B. longum clade. Heatmap shows presence (light) and absence (dark) of GH and PL genes, and the size of the circles represent the number of these genes per genome of a particular strain.

[0064] Figure 8 - Growth of B. longum transitional strain NCC5001 was promoted in a complex gut microbiota community by pectin (sugar beet) and arabinogalactan (larch wood). P **** < 0.0001, *** <0.001 , ** <0.01, * <0.05, one-way ANOVA with uncorrected Fisher's LSD.

[0065] Figure 9 - Growth of B. longum transitional strain NCC5002 was promoted in a complex gut microbiota community by arabinogalactan (larch wood) and starch (potato), p **** < 0.0001 , *** <0.001 , ** <0.01 , * <0.05, one-way ANOVA with uncorrected Fisher's LSD. Figure 10 - Representative CAZyme sequences

[0066] Figure 11 - Schematic representation of the organization of the genes implicated in the degradation and the metabolization of fucosylated human milk oligosaccharides in the B. longum transitional strains, compared to B. longum subsp. infantis ATCC 15697 and B. kashiwanohense DSM 21854. Values represent percentage (%) of identity between the different genes.

[0067] Figure 12 - Growth of B. longum transitional strains and B. longum subsp. infantis LMG 11588 on glucose, 2’-FL or 3-FL as sole carbon source (0.5% final). Significant differences between 2’-FL and 3-FL growth for each strain were calculated using one-way ANOVA, followed by a Sidak’s multiple comparison test (ns=non-significant, * p-value <0.05, ** p-value <0.01).

[0068] Figure 13 - Growth ratios of 3-FL over 2’-FL of B. longum transitional strains and B. longum subsp. infantis LMG 11588.

[0069] Figure 14 - Schematic of model of early life viral airway infection and pollution enhanced allergic airway inflammation.

[0070] Figure 15 - Early life nutritional intervention with synbiotic (8. infantis + 6HMOs) reduces virus- induced lung pathology.

[0071] Figures 16 and 17 - Early life nutritional intervention with synbiotic (B. infantis + 6HMOs) promotes sustained immune benefits into adulthood.

[0072] Figure 18 - Early life nutritional intervention with synbiotic (8. infantis + 5HMOs) promotes anti-viral immune responses at peak of infection and reduces virus-induced lung inflammation.

[0073] Figure 19 - Schematics of the experimental set up for a preclinical model for efficacy testing of 8. longum transitional strain in infection model.

[0074] Figure 20 - Kinetics of weight change following airway viral infection with pneumonia virus of mice from 0 to 10 dpi. Each dot represents the mean with error bars indicating standard error of the mean with N=81 experimental group. Statistical difference between different groups was calculated by a 2way ANOVA. *£p value <0.05, "££p value <0.005,$$$’$$$$p value < 0.0001.

[0075] Detailed Description of the invention

[0076] All percentages are by weight unless otherwise stated.

[0077] The terms “about” or “approximatively” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specific value, such as the variation of 1 / -10% or less, 1 / -5% or less, 1 / -1 % or less, and + / 0.1 % or less of and from the specific value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0078] The terms “subject”, “individual” and “patient” are used interchangeably to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include but are not limited to murines, simians, humans, farm animals, sport animals and pets.

[0079] The term “infant” means a human subject under the age of 12 months or an age equivalent non-human animal.

[0080] The terms “young child” or “toddler” as used herein may mean a human subject aged between 12 months and 5 years of age. Suitably, a “young child” may refer to an age equivalent non- human animal.

[0081] The term “child” means a human child aged between three and twelve years. Preferably, the term “child” means a child aged between three and six years.

[0082] A "preterm" or "premature" subject means an infant or young child who was not born at term. Generally it refers to an infant or young child born prior 36 weeks of gestation.

[0083] By the expression "small for gestational age" or "SGA" it is referred to an infant or young child who is smaller in size than normal for their gestational age at birth, most commonly defined as a weight below the 10th percentile for the gestational age. In some embodiments, SGA may be associated with intrauterine growth restriction (IIIGR), which refers to a condition in which a foetus is unable to achieve its potential size.

[0084] By the expression “low birth weight”, it should be understood as any body weight under 2500g at birth.

[0085] The expressions “complementary feeding period”, “complementary period”, “transitional period”, “transitional feeding period” and “weaning period” can be interchangeably used and refer to the period during which the milk, either breast milk or formula, is substituted by other foods in the diet of an infant or a young child. The infant or the young child is typically moved or transitioned gradually from exclusive milk-feeding, either breast feeding or formula feeding, to mixed diet comprising milk and / or solid foods. The transitional period depends on the infant or young child but typically falls between about 4 months and about 18 months of age, such as between about 6 and about 18 months of age, but can in some instances extend up to about 24 months or more. For humans, the weaning period typically starts between 4 and 6 months of age and is considered completed once the infant and / or the young child is no longer fed with breast milk or infant formula, typically at about 24 months of age. In some embodiments, the weaning period is between 4 and 24 months.

[0086] The expressions “composition” or “nutritional composition” refer to any kind of composition or formulation that provides a nutritional benefit to an individual and that may be safely consumed by a human or an animal. A nutritional composition may be in solid (e.g. powder), semi-solid or liquid form and may comprise one or more macronutrients, micronutrients, food additives, water, etc. For instance, the nutritional composition may comprise the following macronutrients: a source of proteins, a source of lipids, a source of carbohydrates and any combination thereof. Furthermore, the nutritional composition may comprise the following micronutrients: vitamins, minerals, fiber, phytochemicals, antioxidants, prebiotics, probiotics, bioactives, metabolites (e.g. butyrate, Docosahexaenoic acid (DHA), Eicosapentaenoic acid (EPA), Gamma-Linolenic acid (GLA)) and any combination thereof. The composition may also contain food additives such as stabilizers (when provided in liquid or solid form) or emulsifiers (when provided in liquid form). The amount of the various ingredients (e.g. the oligosaccharides) can be expressed in g / 100 g of composition on a dry weight basis when it is in a solid form, e.g. a powder, or as a concentration in g / L of the composition when it refers to a liquid form (this latter also encompasses liquid composition that may be obtained from a powder after reconstitution in a liquid such as milk, water, e.g. a reconstituted infant formula or follow-on / follow-up formula or infant cereal product or any other formulation designed for infant or young child nutrition). Generally, a nutritional composition can be formulated to be taken enterally, orally, parenterally, or intravenously, and it usually includes one of more nutrients selected from: a lipid or fat source, a protein source, and a carbohydrate source. Preferably, a nutritional composition is for oral use.

[0087] In a particular embodiment, the nutritional composition is a “synthetic nutritional composition”. The expression “synthetic nutritional composition” means a mixture obtained by chemical and / or biological means.

[0088] The expression "infant formula" as used herein refers to a foodstuff intended for particular nutritional use by infants during the first months of life and satisfying by itself the nutritional requirements of this category of person (Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant formulae and follow-on formulae). It also refers to a nutritional composition intended for infants and as defined in Codex Alimentarius (Codex STAN 72-1981) and Infant Specialities (incl. Food for Special Medical Purpose). The expression "infant formula" encompasses both “starter infant formula” and “follow-up formula” or “follow-on formula”.

[0089] A “follow-up formula” or “follow-on formula” is given from the 6th month onwards. It constitutes the principal liquid element in the progressively diversified diet of this category of person.

[0090] The expression “baby food” means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.

[0091] The expression “infant cereal composition” means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.

[0092] The expression “growing-up milk” (or GUM) refers to a milk-based drink generally with added vitamins and minerals, that is intended for young children or children.

[0093] The terms “fortifier” refers to liquid or solid nutritional compositions suitable for fortifying or mixing with human milk, infant formula, growing-up milk or human breast milk fortified with other nutrients. Accordingly, the fortifier can be administered after dissolution in human breast milk, in infant formula, in growing-up milk or in human breast milk fortified with other nutrients or otherwise it can be administered as a stand-alone composition. When administered as a stand-alone composition, the milk fortifier can be also identified as being a “supplement”.

[0094] The term “metabolize” is used herein to mean that a substrate can by broken down, adsorbed and / or utilized by a microorganism. For example, the substrate may promote and / or contribute to the growth and / or survival of the microorganism.

[0095] Suitably, the term “capable of metabolizing the glycan substrate” may mean that the B. longum transitional strain encodes at least one CAZyme which is capable of utilizing the glycan substrate. For example, the CAZyme may be capable of catalyzing the hydrolysis of a glycosidic bond within the glycan substrate. Suitably, the B. longum transitional strain may encode at least one, at least two, at least three, at least four or at least five CAZymes that are capable of utilizing the glycan substrate. Suitably, the term “capable of metabolizing the glycan substrate” may mean that the glycan substrate (or a fiber or ingredient comprising the glycan substrate) is capable of promoting growth and / or survival of the B. longum transitional strain (e.g. when added to an anaerobic culture of the B. longum transitional strain). Growth and / or survival of the B. longum transitional strain may be determined by measuring the abundance of 16S rDNA - for example using PCR methods. An illustrative assay for measuring growth of a B. longum transitional strain in the presence of glycan substrates (e.g. in the form of fiber) is provided in the present examples. Suitably, the glycan substrate is capable of being metabolized by the B longum transitional microorganism. Suitably, the glycan substrate may be capable of promoting growth and / or survival of the B. longum transitional strain. Glycan substrates capable of promoting growth and / or survival of the B. longum transitional strain may be determined by e.g. anaerobic culture of the B. longum transitional strain with the glycan substrate to be tested. Growth and / or survival of the B. longum transitional strain may be determined by measuring bacteria cell number, cell density (e.g. measured by optical density) and / or the abundance of 16S rDNA - for example using PGR methods. An illustrative assay for measuring growth of a B. longum transitional strain in the presence of glycan substrates is provided in the Examples. A glycan substrate capable of promoting growth and / or survival of the B. longum transitional strain may increase the number of B. longum transitional bacteria in an anaerobic culture by at least 20%, at least 30%, at least 40%, at least 50%, at least 75% or at least 100% compared to the number of B. longum transitional bacteria in a control anaerobic culture which does not comprise the HMO. Suitably, a glycan substrate capable of promoting growth and / or survival of the B. longum transitional strain may increase the number of B. longum transitional bacteria in an anaerobic culture by a statistically significant amount (e.g. p-value <0.05 as determined by one-way ANOVA) compared to the number of B. longum transitional bacteria in a control anaerobic culture which does not comprise the glycan substrate.

[0096] A “glycan substrate” refers to a glycan that can be metabolized by a microorganism. A glycan substrate may be, for example, a glycoconjugate, oligo- or polysaccharide. Glycoconjugate glycans may comprise N-linked glycans or O-linked glycans within glycoproteins and proteoglycans, or glycolipids. For example, an O-linked glycan may comprise a protein or peptide where the oxygen atom of a serine or threonine residue is linked to a monosaccharide, oligo- or polysaccharide as in the case with glycosaminoglycans (GAGs). Further examples of “glycan substrates” are cellulose, which is a glycan composed of (3-1 ,4-linked D-glucose, and chitin, which is a glycan composed of (3-1 ,4-linked N-acetyl-D-glucosamine. Glycans may be homo- or heteropolymers of monosaccharide residues and can be linear or branched. “Glycan substrate” as used herein encompasses, for example, oligosaccharides and polysaccharides.

[0097] The “oligosaccharide” may refer to a carbohydrate that has greater than 2 but relatively few monosaccharide units (typically 3, 4, 5, 6, and up to 10). Exemplary oligosaccharides include, but are not limited to, fructo-oligosaccharides, galacto-oligosaccharides (raffinose, stachyose, verbascose), maltooligosaccharides, gentio-oligosaccharides, cellooligosaccharides, milk oligosaccharides (e.g., those present in secretions from mammary glands), isomaltooligosaccharides, lactosucrose, mannooligosaccharides, melibiose-derived oligosaccharides, pectic oligosaccharides, xylo-oligosaccharides. The term “polysaccharide” may refer to a carbohydrate that has more than ten monosaccharide units. Exemplary polysaccharides include, but are not limited to, starch, arabinogalactan, laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan and galactomannan. It is to be understood that there is not a precise cut-off or distinction between the terms oligosaccharide and polysaccharide, nor is such a distinction necessary to practice the invention.

[0098] The term “glycosaminoglycan” (GAG) or mucopolysaccharide refers to long linear polysaccharides consisting of repeating disaccharide units (i.e. two-sugar units). The repeating two-sugar unit consists of a uronic sugar and an amino sugar, with the exception of keratan, where in the place of the uronic sugar it has galactose. GAGs are classified into four groups based on core disaccharide structures.

[0099] “Mucins”, as used herein, may refer to a family of high molecular weight, heavily glycosylated proteins (glycoconjugates). Mucins' key characteristic is their ability to form gels; therefore they are a key component in most gel-like secretions, serving functions from lubrication to cell signaling to forming mechanical and chemical barriers.

[0100] The term “HMO” or “HMOs” refers to human milk oligosaccharide(s). These carbohydrates are highly resistant to enzymatic hydrolysis, indicating they may display essential functions not directly related to their caloric value. It has been especially illustrated they play a vital role in the early development of infants and young children, such as the maturation of the immune system. Many different kinds of HMOs are found in the human milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N- acetylneuraminic acid), fucose and / or N-acetylglucosamine with many and varied linkages between them, thus accounting for the enormous number of different oligosaccharides in human milk - over 130 such structures have been identified so far. Almost all of them have a lactose moiety at their reducing end while sialic acid and / or fucose (when present) occupy the terminal position at the non-reducing ends. Depending on the presence of fucose and sialic acid in the oligosaccharide structure, the HMOs can be divided as non-fucosylated (neutral) or fucosylated (neutral) and sialylated (acidic) and non-sialylated molecules, respectively.

[0101] The expression “fucosylated oligosaccharide” refers to an oligosaccharide having a fucose residue. It has a neutral nature. Some examples are 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), difucosyllactose (DiFL), lacto-N-fucopentaose (e.g. lacto-N-fucopentaose I, lacto-N- fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto- N-hexaose I, difucosyllacto-N-neohexaose II and any combination thereof. Fucosylated oligosaccharides represents the largest fraction of human milk with 2’-FL constituting up to 30% of the total HMOs. Fucosylated oligosaccharides are thought to reduce the risk of infections and inflammations and to boost growth and metabolic activity of specific commensal microbes reducing inflammatory response.

[0102] The expression “N-acetylated oligosaccharide(s)” encompasses both “N-acetyl-lactosamine” and “oligosaccharide(s) containing N-acetyl-lactosamine”. They are neutral oligosaccharides having an N-acetyl-lactosamine residue. Suitable examples are LNT (lacto-N-tetraose), para- lacto-N-neohexaose (para-LNnH), LNnT (lacto-N-neotetraose), DSLNT (disialyllacto-N- tetraose), and any combinations thereof. Other examples are lacto-N-hexaose, lacto-N- neohexaose, para- lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N- neooctaose, iso- lacto-N-octaose, para- lacto-N-octaose and lacto-N-decaose.

[0103] The expressions “at least one fucosylated oligosaccharide” and “at least one N-acetylated oligosaccharide” should be understood as “at least one type of fucosylated oligosaccharide” and “at least one type of N-acetylated oligosaccharide”.

[0104] The term “sialylated oligosaccharide” refers to an oligosaccharide having a charged sialic acid residue. It has an acidic nature. Some examples are 3’-sialyllactose (3-SL), 6’-sialyllactose (6- SL), sialyllacto-N-tetraose (Lst - e.g. Lst-a, Lst-b or Lst-c).

[0105] Suitably, the term “capable of metabolizing the HMO” may mean that the B. longum transitional strain encodes at least one CAZyme which is capable of utilizing the HMO. For example, the CAZyme may be capable of catalyzing the hydrolysis of a glycosidic bond within the HMO. Suitably, the B. longum transitional strain may encode at least one, at least two, at least three, at least four or at least five CAZymes that are capable of utilizing the HMO. Suitably, the term “capable of metabolizing the HMO” may mean that the HMO is capable of promoting growth and / or survival of the B. longum transitional strain (e.g. when added to an anaerobic culture of the B. longum transitional strain). Growth and / or survival of the B. longum transitional strain may be determined by measuring the abundance of 16S rDNA - for example using PCR methods.

[0106] The term fibers is used herein to refer to carbohydrates that are indigestible by a human or animal. Such fibers are also discussed in relation to carbohydrates herein. Suitably, the fiber can be fermented by one or more B. longum transitional microorganisms provided in the present use or composition and / or within one or more regions in the gastrointestinal tract within an organism, such as a human or non-human animal. As used herein, the expressions “fiber” or “fibers” or “dietary fiber” or “dietary fibers” within the context of the present invention indicate the indigestible portion, in small intestine, of food derived from plants which comprises two main components: soluble fiber, which dissolves in water and insoluble fiber. Mixtures of fibers are comprised within the scope of the terms above mentioned. Soluble fiber is readily fermented in the colon into gases and physiologically active byproducts and can be prebiotic and viscous. Insoluble fiber does not dissolve in water, is metabolically inert and provides bulking, or it can be prebiotic and metabolically ferment in the large intestine. Chemically, dietary fiber consists of carbohydrate polymers with three or more monomeric units which are not hydrolyzed by endogenous enzymes in the small intestine such as arabinoxylans, cellulose, and many other plant components such as resistant starch, resistant dextrins, inulin, lignin, chitins, pectins, arabinans, arabinogalactans, galactans, xylans, beta-glucans, and oligosaccharides. Non-limiting examples of dietary fibers are: prebiotic fibers such as Fructooligosaccharides (FOS), inulin, galacto-oligosaccharides (GOS), fruit fiber, vegetable fiber, cereal fiber, resistant starch such as high amylose corn starch.

[0107] As used herein, “added fiber” or “added dietary fiber” indicates an ingredient mainly or totally constituted by fiber which is added to the complementary nutritional composition and whose content in fiber contributes to the total fiber content of the composition. The total fiber content of the complementary nutritional composition is provided by the sum of amount of fiber naturally present in ingredients used in the recipe (for example from whole grain cereal flour) plus amount of added fiber.

[0108] The term “prebiotic” means non-digestible carbohydrates that beneficially affect the host by selectively stimulating the growth and / or the activity of healthy bacteria such as bifidobacteria in the colon of humans (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995;125:1401-12).

[0109] The term “probiotic” means microbial cell preparation or components of microbial cells with a beneficial effect on the health or well-being of the 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 according to the present invention are generally bacteria.

[0110] The term “cfu” should be understood as colony forming unit.

[0111] The “gut microbiota” is the composition of microorganisms (including bacteria, archaea and fungi) that live in the digestive tract.

[0112] The term “gut microbiome” may encompass both the “gut microbiota” and their “theater of activity”, which may include their structural elements (nucleic acid, proteins, lipids, polysaccharides), metabolites (signaling molecules, toxins, organic and inorganic molecules) and molecules produced by coexisting hosts and structured by the surrounding environmental conditions (Berg, G., et al., 2020. Microbiome, 8(1), pp.1-22).

[0113] The term “SOFA” means short chain fatty acid(s).

[0114] The expression “increasing SCFA production” means that the amount of systemic and / or colonic SCFA, is higher in an individual fed with the nutritional composition according to the present invention in comparison with a standard. The SCFA production may be measured by techniques known by the skilled person such as by Gas-Liquid Chromatography.

[0115] In the present context, the term “gastrointestinal tract” includes the mouth, pharynx, oesophagus, stomach, small intestine, large intestine, rectum and anus. The term “intestine” includes the small intestine, the large intestine and rectum.

[0116] In the present context, the term “respiratory tract” refers to the passage formed by the nose, nasal cavity, pharynx, larynx, trachea, bronchi and the lungs through which air passes during breathing.

[0117] Composition

[0118] The present inventors have surprisingly found that the combination of a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT) in early life provides beneficial effects on preventing and / or reducing the risk of developing infection.

[0119] That the specific combination of a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) is effective for preventing and / or reducing the risk of developing infection is surprising.

[0120] Accordingly, in one aspect, the invention provides a composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyl lactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0121] In a further aspect, the invention provides a nutritional composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'- fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0122] In a further aspect, the invention provides a combination comprising or consisting of a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'- fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT). Preferably, the combination consists of a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyl lactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0123] In one embodiment, the composition further comprises Bifidobacterium longum subsp. infantis and / or Bifidobacterium lactis. Suitably, the composition further comprises Bifidobacterium longum subsp. infantis. Suitably, the composition further comprises Bifidobacterium lactis. Preferably, the composition further comprises Bifidobacterium longum subsp. infantis and Bifidobacterium lactis.

[0124] In one embodiment, the combination comprises or consists of a Bifidobacterium longum transitional microorganism, a HMO mixture, and:

[0125] (i) Bifidobacterium longum subsp. infantis’, and / or

[0126] (ii) Bifidobacterium lactis., wherein the HMO mixture consists of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto- N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3- fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT). Preferably, the combination consists of a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'- fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0127] Suitably, the combination includes Bifidobacterium longum subsp. infantis. Suitably, the combination includes Bifidobacterium lactis. Preferably, the combination includes Bifidobacterium longum subsp. infantis and Bifidobacterium lactis.

[0128] Bifidobacterium longum transitional microorganism

[0129] Bifidobacterium longum subsp microorganisms of a clade that is present in the gut microbiome of the transitional feeding period of mammals, particularly humans, have previously been identified. B. longum microorganisms belonging to this clade are referred to herein as Bifidobacterium longum transitional (B. longum transitional) and are also known in the art as B. longum subsp. iuvenis. B. longum transitional strains NCC 5000, NCC 5001 , NCC 5002, NCC 5003 and NCC 5004 were deposited with the Collection nationale de cultures de microorganisms (CNCM), Institute Pasteur (INSTITUT PASTEUR, 25 RUE DU DOCTEUR ROUX, F-75724 PARIS CEDEX 15, FRANCE) by SOCIETE DES PRODUITS NESTLE S.A according to Budapest Treaty on 11th of May 2021 receiving the deposit numbers CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687, respectively. In US provisional patent application 63 / 216127, it was shown that the B. longum transitional microorganisms are greater in relative abundance during the transitional feeding period (e.g. weaning period) than either 8. longum subsp. infantis (B. infantis) or 8. longum subsp longum. Indeed, the relative abundance of 8. longum subsp. infantis decreases at the beginning of the transitional feeding period until the end of the transitional feeding period while B. longum subsp. longum begins to increase in abundance. Vatanen et al. demonstrated that this distinct Bifidobacterium longum clade expanded with introduction of solid foods and harbored enzymes for utilizing both breast milk and solid food substrates (Vatanen et a!:, 2022, Cell 185, 1-18; published online 1 November 2022; https: / / doi.Org / 10.1016 / j.cell.2022.10.011).

[0130] Suitably, the 8. longum transitional microorganism may encode one or more CAZymes selected from the groups recited in Table 1. Suitably, the 8. longum transitional microorganism may encode one or two CAZymes selected from the group recited in Table 1 .

[0131] Suitably, the 8. longum transitional microorganism encodes at least one CAZyme selected from the group recited in Table 1 and one or more of the CAZymes selected from the groups recited in Table 2 and 3. For example, the B. longum transitional microorganism may encode at least 2, at least 5, at least 10, at least 20 or at least 30 of the CAZymes selected from the groups recited in Table 2 and 3.

[0132] Suitably, B. longum transitional microorganism encodes (i) at least one CAZyme selected from the group recited in Table 1 and (ii) each of the CAZymes recited in Table 3 or each of the CAZymes recited in Table 3 apart from GH5_44.

[0133] Suitably, 8. longum transitional microorganism encodes (i) at least one CAZyme selected from the group recited in Table 1 and (ii) each of the CAZymes recited in Table 3 or each of the CAZymes recited in Table 3 apart from GH25.

[0134] Suitably, the 8. longum transitional microorganism does not encode one or more of the CAZymes recited in Table 4. Suitably, the B. longum transitional microorganism does not encode any of the CAZymes recited in Table 4. The B. longum transitional microorganism of the present invention advantageously harbors genes coding for CAZymes allowing cleavage of sialic residues from glycans such as sialilated oligosachharides, glycoproteins and glycolipids. This allows effective utilization of the sialylated oligosaccharides that are present in the breast milk at weaning and hence can participate to an appropriate development of the gut microbiome of an infant and / or a young child. It may also help in preventing presence of enteropathogens.

[0135] In some embodiments, a B. longum transitional microorganism comprises a sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene having at least 60% identity with BLON_2348 gene present in Bifidobacterium longum subsp. infantis ATCC 15697.

[0136] In some embodiments, a B. longum transitional microorganism comprises sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene having about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% identity with BLON_2348 gene present in B. longum subsp. infantis ATCC 15697.

[0137] In some embodiments, a B. longum transitional microorganism comprises sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with BLON_2348 gene present in B. longum subsp. infantis ATCC 15697.

[0138] In some embodiments, the B. longum transitional microorganism used according to the present invention comprises a glycosyl hydrolase family 95 (GH95, a-L-galactosidase; a-L- fucosidase; a-1 ,2-L-fucosidase) gene having at least 60% of identity with BLON_2335 gene present in B. longum subsp. infantis ATCC 15697 and / or a glycosyl hydrolase family 29 (GH29, a-L-fucosidase; a-1 ,3 / 1 ,4-L-fucosidase;a-1 ,2-L-fucosidase) having at least 60 % identity with BLON_2336 gene present in B. longum subsp. infantis ATCC 15697. In some embodiments, the B. longum transitional microorganism used according to the present invention comprises a sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene having at least 60% identity with BLON_2348 gene present in B. longum subsp. infantis ATCC 15697.

[0139] In some embodiments, the B. longum transitional microorganism preferentially utilizes 3- fucosyllactose (3-FL) over 2’ -fucosyllactose (2’-FL). Suitably, B. longum transitional microorganism may preferentially utilize 3-FL over 2’-FL in a ratio between 0.1 :5, preferably in a ratio between 0.1 :4, more preferably in a ratio between 0.2:2.

[0140] In some embodiments, the B. longum transitional microorganism used in the present invention utilizes 3-FL more efficiently than 2’-FL, as demonstrated by a better growth, for example as shown in the present Examples.

[0141] In some embodiments, a B. longum transitional microorganism has an Average Nucleotide Identity (ANI) of at least 96% with at least one B. longum strain selected in the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687, and CMCC- P0001 (ATCC BAA-2753), and any combination thereof. In some embodiments, a Bifidobacterium longum transitional microorganism has an ANI of about 96%, 96.1 %, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1 %, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.6 %, 98.7 %, 98.8 %, 98.9 %, 99 %, 99.1 %, 99.2 %, 99.3 %, 99.4 %, 99.5 %, 99.6 %, 99.7 %, 99.8 %, 99.9 %, or 100 % with at least one Bifidobacterium longum strain selected in the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687 and CMCC-P0001 (ATCC BAA-2753), and any combination thereof. In some embodiments, a Bifidobacterium longum transitional microorganism has an ANI of at least 96%, of at least 96.1%, of at least 96.2%, of at least 96.3%, of at least 96.4%, of at least 96.5%, of at least 96.6%, of at least 96.7%, of at least 96.8%, of at least 96.9%, of at least 97%, of at least 97.1%, of at least 97.2%, of at least 97.3%, of at least 97.4%, of at least 97.5%, of at least 97.6%, of at least 97.7%, of at least 97.8%, of at least 97.9%, of at least 98%, of at least 98.1%, of at least 98.2%, of at least 98.3%, of at least 98.4%, of at least 98.5%, of at least 98.6%, of at least 98.6 %, of at least 98.7 %, of at least 98.8 %, of at least 98.9 %, of at least 99 %, of at least 99.1 %, of at least 99.2 %, of at least 99.3 %, of at least 99.4 %, of at least 99.5 %, of at least 99.6 %, of at least 99.7 %, of at least 99.8 %, of at least 99.9 % with at least one Bifidobacterium longum strain selected in the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687 and CMCC-P0001 (ATCC BAA-2753), and any combination thereof. In some embodiments, a B. longum transitional microorganism has an Average Nucleotide Identity (ANI) of at least 98% with at least one B. longum strain selected in the group consisting of CNCM 1-5683, CNCM 1-5684, CNCM 1-5685, CNCM 1-5686 and CNCM 1-5687, and any combination thereof. Suitably, the B. longum transitional microorganism encodes one or more CAZymes selected from the group recited in Table 1 and has an ANI of at least 98% with at least one B. longum strain selected in the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687, and any combination thereof. Suitably, the B. longum transitional microorganism encodes one or more GH31 CAZymes and has an ANI of at least 98% with at least one B. longum strain selected in the group consisting of CNCM I- 5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687, and any combination thereof. In some embodiments, a B. longum transitional microorganism has an ANI of about 98% to 100% with at least one B. longum strain selected in the group consisting of CNCM I- 5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687, and any combination thereof. In some embodiments, a B. longum transitional microorganism has an ANI of at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.6 %, 98.7 %, 98.8 %, 98.9 %, 99 %, 99.1 %, 99.2 %, 99.3 %, 99.4 %, 99.5 %, 99.6 %, 99.7 %, 99.8 %, 99.9 %, or 100 % with at least one B. longum strain selected in the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687, and any combination thereof. In some embodiments, a B. longum transitional microorganism has an ANI of at least 98.6%, of at least 98.6 %, of at least 98.7 %, of at least 98.8 %, of at least 98.9 %, of at least 99 %, of at least 99.1 %, of at least 99.2 %, of at least 99.3 %, of at least 99.4 %, of at least 99.5 %, of at least 99.6 %, of at least 99.7 %, of at least 99.8 %, of at least 99.9 % or of at least 100% with at least one B. longum strain selected in the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687, and any combination thereof.

[0142] Methods for sequencing microbial genomes are well known in the art (see e.g. Segerman; Front. Cell. Infect. Microbiol.; 2020; 10; Article 527102 & Donkor; Genes; 2013; 4(4); 556-572). By way of example, metagenomics methods may be used. Suitable metagenomics methods may be performed using shotgun sequencing data, for example. Suitable metogenomics methods are known in the art and include MetaPhlAn 3.0, for example (see Beghini et al.; eLife 2021 ;10: e65088; https: / / huttenhower.sph.harvard.edu / metaphlan).

[0143] The “Average Nucleotide Identity (ANI)” is a term of art that refers to a distance-based approach to delineate species based on pair-wise comparisons of their genome sequences and is an in silico alternative to the traditional DNA-DNA hybridization (DDH) techniques that have been used for phylogenetic definition of a species (Goris et al., 2007, “DNA-DNA hybridization values and their relationship to whole-genome sequence similarities”, Int. J. Syst. Evol. Microbiol. 57: 81-91). Based on DDH, strains with greater than 70% relatedness would be considered to belong to the same species (see e.g., Wayne et al., 1987, Report of the Ad- Hoc-Committee on Reconciliation of Approaches to Bacterial Systematics. Int J Syst Bacteriol 37: 463-464). ANI is similar to the aforementioned 70% DDH cutoff value and can be used for species delineation. ANI has been evaluated in multiple labs and has become the gold standard for species delineation (see e.g., Kim et al., 2014, “Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes”, Int. J. Syst. Evol. Mier. 64: 346-351 ; Richter et al., 2009, “Shifting the genomic gold standard for the prokaryotic species definition”, P Natl Acad Sci USA 106: 19126-19131 ; and Chan et al., 2012, “Defining bacterial species in the genomic era: insights from the genus Acinetobacter”, Bmc. Microbiol. 12)).

[0144] The ANI of the shared genes between two strains is known to be a robust means to compare genetic relatedness among strains, and that ANI values of about 95% correspond to the 70% DNA-DNA hybridization standard for defining a species. See, e.g., Konstantinidis and Tiedje, Proc Natl Acad Sci USA, 102(7) :2567-72 (2005); and Goris et al., Int Syst Evol Microbiol. 57(Pt 1 ):81 -91 (2007). The ANI between two bacterial genomes is calculated from pair-wise comparisons of all sequences shared between any two strains and can be determined, for example, using any of a number of publicly available ANI tools, including but not limited to OrthoANI with usearch (Yoon et al. Antonie van Leeuwenhoek 110:1281-1286 (2017)); ANI Calculator, JSpecies (Richter and Rossello-Mora, Proc Natl Acad Sci USA 106:19126-19131 (2009)); and JSpeciesWS (Richter et al., Bioinformatics 32:929-931 (2016)). Other methods for determining the ANI of two genomes are known in the art. See, e.g., Konstantinidis, K. T. and Tiedje, J. M., Proc. Natl. Acad. Sci. U.S.A., 102: 2567-2572 (2005); and Varghese et al., Nucleic Acids Research, 43(14):6761-6771 (2015). In a particular embodiment, the ANI between two bacterial genomes can be determined, for example, by averaging the nucleotide identity of orthologous genes identified as bidirectional best hits (BBHs). Protein-coding genes of a first genome (Genome A) and second genome (Genome B) are compared at the nucleotide level using a similarity search tool, for example, NSimScan (Novichkov et al., Bioinformatics 32(15): 2380-23811 (2016)). The results are then filtered to retain only the BBHs that display at least 70% sequence identity over at least 70% of the length of the shorter sequence in each BBH pair. The ANI of Genome A to Genome B is defined as the sum of the percent identity times the alignment length for all BBHs, divided by the sum of the lengths of the BBH genes. These and ANI determination techniques are known in the art. Suitably, a B. longum transitional microorganism selected from the group consisting of CNCM 1-5683, CNCM 1-5684, CNCM 1-5685, CNCM 1-5686 and CNCM 1-5687, represents the reference genome to which a microbial genome is compared.

[0145] Suitably, a Bifidobacterium longum microorganism selected from the group consisting of CNCM 1-5683, CNCM 1-5684, CNCM 1-5685, CNCM 1-5686, CNCM 1-5687 and CMCC-P0001 (ATCC BAA-2753), represents the reference genome to which a microbial genome is compared.

[0146] Genome sequences for B. longum transitional strains NCC 5000 (CNCM I-5683), NCC 5001 (CNCM I-5684), NCC 5002 (CNCM I-5685), NCC 5003 (CNCM I-5686) and NCC 5004 (CNCM I-5687) are available via Joint Genome Project (JGI) Study number: Gs0156595 (https: / / aenome.iqi.doe.gov / portal / ). Analysis project numbers and taxon numbers for each genome are as follows:

[0147] In some embodiments, the B. longum transitional microorganism for use in the present invention is isolated from a human.

[0148] In some other embodiments, the B. longum transitional microorganism is not of the subspecies B. longum subsp. longum or B. longum subsp. infantis.

[0149] Suitably, the B. longum transitional microorganism is provided as a probiotic. Suitably, the B. longum transitional microorganism is provided in a composition.

[0150] The composition or combination according to the invention may contain from 103to 1012cfu of the B. longum transitional microorganism, more preferably between 107and 1012cfu such as between 108and 1O10cfu of the B. longum transitional microorganism per g of composition or combination on a dry weight basis. Suitably, the B. longum transitional microorganism is administered to the subject in an amount of at least about 106cfu / day, at least about 107cfu / day, or at least about 108cfu / day. Suitably, the B. longum transitional microorganism is administered to the subject in an amount of about 1012cfu / day or less, about 1011cfu / day or less, or about 1O10cfu / day or less. In one embodiment, the B. longum transitional microorganism is viable.

[0151] HMO mixture

[0152] The composition or combination of the invention comprises a HMO mixture.

[0153] In some embodiments, the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, and 3SL.

[0154] In some embodiments, the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL and 3-FL.

[0155] In some embodiments, the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL and LNnT.

[0156] In some embodiments, the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL, 3-FL and LNnT.

[0157] In one embodiment, the HMO mixture comprises 2’-FL in an amount of from 16 wt% to 85 wt%. Suitably, the HMO mixture may comprise 2’-FL in an amount of from 31 wt% to 82 wt%, preferably from 41wt% to 70 wt%. Suitably, the HMO mixture may comprise 2’-FL in an amount of from 16 wt% to 69 wt%, preferably from 22 wt% to 59 wt%. Suitably, the HMO mixture may comprise 2’-FL in an amount of from 34 wt% to 85 wt%, preferably from 40 wt% to 71 wt%. Suitably, the HMO mixture may comprise 2’-FL in an amount of from 20 wt% to 60 wt%, preferably from 22 wt% to 55 wt%.

[0158] In one embodiment, the HMO mixture comprises LNT in an amount of from 4 wt% to 40 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 10 wt% to 27 wt%, preferably from 14 wt% to 23 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 9 wt% to 24 wt%, preferably 12 wt% to 21 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 10 wt% to 40 wt%, preferably 12 wt% to 26 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 4 wt% to 30 wt%, preferably 6 wt% to 20 wt%.

[0159] In one embodiment, the HMO mixture comprises DFL in an amount of from 1 wt% to 14 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 4 wt% to 11 wt%, preferably from 6 wt% to 10 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 2 wt% to 10 wt%, preferably from 3 wt% to 8 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 4 wt% to 14 wt %, preferably from 5 wt% to 10 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 1 wt% to 12 wt %, preferably from 2 wt% to 8 wt%.

[0160] In one embodiment, the HMO mixture comprises 6SL and 3SL combined in an amount of from 7 wt% to 34 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 9 wt% to 34 wt%, preferably from 11 wt% to 29 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 8 wt% to 26 wt%, preferably from 11 wt% to 22 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 9 wt% to 31 wt%, preferably from 10 wt% to 28 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 7 wt% to 23 wt%, preferably from 8 wt% to 22 wt%.

[0161] In one embodiment, the HMO mixture comprises 3-FL in an amount of from 10 wt% to 50 wt%. Suitably, the HMO mixture may comprise 3-FL in an amount of from 18 wt% to 50 wt%, preferably from 11 wt% to 43 wt%. Suitably, the HMO mixture may comprise 3-FL in an amount of from 10 wt% to 50 wt%, preferably from 13 wt% to 46 wt%.

[0162] In one embodiment, the HMO mixture comprises LNnT in an amount of from 6 wt% to 30 wt%. Suitably, the HMO mixture may comprise LNnT in an amount of from 6 wt% to 30 wt%, preferably from 7 wt% to 22 wt%. Suitably, the HMO mixture may comprise LNnT in an amount of from 3 wt% to 25 wt%, preferably from 5 wt% to 20 wt%.

[0163] In some embodiments, the HMO mixture consists essentially of: i. 31 wt% to 82 wt% of 2’-FL; ii. 10 wt% to 27 wt% of LNT ; iii. 4 wt% to 11 wt% of DFL; and iv. 9 wt% to 34 wt% of 6SL and 3SL combined.

[0164] In some preferred embodiments, the HMO mixture consists essentially of: i. 41 wt% to 70 wt% of 2’-FL; ii. 14 wt% to 23 wt% of LNT ; iii. 6 wt% to 10 wt% of DFL; and iv. 11 wt% to 29 wt% of 6SL and 3SL combined.

[0165] In some embodiments, the HMO mixture consists essentially of: i. 16 wt% to 69 wt% of 2’-FL; ii. 9 wt% to 24 wt% of LNT ; iii. 2 wt% to 10 wt% of DFL; iv. 8 wt% to 26 wt% of 6SL and 3SL combined; and v. 18 wt% to 50 wt% of 3-FL.

[0166] In some preferred embodiments, the HMO mixture consists essentially of: i. 22 wt% to 59 wt% of 2’-FL; ii. 12 wt% to 21 wt% of LNT ; iii. 3 wt% to 8 wt% of DFL; iv. 11 wt% to 22 wt% of 6SL and 3SL combined; and v. 11 wt% to 43 wt% of 3-FL.

[0167] In some embodiments, the HMO mixture consists essentially of: i. 34 wt% to 85 wt% of 2’-FL; ii. 10 wt% to 40 wt% of LNT ; iii. 4 wt% to 14 wt% of DFL; iv. 9 wt% to 31 wt% of 6SL and 3SL combined; and v. 6 wt% to 30 wt% of LNnT.

[0168] In some preferred embodiments, the HMO mixture consists essentially of: i. 40 wt% to 71 wt% of 2’-FL; ii. 12 wt% to 26 wt% of LNT ; iii. 5 wt% to 10 wt% of DFL; and iv. 10 wt% to 28 wt% of 6SL and 3SL combined; and v. 7 wt% to 22 wt% of LNnT.

[0169] In some embodiments, the HMO mixture consists essentially of: i. 20 wt% to 60 wt% of 2’-FL; ii. 4 wt% to 30 wt% of LNT ; iii. 1 wt% to 12 wt % of DFL; iv. 7 wt% to 23 wt% of 6SL and 3SL combined; v. 10 wt% to 50 wt% of 3-FL; and vi. 3 wt% to 25 wt% of LNnT.

[0170] In some preferred embodiments, the HMO mixture consists essentially of: i. 22 wt% to 55 wt% of 2’-FL; ii. 6 wt% to 20 wt% of LNT ; iii. 2 wt% to 8 wt % of DFL; iv. 8 wt% to 22 wt% of 6SL and 3SL combined; v. 13 wt% to 46 wt% of 3-FL and vi. 5 wt% to 20 wt% of LNnT. When the composition or combination is in liquid form, the total HMO concentration is typically in the range from 0.5 to 10 g / L, preferably in the range from 1 to 7.5 g / L. Specific examples of the concentration level of total HMO, when the composition or combination is in liquid form, include 1 to 5 g / L, 1 to 4 g / L, 2 to 5 g / L, 1 to 3 g / L or 2 to 4 g / L.

[0171] When the composition or combination is in solid form, the total HMO concentration is typically in the range from 0.35 to 7 wt% (g total HMO / 100 g dry composition), preferably in the range from 0.35 to 5 wt%. Specific examples of the concentration level of total HMO, when the composition or combination is in dry form, include 0.5 to 3.5 wt% (g total HMO per 100 g dry composition), 0.5 to 2.5 wt%, 1 to 3.5 wt%, 0.5 to 2 wt% or 1 to 2.5 wt%.

[0172] Suitably, the B. longum transitional microorganism is capable of metabolising one or more HMOs of the HMO mixture. Suitably, the B. longum transitional microorganism is capable of metabolising the HMOs contained in the HMO mixture. Suitably, the HMO mixture may be capable of promoting growth and / or survival of the B. longum transitional strain as described herein.

[0173] Bifidobacterium longum subsp. infantis

[0174] Bifidobacterium longum is a bacterium of the Bifidobacterium genus which is present in the human gastrointestinal tract. In 2002, three previously distinct species of Bifidobacterium, B. infantis, B. longum, and B. suis, were unified into a single species named B. longum with the biotypes infantis, longum, and suis, respectively (Sakata, S., et al., 2002. International journal of systematic and evolutionary microbiology, 52(6), pp.1945-1951).

[0175] Any suitable Bifidobacterium longum subsp. infantis strain may be used in the present invention. Such strains will be well-known to the skilled person. 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).

[0176] The Bifidobacterium longum subsp. infantis may be a strain having at least 99% (suitably, at least 99.9%) ANI to Bifidobacterium longum subsp. infantis strain known to the skilled person.

[0177] Suitably, the Bifidobacterium longum subsp. infantis has 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%) ANI to Bifidobacterium longum subsp. infantis LMG 11588 (also known as Bifidobacterium longum subsp. infantis NCC3039 or Bifidobacterium longum subsp. infantis ATCC 17930). Preferably, the Bifidobacterium longum subsp. infantis has at least 99.9% AN I to Bifidobacterium longum subsp. infantis LMG 11588.

[0178] An example of a microorganism genome that has at least 99.9% ANI with B. longum subsp. infantis LMG 11588 can be found in PATRIC (https: / / www.patricbrc.org), genome ID 1678.111. Hence, suitably the B. longum subsp. infantis having the PATRIC genome ID 1678.111 may be used in the present invention.

[0179] Bifidobacterium longum subsp. infantis LMG 11588 is sold by the Belgian Coordinated Collections of Microorganisms (BCCM) under the LMG accession number LMG 11588.

[0180] Bifidobacterium longum subsp. infantis ATCC 15697 is sold by the American Type Culture Collection (ATCC) under the accession number ATCC 15697.

[0181] The composition or combination according to the invention may contain from 103to 1012cfu of Bifidobacterium longum subsp. infantis, more preferably between 107and 1012cfu such as between 108and 1O10cfu of Bifidobacterium longum subsp. infantis per g of composition or combination on a dry weight basis. Suitably, the Bifidobacterium longum subsp. infantis is administered to the subject in an amount of at least about 106cfu / day, at least about 107cfu / day, or at least about 108cfu / day. Suitably, the Bifidobacterium longum subsp. infantis is administered to the subject in an amount of about 1012cfu / day or less, about 1011cfu / day or less, or about 1O10cfu / day or less.

[0182] In one embodiment, the Bifidobacterium longum subsp. infantis is viable.

[0183] Bifidobacterium lactis

[0184] Any suitable Bifidobacterium animalis subsp. lactis (B. lactis) strain may be used in the present invention. Such strains will be well-known to the skilled person. Suitable strains include Bifidobacterium lactis CNCM 1-3446.

[0185] The Bifidobacterium lactis may be a strain having at least 99% (suitably, at least 99.9%) ANI to Bifidobacterium lactis strain known to the skilled person.

[0186] Suitably, the Bifidobacterium lactis has 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%) ANI to Bifidobacterium lactis CNCM 1-3446. Preferably, the Bifidobacterium lactis has at least 99.9% ANI to Bifidobacterium lactis CNCM 1-3446.

[0187] Bifidobacterium lactis CNCM 1-3446 was deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur (INSTITUT PASTEUR, 25 RUE DU DOCTEUR ROUX, F-75724 PARIS CEDEX 15, FRANCE) by NESTEC S.A. (NESTEC S.A., AVENUE NESTLE 55, CH-1800 VEVEY) according to the Budapest Treaty on 7thJune 2005 receiving the deposit number CNCM 1-3446.

[0188] The composition or combination according to the invention may contain from 103to 1012cfu of Bifidobacterium lactis, more preferably between 107and 1012cfu such as between 108and 101° cfu of Bifidobacterium lactis per g of composition or combination on a dry weight basis. Suitably, the Bifidobacterium lactis is administered to the subject in an amount of at least about 106cfu / day, at least about 107cfu / day, or at least about 108cfu / day. Suitably, the Bifidobacterium lactis is administered to the subject in an amount of about 1012cfu / day or less, about 1011cfu / day or less, or about 101° cfu / day or less.

[0189] In one embodiment, the Bifidobacterium lactis is viable.

[0190] Therapeutic use

[0191] Preventing, reducing the risk of and / or treating an infection

[0192] In a further aspect, the invention provides a composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in preventing, reducing the risk of and / or treating an infection in a subject.

[0193] In a further aspect, the invention provides a composition comprising a Bifidobacterium longum transitional microorganism, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in preventing, reducing the risk of and / or treating an infection in a subject.

[0194] In a further aspect, the invention provides a nutritional composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'- fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in preventing, reducing the risk of, and / or treating an infection in a subject.

[0195] In a further aspect, the invention provides a nutritional composition comprising a Bifidobacterium longum transitional microorganism, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis, and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT) for use in preventing, reducing the risk of, and / or treating an infection in a subject.

[0196] In a further aspect, the invention provides the use of a composition according to the invention for the manufacture of a medicament for preventing, reducing the risk of and / or treating an infection in a subject.

[0197] In a further aspect, the invention provides the use of a nutritional composition according to the invention for the manufacture of a medicament for preventing, reducing the risk of and / or treating an infection in a subject.

[0198] In a further aspect, the invention provides a method of preventing, reducing the risk of and / or treating an infection in a subject, wherein the method comprises administering a composition according to the invention to the subject.

[0199] In a further aspect, the invention provides a method of preventing, reducing the risk of and / or treating an infection in a subject, wherein the method comprises administering a nutritional composition according to the invention to the subject.

[0200] Preferably, the composition or nutritional composition is for use in preventing and / or reducing the risk of an infection in a subject.

[0201] The invention also provides a combination of a Bifidobacterium longum transitional microorganism and a HMO mixture for use in preventing, reducing the risk of and / or treating an infection in a subject; wherein the HMO mixture consists of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0202] In a further aspect, the invention provides a combination of a Bifidobacterium longum transitional microorganism, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis, and a HMO mixture for use in preventing, reducing the risk of and / or treating an infection in a subject; wherein the HMO mixture consists of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3- fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0203] In a further aspect, the invention provides the use of a combination according to the invention for the manufacture of a medicament for preventing, reducing the risk of and / or treating an infection in a subject. In a further aspect, the invention provides a method of preventing, reducing the risk of and / or treating an infection in a subject, wherein the method comprises administering a combination according to the invention to the subject.

[0204] Preferably, the composition or nutritional composition is for use in preventing and / or reducing the risk of an infection in a subject.

[0205] The Bifidobacterium longum transitional microorganism may be a Bifidobacterium longum transitional microorganism as described herein.

[0206] The HMO mixture may be a HMO mixture as described herein.

[0207] The Bifidobacterium longum subsp. infantis may be a Bifidobacterium longum subsp. infantis as described herein.

[0208] The Bifidobacterium lactis may be a Bifidobacterium lactis as described herein.

[0209] The combination (e.g. of a Bifidobacterium longum transitional microorganism and a HMO mixture) may be provided in any form as described herein. For example, the combination may be provided in a composition as described herein.

[0210] The B. longum transitional microorganism and HMO mixture may be administered separately, simultaneously or sequentially.

[0211] Suitably, the B. longum transitional microorganism and HMO mixture may be administered in a combined composition.

[0212] The B. longum transitional microorganism, HMO mixture, Bifidobacterium longum subsp. infantis and / or Bifidobacterium lactis may be administered separately, simultaneously or sequentially.

[0213] Suitably, the B. longum transitional microorganism, HMO mixture, Bifidobacterium longum subsp. infantis and Bifidobacterium lactis may be administered in a combined composition.

[0214] Suitably, a combination of a B. longum transitional microorganism (and optionally Bifidobacterium longum subsp. infantis and / or Bifidobacterium lactis) and a HMO mixture may be referred to as a “synbiotic”.

[0215] “Infection”, as used herein, may refer to a disease or disorder caused by an infectious agent or pathogen (including symptoms thereof). “Preventing”, as used herein, may refer to administering the composition and / or combination and / or prebiotic of the invention to a subject who has not yet contracted an infection and / or who is not showing any symptoms of the infection to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease. The subject may have a predisposition for, or be thought to be at risk of developing, the disease.

[0216] “Reducing the risk of an infection”, may refer to administering the composition and / or combination and / or prebiotic of the invention to a subject who has not yet contracted an infection and / or who is not showing any symptoms of the infection to reduce the likelihood of the infant or young child developing a disease caused by infectious agent or pathogen. The administration may prevent or impair the cause of the disease or reduce or prevent development of at least one symptom associated with the disease. The subject may have a predisposition for, or be thought to be at risk of developing, the disease.

[0217] By the expressions “treating” or “treatment”, it is meant a decrease of the duration and / or of the severity of a physical state, a condition or their consequences (e.g. a decrease or elimination of symptoms of the condition). Treatment also encompasses to reduce, alleviate or eliminate one or more symptoms associated with the disease, disorder or condition which is being treated and / or to slow down, reduce or block the progression of the disease, disorder or condition which is being treated.

[0218] The prevention and / or the treatment of a physical state, a condition or their consequences can occur during the treatment (i.e. during the administration of the composition of the present invention, either immediately after the start of the administration or some time after, e.g. some days or weeks after the start). But it can also encompass the prevention and / or the treatment later in life. The term “later in life” encompasses the effect after the termination of the intervention or treatment. The effect “later in life” can be from 1 week to several months, or even years, for example from 2 to 4 weeks, from 2 to 6 weeks, from 2 to 8 weeks, from 1 to 6 months or from 2 to 12 months. Suitably, the effect “later in life” can be from 12 months to 12 years, such as from 2 years to 10 years, or from 4 years to 5 years, after the termination of the intervention or treatment. Suitably, the effect “later in life” lasts until the subject is at least 5 years of age, such as at least 10 years of age, at least 20 years of age or at least 30 years of age.

[0219] The present use to prevent and / or reduce the risk of an infection may be referred to as a prophylactic use to delay or prevent the onset of the symptoms of the infection and / or reduce the number or severity of symptoms of the infection. Suitably, administering the composition and / or combination and / or prebiotic of the invention to a subject may reduce the magnitude and / or amount of symptoms of an infection caused by the infectious agent or pathogen.

[0220] Suitably, the present composition and / or combination and / or prebiotic may be administered to an infant, young child or child.

[0221] Suitably, the present composition and / or combination and / or prebiotic may prevent and / or reduce the risk of an infection in a subject.

[0222] Suitably, the present composition and / or combination and / or prebiotic may be administered to a subject and prevent and / or reduce the risk of an infection in the subject.

[0223] Suitably, the composition and / or combination and / or prebiotic is not for use to reduce or prevent the presence of enteropathogens. Suitably, the composition and / or combination and / or prebiotic is not for use to reduce or prevent the presence of enteropathogens in the gut of a subject.

[0224] Suitably, the composition and / or combination and / or prebiotic may increase the levels of IL-6 in the subject.

[0225] IL-6 is secreted by macrophages in response to pathogen-associated molecular patterns (PAMPs). As such, IL-6 is an important component of fever and of the acute phase response. In addition, IL-6 is responsible for stimulating acute phase protein synthesis, as well as the production of neutrophils in the bone marrow. It supports the growth of B cells and is antagonistic to regulatory T cells. IL-6 has been shown to have an important role in preventing and / or controlling a number of infections including, for example, vaccinia virus and Listeria monocytogenes (Kopf et a / ; 1994; Nature; 368; 339-342); herpes simplex virus (LeBlanc et a / ; 1999; J Virol; 73(10)); influenza virus (Pyle et al , 2017; PLoS Pathogens; 13(9), Dienz et al:, 2012; Mucosal Immunol; 5(3); 258-266, Gou et al; 2019; Front Immunol; 10:3102); enteric bacterial pathogens (Dann et al:, 2008; J Immunol; 180(10); 6816-6826); Escherichia coli (Dalrymple et al:, 1996; Infect Immun; 64(8): 3231-3235); Pulmonary Aspergillosis (Cenci et al:, 2001 ; J Infect Dis; 184(5); 610-617) and Candida albicans (van Enckevort et al:, 1999; Med Mycol; 37(6): 419-426).

[0226] Suitably, the composition and / or combination and / or prebiotic may increase the levels of shortchain fatty acids (SFCA) in the subject.

[0227] Suitably, the SCFA may be selected from acetate (Ethanoate, C1 :0), butyrate (Butanonate, C4:0) and / or propionate (Propanoate, C3:0). SCFAs are produced when dietary fiber is fermented in the colon. SCFAs have diverse physiological roles in body functions; they can affect the production of lipids, energy and vitamins; affect appetite and cardiometabolic health; and have roles in lowering blood pressure in experimental models.

[0228] SCFAs have been shown to have an important role in preventing and / or controlling a number of infections and immune responses (Kim et al.; Cell Host & Microbe; 2016; 20(2); 202-214). For example, SCFAs have been shown to have a protective affect against RSV (Antunes et al.; Nat Comm; 2019; 10; 3273); influenza virus (Trompette; Immunity; 2018; 48(5); 992-1005 and Moriyama and Ichinobe; PNAS; 2018; 16(8); 3118-3125); viral bronchiolitis (Lynch et al.; J Exp Med; 2018; 215(2); 537-557) and general microbe infection (Schulthess etal.’, Immunity; 2019; 50(2); 432-445). Notably, SCFA produced in the gut impacts systemic levels and local SFCA levels in other local organs, for example, the lungs.

[0229] The cytokine and SCFA effects mediated by the present composition, combination and / or prebiotic may be systemic. As such, the cytokine effects (e.g. increase in the levels of IL-6 and / or SCFAs) may systemically prevent or reduce the risk of an infection as described herein. The cytokine effects may occur locally in the gut, the lungs and / or the skin of the subject. Suitably, the SCFA effects may systemically prevent or reduce the risk of an infection as described herein. The SCFA effects may occur locally in the gut, the lungs and / or the skin of the subject. Suitably, the cytokine or SCFA effects may occur in the gut of the subject. Suitably, the cytokine or SCFA effects may occur in the lungs of the subject. Accordingly, the cytokine or SCFA effects may prevent or reduce the risk of an infection in a particular organ or system.

[0230] Suitably, the composition and / or combination and / or prebiotic may modulate the permeability of the gut epithelial barrier of the subject. Suitably, the composition and / or combination and / or prebiotic may decrease the permeability of the gut epithelial barrier. Increased permeability of the gut epithelial barrier may be associated with an increase crossing of e.g. pathogens across the intestinal epithelium. Accordingly, decreased permeability of the gut epithelial barrier may be associated with a decreased crossing of e.g. pathogens across the intestinal epithelium.

[0231] The composition and / or combination and / or prebiotic may reduce and / or prevent an exacerbation of symptoms of an infection. For example, the composition and / or combination and / or prebiotic may reduce and / or prevent an exacerbation of symptoms caused by inflammation. The inflammation may be - for example - a pro-inflammatory response to an existing infection. The existing infection may be the present infection or a separate infection caused by a different infectious agent or pathogen. For example, the present examples show that a B. longum transitional microorganism reduced the level of increased permeability in a model of gut epithelial barrier function following a pro-inflammatory insult. Without wishing to be bound be theory, it is considered that the reduced gut epithelial barrier permeability following an inflammatory insult may reduce the number / levels of pathogens that pass through the gut epithelial barrier during an inflammatory episode and thus prevent and / or reduce the risk of an infection; and / or prevent and / or reduce the risk of an exacerbation of symptoms of an existing infection.

[0232] The combination or prebiotic for use in the present invention may be provided in the form of a composition.

[0233] The composition of the invention may suitably be administered to an individual, for example an infant or a young child, in any suitable form such as a nutritional composition in a dosage unit (for example a tablet, a capsule, a sachet of powder, etc). The composition may be in powder, semi-liquid or liquid form. The composition may be added to a nutritional composition, an infant formula, a food composition, a supplement, a baby food, a follow-up formula, a growing-up milk, an infant cereal or a fortifier. In some embodiments, the composition of the present invention is an infant formula, a baby food, an infant cereal, a growing-up milk, a supplement or fortifier that may be intended for infants, young children or children.

[0234] By way of example, the composition may comprise further components which may be beneficial in preventing and / or reducing the risk of an infection. In addition, or alternatively, the composition may comprise further components may be beneficial during the weaning period.

[0235] For example, the composition may comprise a further probiotic - such as a probiotic with known effects on preventing and / or reducing the risk of an infection - (e.g. B. lactis, L. rhamnosus, B. infantis), formula (e.g. partially hydrolysed formulae, extensively hydrolysed formulae, amino acid-based formulae, or intact formulae), baby food (with or without milk fat), milk fat, cereals, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), butyrate and / or gamma-linolenic acid (GLA).

[0236] Suitably, the further probiotic may be a B.infantis microorganism.

[0237] The infection may be a viral, bacterial or fungal infection.

[0238] Viral infection

[0239] Viral respiratory infections, such as respiratory syncytial virus (RSV), affect nearly 90% of children by the age of two (Karpinnen et al, Clin Microbiol Infect, 2016;22;208.e1-e6). Such viral respiratory infections in infants and young children often lead to bronchiolitis, an inflammatory bronchial reaction in infants and young children (Pickles et al, J Pathol, 2015;235;266-276). Severe RSV-induced bronchiolitis is a major cause of morbidity and mortality in infants globally (Nair et al, Lancet, 2010;375;9725;2545-1555).

[0240] The viral infection may be a viral gastrointestinal infection or a viral respiratory tract infection. The viral gastrointestinal infection may be a viral intestinal infection or a viral stomach infection.

[0241] In a preferred embodiment, the viral infection is a viral respiratory tract infection. The viral respiratory tract infection may be a viral infection in the upper respiratory tract or in the lower respiratory tract.

[0242] The disease associated with the viral infection will typically be 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 diarrhoea. Typically, when the viral infection is in the respiratory tract, the disease associated with the respiratory tract infection is 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 often, the disease associated with the viral respiratory tract infection is common cold, influenza (flu), bronchitis, bronchiolitis or pneumonia.

[0243] Accordingly, in a preferred embodiment of the invention the composition, combination and / or prebiotic of the invention is for use in treating and / or preventing a disease associated with a viral respiratory tract infection selected from the group consisting of common cold, influenza (flu), bronchitis, bronchiolitis and pneumonia. In a more preferred embodiment, the disease associated with the respiratory tract infection is selected from the group consisting of bronchiolitis and pneumonia, in particular 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 tract infection is bronchiolitis, in particular RSV- induced bronchiolitis.

[0244] The symptoms most often associated with the viral infection, and which may be reduced by the composition of the invention, are irritation in the lungs, congestion in the lungs, excessive mucus production, fever, cough, wheezing, breathlessness, abdominal cramps, diarrhoea or vomiting.

[0245] The above-mentioned infections may be caused by a variety of different viruses, including respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus such as influenza virus A (IVA) and / or influenza virus B (IVB), rhinovirus (RV), adenovirus (ADV), metapneumovirus (MPV), bocavirus (BoV), coronavirus (CoV), myxovirus, herpesvirus, enterovirus (EV), parachovirus (PeV) or a combination thereof.

[0246] For example, the infection may be selected from an influenza virus, respiratory syncytial virus, rhinovirus, parainfluenza viruses, metapneumovirus, coronavirus, adenovirus, and bocavirus infection.

[0247] Suitably the infection may be an influenza virus, respiratory syncytial virus or rhinovirus infection. In a typical embodiment of the invention, the viral respiratory tract infection is caused by respiratory syncytial virus (RSV).

[0248] Influenza virus is the infectious agent that causes influenza (flu). Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. These symptoms begin from one to four days after exposure to the virus (typically two days) and last for about 2-8 days. Diarrhea and vomiting can occur, particularly in children. There are four types of influenza virus, termed influenza viruses A, B, C, and D. Aquatic birds are the primary source of Influenza A virus (IAV), which is also widespread in various mammals, including humans and pigs. Influenza B virus (IBV) and Influenza C virus (ICV) primarily infect humans, and Influenza D virus (IDV) is found in cattle and pigs. IAV and IBV circulate in humans and cause seasonal epidemics, and ICV causes a mild infection, primarily in children. IDV can infect humans but is not known to cause illness. In humans, influenza viruses are primarily transmitted through respiratory droplets produced from coughing and sneezing. T ransmission through aerosols and intermediate objects and surfaces contaminated by the virus also occur.

[0249] Respiratory syncytial virus (RSV) a negative-sense, single-stranded RNA virus. It is the single most common cause of respiratory hospitalization in infants, with infection rates typically higher during the cold winter months, causing bronchiolitis. RSV is spread through contaminated air droplets and can cause outbreaks both in the community and in hospital settings. Following initial infection via the eyes or nose, the virus will infect the epithelial cells of the upper and lower airway, causing inflammation, cell damage, and airway obstruction.

[0250] Rhinovirus is the most common viral infectious agent in humans and is the predominant cause of the common cold. The three species of rhinovirus (A, B, and C) include around 160 recognized types of human rhinovirus that differ according to their surface proteins (serotypes). They are lytic in nature and are among the smallest viruses, with diameters of about 30 nanometers. Symptoms of rhinovirus infection may include sore throat, runny nose, nasal congestion, sneezing and cough; sometimes accompanied by muscle aches, fatigue, malaise, headache, muscle weakness, or loss of appetite.

[0251] Using a pneumonia Virus of Mice (PVM) model of human RSV infection, the present inventors have surprisingly found that synbiotic interventions (namely, Bifidobacterium longum transitional microorganism, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis and a mix of HMOs as well as Bifidobacterium longum subsp. infantis in combination with a mix of HMOs) in early life provides protection from virus-induced bronchiolitis. These findings support the use of the synbiotic in providing protection against and treatment of viral infections, in particular viral bronchiolitis, in early life and uncover functional benefits of the synbiotics to mount effective anti-viral immune responses associated with faster disease resolution.

[0252] Thus, the composition, combination and / or prebiotic of the invention is, in particular, effective for use in the treatment and / or prevention of a viral infection in a subject.

[0253] The composition, combination and / or prebiotic of the invention is particularly effective in treating, preventing, reducing the risk of contracting and / or reducing the symptoms of a viral infection caused by RSV. Thus, composition of the invention is particularly preferred for use in treating, preventing, reducing the risk of contracting and / or reducing the symptoms of RSV- induced bronchiolitis or RSV-induced pneumonia.

[0254] The composition, combination and / or prebiotic of the invention is useful for treating and / or preventing viral infections, in particular respiratory tract infection in a human of any age. Thus, the human to be treated with the composition of the invention may be selected from the group consisting of 0 to <1 year (infants), 1 to <3 years (young children) and 3 to <6 years (children), including 3 to <5 years (pre-schoolers).

[0255] Sustained immune benefit

[0256] Viral infections can also interfere with the normal functioning of the host and may lead to more severe infection-related disorders, including immunopathology following respiratory viral infection (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 later in life. For example, uncontrolled inflammatory responses following viral infection of the respiratory tract can lead to pathological airway smooth muscle remodelling, a hallmark feature of asthma reported to commence in early life (O’Reilly et al, JACI, 2013; 131 ; 1024-1032) as well as playing a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD; Yan F et al, J Transl Med, 2018;16;262-270). Thus, severe viral airway infections in early life represent a major independent risk factor for subsequent development of respiratory diseases such as allergic airway disease (e.g. asthma; Feldman et al, Am J Respir Crit Care Med, 2015;191 ;34-44) and chronic obstructive pulmonary disease (Savran O et al. Int J Chron Obstruct Pulmon Dis. 2018; 13: 683-693) in later life.

[0257] Thus, the composition and / or combination of the invention is, in particular, effective for use in promoting a sustained immune benefit in a subject.

[0258] Accordingly, in a further aspect, the invention provides a composition according to the invention for use in promoting a long-term immune benefit in a subject.

[0259] In a further aspect, the invention provides the use of a composition according to the invention for the manufacture of a medicament for promoting a long-term immune benefit in a subject.

[0260] In a further aspect, the invention provides a method of promoting a long-term immune benefit in a subject, the method comprising administering to the subject a composition according to the invention.

[0261] In a further aspect, the invention provides a combination according to the invention for use in promoting a long-term immune benefit in a subject.

[0262] In a further aspect, the invention provides the use of a combination according to the invention for the manufacture of a medicament for promoting a long-term immune benefit in a subject.

[0263] In a further aspect, the invention provides a method of promoting a long-term immune benefit in a subject, the method comprising administering to the subject a combination according to the invention.

[0264] Promoting a long-term immune benefit comprises: i. promoting long-term respiratory health; ii. preventing and / or reducing the risk of allergen sensitisation; and / or iii. preventing and / or reducing the risk of developing a respiratory condition later in life.

[0265] Suitably, promoting a long-term immune benefit refers to promoting long-term respiratory health. Suitably, promoting a long-term immune benefit refers to preventing and / or reducing the risk of allergen sensitisation. Suitably, promoting a long-term immune benefit refers to preventing and / or reducing the risk of developing a respiratory condition later in life.

[0266] As used herein, the phrase “long-term” encompasses the effect after the termination of the intervention or treatment. The effect “long-tern” can be from 1 week to several years, for example from 2 to 4 weeks, from 2 to 6 weeks, from 2 to 8 weeks, from 1 to 6 months, from 2 to 12 months, from 12 months to 12 years, such as from 2 years to 10 years, or from 4 years to 5 years, after the termination of the intervention or treatment. Hence, the effect “long term” may be present when the subject has reached an age of 3 years or more, preferably from 3 to 12 years, more preferably from 3 to 10 years, even more preferably from 3 to 8, most preferably from 3 to 6 years, in particular from 3 to 5 years or from 3 to 4 years. Suitably, the long-term benefit lasts until the subject is at least 5 years of age, such as at least 10 years of age, at least 20 years of age or at least 30 years of age.

[0267] Suitably, the composition and / or combination of the invention may prevent a complication associated with a viral infection of the respiratory tract. Suitably, this effect may be the longterm prevention of a complication associated with a viral infection of the respiratory tract. Such complications may be those associated with the immune systems, such as the inflammatory response, and include immunopathology following respiratory viral infection, such as longterm alterations in the immune system (e.g. long-term alterations in inflammatory responses) and pathological airway smooth muscle remodelling. These complications may in turn predispose the subject to subsequent allergic or inflammatory diseases later in life, such as allergic respiratory diseases or chronic inflammatory diseases of the respiratory tract.

[0268] Allergic sensitization and respiratory conditions

[0269] Given that viral respiratory tract infections in early life represent a major independent risk factor for subsequent asthma, recurrent wheeze and chronic obstructive pulmonary disease later in life (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 composition and / or combination of the invention is also effective use in preventing and / or reducing the risk of developing respiratory conditions, such as chronic inflammatory diseases of the respiratory tract and allergic airway diseases.

[0270] Since viral infections, in particular infection with RSV, is associated with subsequent development of allergic airway diseases, such as asthma later in life (Feldman et al. 2015 Am J Respir Crit Care Med, 191 ;34-44), the composition and / or combination of the invention is also effective for use in preventing and / or reducing the risk of allergen sensitisation and / or developing an allergic respiratory tract disease in a subject.

[0271] Accordingly, in a further aspect the invention provides a composition according to the invention for use in i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0272] In a further aspect, the invention provides a composition according to the invention for use in preventing and / or reducing the risk of developing asthma in a subject. In a further aspect, the invention provides the use of a composition according to the invention for the manufacture of a medicament for i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0273] In a further aspect, the invention provides the use of a composition according to the invention for the manufacture of a medicament for preventing and / or reducing the risk of developing asthma in a subject.

[0274] In a further aspect, the invention provides a method of i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject, the method comprising administering to the subject a composition according to the invention.

[0275] In a further aspect, the invention provides a method of preventing and / or reducing the risk of developing asthma in a subject, the method comprising administering to the subject a composition according to the invention.

[0276] Accordingly, in a further aspect the invention provides a combination according to the invention for use in i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0277] In a further aspect, the invention provides a combination according to the invention for use in preventing and / or reducing the risk of developing asthma in a subject.

[0278] In a further aspect, the invention provides the use of a combination according to the invention for the manufacture of a medicament for i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0279] In a further aspect, the invention provides the use of a combination according to the invention for the manufacture of a medicament for preventing and / or reducing the risk of developing asthma in a subject.

[0280] In a further aspect, the invention provides a method of i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject, the method comprising administering to the subject a combination according to the invention. In a further aspect, the invention provides a method of preventing and / or reducing the risk of developing asthma in a subject, the method comprising administering to the subject a combination according to the invention.

[0281] Respiratory conditions include chronic inflammatory diseases of the respiratory tract and allergic respiratory diseases.

[0282] Chronic inflammatory diseases of the respiratory tract include chronic obstructive pulmonary disease (COPD) and asthma, including allergic asthma and non-allergic asthma.

[0283] COPD is the term for a collection of lung diseases including chronic bronchitis, emphysema and chronic obstructive airways disease. People with COPD have difficulties breathing, primarily due to the narrowing of their airways.

[0284] Asthma is a chronic respiratory condition marked by inflammation and bronchospasm, causing difficulty in breathing. It is usually associated with an allergic reaction or other forms of hypersensitivity. Inflammation and narrowing of the small airways in the lungs cause asthma symptoms, which can be any combination of cough, wheeze, shortness of breath and chest tightness. Asthma often develops during childhood, particularly at the preschool stage (3 years to 5 years old).

[0285] Allergic respiratory tract diseases include recurrent wheeze and asthma, including allergic asthma.

[0286] For i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject, the composition of the invention is preferably administered to a human having an age from 0 to <3 years, preferably from 0 to 2 years, more preferably from 0 to <1 year, such as from 0 to 6 months. This, in turn, prevents and / or reduces the risk of developing a respiratory condition when the subject has reached an age of 3 years or more, preferably from 3 to 12 years, more preferably from 3 to 10 years, even more preferably from 3 to 8, most preferably from 3 to 6 years, in particular from 3 to 5 years or from 3 to 4 years.

[0287] Since viral infections, in particular infection with RSV, is often associated with bacterial coinfection (Thorburn et al, Thorax, 2006;61 (7);611-615) or secondary infection (Sande et al, Nature Communications, 2019;10;2218), including antibiotic use, the composition and / or combination of the invention is also effective for use in preventing or reducing the risk of a bacterial co-infection and / or a bacterial secondary infection associated with respiratory viral infection in a mammal, in particular a human. Pathogenic bacteria typically involved in coinfections or secondary infections include Staphylococcus aureus, Streptococcus pneumoniae and / or Haemophilus influenza. Prebiotic

[0288] The invention further provides a prebiotic for use in preventing, reducing the risk of and / or treating an infection in a subject by promoting the growth of a Bifidobacterium longum transitional microorganism in the gut of the subject, wherein the prebiotic is a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'- sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0289] In a further aspect, the invention provides the use of a prebiotic for the manufacture of a medicament for preventing, reducing the risk of and / or treating an infection in a subject by promoting the growth of a Bifidobacterium longum transitional microorganism in the gut of the infant or young child, wherein the prebiotic is a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'- sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0290] In a further aspect, the invention provides a method of preventing, reducing the risk of and / or treating an infection in a subject by promoting the growth of a Bifidobacterium longum transitional microorganism in the gut of the subject, wherein the method comprises administering a prebiotic to the infant or young child, wherein the prebiotic is a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'- sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0291] The HMO mixture may be as defined herein.

[0292] Preferably, the prebiotic is for use in preventing and / or reducing the risk of an infection in a subject.

[0293] Preferably, the subject is an infant or a young child.

[0294] In some embodiments, the composition and / or combination of the invention may further comprise a prebiotic (i.e. in addition to the HMO mixture as described herein). Suitably, the prebiotic is a glycan substrate.

[0295] Glycan Substrate Carbohydrate-Active Enzymes (CAZymes)

[0296] The B. longum transitional microorganisms encode a profile of Carbohydrate-Active Enzymes (CAZymes). Without wishing to be bound by theory, it is considered that targeting these CAZymes by, for example, providing the HMO mixture as described herein and / or suitable glycan substrates in the form of a prebiotic, may promote the growth and / or survival of the Bifidobacterium longum transitional microorganisms in the gut microbiota of an infant or young child.

[0297] Suitably, promoting the growth and / or survival of the B longum transitional microorganism may refer to increasing the number and / or concentration of the B longum transitional microorganism in the gut microbiota.

[0298] In particular, the CAZymes encoded by each of the Bifidobacterium longum transitional strains NCC 5000, NCC 5001 , NCC 5002, NCC 5003 and NCC 5004, which were deposited with the Institute Pasteur according to Budapest Treaty on 11thof May 2021 receiving the deposit numbers CNCM I-5683, CNCM I-5684, CNCM I.5685, CNCM I-5686 and CNCM I-5687, respectively; have been determined.

[0299] Carbohydrate-active enzymes (CAZymes) are responsible for the synthesis and breakdown of glycoconjugates, oligo- and polysaccharides. They typically correspond to 1-5% of the genes in the living organism. Glycoconjugates, oligo- and polysaccharides play essential roles in many biological functions, for example as structure and energy reserve components and in many intra- and intercellular events. The Carbohydrate Active Enzyme (CAZy) classification is a sequence-based family classification system that correlates with the structure and molecular mechanism of CAZymes (www.cazy.org).

[0300] CAZymes include glycoside hydrolyases (GH), glycosyltransferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE), and carbohydrate-binding module families (CBM)

[0301] Suitably, the CAZyme may be a glycoside hydrolyase (GH). GHs catalyze the hydrolysis of glycosidic bonds between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety. In most cases, the hydrolysis of the glycosidic bond is catalyzed by two amino acid residues of the enzyme: a general acid (proton donor) and a nucleophile / base. Depending on the spatial position of these catalytic residues, hydrolysis occurs via overall retention or overall inversion of the anomeric configuration.

[0302] A GH classification system is provided by the CAZy classification. Herein, GHs are divided into families based on molecular function (e.g., GH1 , GH2, GH3, GH4, etc.). These families are then further divided into subfamilies based on subgroups found within a family that share a more recent ancestor and, typically more uniform in molecular function (e.g., GH13_1 , GH13_2, GH13_3, GH13_4, etc.).

[0303] T able 1 provides details of CAZymes that are unique to the Bifidobacterium longum transitional strains (i.e., not encoded by Bifidobacterium longum suis / suillum, Bifidobacterium longum longum or Bifidobacterium longum subsp. infantis strains). Table 1 also provides a summary of the glycan substrate metabolized by each CAZyme and illustrative dietary fiber sources / ingredients.

[0304] Table 1

[0305] Table 2 provides details of CAZymes that were present in at least one Bifidobacterium longum transitional strain but not present in at least one of the groups selected from the Bifidobacterium longum subsp. suis / suillum, Bifidobacterium longum subsp. longum or

[0306] Bifidobacterium longum subsp. infantis strains presented in Figure 7. Table 2 also provides a summary of the glycan substrate metabolized by each CAZyme and illustrative dietary fiber sources / ingredients.

[0307] Table 2

[0308] Table 3 provides details of CAZymes that were present in all Bifidobacterium longum strains analysed (i.e., Bifidobacterium longum transitional, Bifidobacterium longum subsp. suis / suillum, Bifidobacterium longum subsp. longum and Bifidobacterium longum subsp. infantis). Table 3 also provides a summary of the glycan substrate metabolized by each CAZyme and illustrative dietary fiber sources / ingredients. Table 3

[0309] Table 4 provides details of the CAZymes that are not encoded by Bifidobacterium longum transitional strains but are encoded by one or more of Bifidobacterium longum subsp. suis / suillum, Bifidobacterium longum subsp. longum and Bifidobacterium longum subsp. infantis.

[0310] Table 4

[0311] Representative sequences for the CAZymes listed in Tables 1-4 are shown in Figure 7. Suitably, the CAZyme referred to in any of Tables 1-4 may comprise or consist of the corresponding sequence shown in Figure 7. Suitably, the CAZyme may comprise or consist of a variant of the corresponding sequence shown in Figure 7, which variant retains at least one of the functions of the corresponding CAZyme as recited in T able 1 -4. Suitably, the variant may provide each of the functional activities of the corresponding CAZyme as recited in Table 1-4. Suitably, the variant may comprise or consist of an amino acid sequence which has at least 70% sequence identity to the sequence listed in Figure 7, and retains at least one of the functional activities, preferably each of the functional activities, of the corresponding CAZyme as recited in Table 1-4. Suitably, the variant may comprise or consist of an amino acid sequence, which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the corresponding sequence listed in Figure 7. The variant retains at least one of the functional activities, preferably each of the functional activities, of the corresponding CAZyme as recited in Table 1-4.

[0312] Suitably, the prebiotic for use in the present invention may comprise a glycan substrate selected from the groups recited in any of Tables 1 to 3. Suitably, the prebiotic for use in the present invention may comprise a combination of glycan substrates selected from the groups recited in any of Tables 1 to 3.

[0313] The combination of glycan substrates may comprise at least 2, at least 4, at least 10, at least 20, at least 30, at least 40 or at least 50 of the glycan substrates selected from the groups recited in Tables 1 to 3. The combination may comprise each of the glycan substrates recited in Tables 1 to 3.

[0314] Suitably, the prebiotic may comprise one or more glycan substrates selected from the group recited in Table 1 or Table 2.

[0315] The prebiotic may comprise at least 2, at least 4, at least 10, at least 20, or at least 30 of the glycan substrates recited in Tables 1 and 2. The prebiotic may comprise each of the glycan substrates recited in Tables 1 and 2.

[0316] Suitably, the glycan substrate may comprise or consist of pectin, arabinogalactan and / or starch.

[0317] Suitably, the glycan substrate may comprise or consist of pectin.

[0318] Suitably, the glycan substrate may comprise or consist of arabinogalactan.

[0319] Suitably, the glycan substrate may comprise or consist of starch.

[0320] Suitably, the glycan substrate is provided in the form of a dietary fiber. For example, the dietary fiber may be a prebiotic fiber.

[0321] Suitably, the glycan substrate may be comprised in an ingredient, for example a dietary ingredient.

[0322] The ingredient containing one or several glycan substrates may be selected from the group consisting of purified polysaccharide or purified oligosaccharide, a dietary fiber ingredient, a semi-purified food ingredient, a raw food ingredient, a food additive, a HMO, a semi-purified or purified peptido-glycan.

[0323] The semi-purified food ingredient may be a fruit, vegetable or cereal extract.

[0324] The raw food ingredient may be a fruit, vegetable, cereal, algae or microalgae.

[0325] The food additive may be a guar gum or gum arabic.

[0326] Suitably, the peptide-glycan may be a GAG. Suitably, the glycan substrate may be comprised in a purified fiber.

[0327] Illustrative ingredients and / or purified fibers comprising suitable glycan substrates are provided in Tables 1 to 3. In particular, dietary fibers and / or ingredients that comprise a given glycan substrate are identified in the same row as the glycan substrate.

[0328] The pectin may be comprised in fruit or vegetable pectin. Accordingly, suitable ingredients comprising pectin include, but are not limited to, fruits (e.g., apple, pear), vegetables, legumes (peas), and roots (e.g., sugar beet). Suitable purified fibers comprising arabinogalactan include peach pectin. Suitably, the pectin extracted from sugar beet contains arabinan, galactans and arabinogalactans and may be provided as an ingredient.

[0329] The arabinogalactan may be comprised in fruit or vegetable pectin. Illustrative suitable ingredients comprising arabinogalactan include, but are not limited to, fruits, vegetables, whole grain cereals and sea weed dietary fiber. Suitable purified fibers comprising arabinogalactan include peach pectin, larch wood arabinogalactan, and Arabic gum. Suitably, the arabinogalactan may be provided in larch wood arabinogalactan.

[0330] The starch may be comprised in resistant-starch from cereals (whole grains), legumes, vegetables (e.g., corn) and roots (e.g., potato). Illustrative suitable ingredients comprising starch include, but are not limited to, corn. Suitable purified fibers comprising starch include high amylose starch and resistant dextrin. Suitably, the starch may be provided in a potato, corn or other ingredient. Suitably, the starch may be comprised in a potato ingredient.

[0331] Human milk oligosaccharide (HMO)

[0332] Suitably, the prebiotic comprises one or more additional HMO(s). Suitably, the additional HMO(s) is / are different to those provided in the HMO mixture as described herein.

[0333] Suitably, the additional HMO(s) is / are capable of being metabolized by the B longum transitional microorganism. Suitably, the additional HMO(s) may be capable of promoting growth and / or survival of the B. longum transitional strain. HMOs capable of promoting growth and / or survival of the B. longum transitional strain may be determined by e.g. anaerobic culture of the B. longum transitional strain with the HMO to be tested. Growth and / or survival of the B. longum transitional strain may be determined by measuring bacteria cell number, cell density (e.g. measured by optical density) and / or the abundance of 16S rDNA - for example using PCR methods. An illustrative assay for measuring growth of a B. longum transitional strain in the presence of HMOs is provided in present Example 6. An HMO capable of promoting growth and / or survival of the B. longum transitional strain may increase the number of B. longum transitional bacteria in an anaerobic culture by at least 20%, at least 30%, at least 40%, at least 50%, at least 75% or at least 100% compared to the number of B. longum transitional bacteria in a control anaerobic culture which does not comprise the HMO. Suitably, a HMO capable of promoting growth and / or survival of the B. longum transitional strain may increase the number of B. longum transitional bacteria in an anaerobic culture by a statistically signifiicant amount (e.g. p-value <0.05 as determined by one-way ANOVA) compared to the number of B. longum transitional bacteria in a control anaerobic culture which does not comprise the HMO.

[0334] The HMO may be a fucosylated oligosaccharide (i.e. an oligosaccharide having a fucose residue; e.g. 3-fucosyllactose (3-FL), difucosyllactose (DiFL), lacto-N-fucopentaose (e.g. lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose II and any combination thereof), an N-acetylated oligosaccharide (e.g. para-lacto-N-neohexaose (para-LNnH), LNnT (lacto-N-neotetraose), DSLNT (disialyllacto-N-tetraose), lacto-N-hexaose, lacto-N-neohexaose, para- lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N- neooctaose, iso- lacto-N-octaose, para- lacto-N-octaose and lacto-N-decaose and any combinations thereof) and / or a sialylated oligosaccharide (e.g. Lst (sialyllacto-N-tetraose), Lst-a, Lst-b or Lst-c)).

[0335] Subject

[0336] In one embodiment, the subject is an infant. In one embodiment, the subject is a young child. In one embodiment, the subject is a child.

[0337] The composition or combination according to the invention is for use in infants, young children or children. It is particularly adapted for infants under 6 months of age.

[0338] In general, formula-fed infants have an underdeveloped immune system compared with adults and are more prone to viral infections than breastfed, and the younger the infant is, the less developed the immune system. Accordingly, the composition or combination is particularly useful for preterm infants and / or low or very low birth weight infants, since these infants are even more vulnerable and prone to viral infections. In another particularly interesting embodiment, the composition or combination is used in infants delivered via Caesarean section. Caesarean section born infants are born in a hospital in an environment having more pathogens against which the antibodies, transferred from the mother to the infant, are not effective against. Further, antibiotic administration is a recommended medical practice for C- section birth in order to prevent infection. Such interventions are potent disruptors of microbial communities (the mother’s or the child’s) and antibiotic treatment in early life is associated with an increased risk of developing immune mediated disorders later in life. Caesarean section born infants have a delayed and less optimal colonization of the large intestinal tract and are therefore also more prone to infections.

[0339] The infants, young children or children may be born term or preterm. In a particular embodiment, the composition or combination of the invention is for use in infants, young children or children that were born preterm. Preterm infants may be at increased risk of poor nutrient utilization, impaired lean body mass growth, fat accumulation in the visceral area and metabolic disease later in life.

[0340] In one embodiment, the subject is an infant, a young child or a child that was born small for gestational age or low birth weight.

[0341] Infants, young children or children with low birth weight may or may not be preterm, and similarly, infants, young children or children who are small for gestational age may or may not be preterm.

[0342] The composition or combination of the present invention may also be used in an infant, a young child or a child that was born by C-section or that was vaginally delivered.

[0343] All infants, young children and children can benefit from the invention as all of them are or can be, at a certain age, susceptible to acquiring an unbalanced intestinal / gut microbiota.

[0344] In some advantageous embodiments of the invention, the composition or combination in for use infants, young children or children having a fragile or unbalanced microbiota or dysbiosis of microbiota, such as preterm infants, infants born by Caesarean-section, infants born small for gestational age or with low birth weight, hospitalized infants / young children / children, infants / young children / children treated or having been treated by antibiotics and / or infants / young children / children suffering or having suffered from gut infection and / or gut inflammation.

[0345] It is indeed foreseen that the composition or combination of the invention may be even more beneficial to infants born with possibly impaired gut microbiota or fragile infants / young children / children (such as prematurely born infants and / or infants born by C-section). It is also foreseen that the composition or combination of the invention may be even more beneficial to infants / young children / children exhibiting intestinal disorders (such as diarrhea, infections or colic), especially after birth, for example, during the first 4 weeks after birth.

[0346] In embodiments of the invention, the infants born prematurely or born by caesarean section or born small for gestational age or with low birth weight, or exhibiting unbalanced or abnormal gut microbiota or suffering or having suffered from gut infection and / or gut inflammation, are targeted by the composition or combination of the present invention, and especially when the infants are 0-6 months of age. Without being bound by the theory, it is believed that younger infants benefit even more from the composition or combination of the invention, especially when the infants have (or are at risk of having) an unbalanced intestinal microbiota and / or have a fragile health condition (as exemplified by the conditions cited above).

[0347] The composition (e.g. nutritional composition) or combination can be administered (or given or fed) at an age and for a period that depends on the needs.

[0348] In one embodiment, the infants or young children are 0-36 months of age, such as 0-12 months or 0-6 months of age. It is foreseen that the composition or combination of the invention may be even more beneficial to infants just after birth (0-4 weeks or 0-8 weeks) as their intestinal tract may be more fragile.

[0349] In some embodiments the composition (e.g. nutritional composition) or combination according to the invention can be for use before and / or during the weaning period.

[0350] In some embodiments the composition (e.g. nutritional composition) or combination according to the invention is for use in a subject at risk and / or in need.

[0351] The subject at risk and / or in need may be bottle-fed and / or formula-fed.

[0352] In one embodiment the composition or combination of the invention is given to the subject as a supplementary composition to the mother's milk. In some embodiments the subject receives the mother's milk during at least the first 2 weeks, first 1 , 2, 4, or 6 months. In one embodiment the composition (e.g. nutritional composition) or combination of the invention is given to the subject after such period of mother's nutrition, or is given together with such period of mother's milk nutrition. In another embodiment the composition or combination is given to the subject as the sole or primary nutritional composition during at least one period of time, e.g. after the 1st, 2ndor 4thmonth of life, during at least 1 , 2, 4 or 6 months. In one embodiment the nutritional composition of the invention is a complete nutritional composition (fulfilling all or most of the nutritional needs of the subject). In another embodiment the nutrition composition of the invention is a supplement or a fortifier intended for example to supplement human milk or to supplement an infant formula or a follow- on formula.

[0353] Nutritional composition

[0354] In some embodiments, the composition of the invention is in the form of a nutritional composition. The nutritional composition according to the invention can be for example an infant formula, a starter infant formula, a follow-on or follow-up formula, a growing-up milk, a baby food, an infant cereal composition, a fortifier such as a human milk fortifier, or a supplement. In some particular embodiments, the composition of the invention is an infant formula, a fortifier or a supplement that may be intended for the first 4 or 6 months of age. In a preferred embodiment the nutritional composition of the invention is an infant formula.

[0355] In some other embodiments, the nutritional composition of the present invention is a fortifier. The fortifier can be a breast milk fortifier (e.g. a human milk fortifier) or a formula fortifier such as an infant formula fortifier or a follow-on / follow-up formula fortifier.

[0356] When the nutritional composition is a supplement, it can be provided in the form of unit doses. In such cases it is particularly useful to define the amount of oligosaccharides and probiotics in terms of daily dose to be administered to the infant or young child.

[0357] When the nutritional composition is a supplement, it may comprise the HMO mixture as described herein and the Bifidobacterium longum subsp microorganism, and no other additional nutrient on top of the excipients necessary to obtain a stable nutritional composition.

[0358] The nutritional composition of the present invention can be in solid (e.g. powder), liquid or gelatinous form. In a specific embodiment the nutritional composition is a supplement, wherein the supplement is in powder form and provided in a sachet, preferably a sachet with 0.1 to 20 g per sachet, for example 1 to 10 g per sachet, or in the form of a syrup, preferably a syrup with a total solid concentration of 5 to 75 g / 100 mL (5 to 75% (w / v)). When the supplement is in powder form, it may comprise a carrier. It is however preferred that the supplement is devoid of a carrier. When the supplement is in the form of a syrup, the components are preferably dissolved or suspended in water acidified with citrate.

[0359] In a particular embodiment the nutritional composition according to the invention is a hypoallergenic composition. In another particular embodiment the composition according to the invention is a hypoallergenic nutritional composition.

[0360] Other ingredients

[0361] The composition or combination according to the present invention may also comprise other types of oligosaccharide(s), polysaccharides and / or a fiber(s) and / or a precursor(s) thereof. The other oligosaccharide and / or fiber and / or precursor thereof may be selected from the list comprising human milk oligosaccharides (HMOs), galacto-oligosaccharides (GOS), fructooligosaccharides (FOS), xylooligosaccharides (XOS), cello-oligosaccharides (COS), arabinoxylans, arabinans, xylans, inulin, polydextrose, beta-glucans, pectins and any combination thereof and any derived preparations thereof (e.g. partial hydrolysis). They may be in an amount between 0 and 10% by weight of composition. In a particular embodiment, the nutritional composition can also contain at least one BMO (bovine milk derived oligosaccharide).

[0362] Additional HMOs which may be included in the nutritional composition according to the present invention may be selected from the group consisting of lacto-N- fucopentaose (e.g. lacto-N- fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N- neohexaose, difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose II, para-lacto-N- neohexaose (para-LNnH), lacto-N-hexaose, lacto- N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N-neooctaose, iso-lacto-N-octaose, para- lacto-N-octaose, lacto-N-decaose, and any combination thereof.

[0363] In some embodiments, the composition or combination according to the invention comprises at least one additional HMO.

[0364] In other embodiments, the composition or combination according to the present invention is devoid of any further HMOs. Thus, the HMO mixture as described herein may be the only HMOs in the composition or combination of the invention.

[0365] The composition or combination of the present invention can further comprise at least one further probiotic (or probiotic strain), such as at least one further probiotic bacterial strain.

[0366] The probiotic microorganisms most commonly used are principally bacteria and yeasts of the following genera: Lactobacillus spp., Lacticaseibacillus spp, Limosilactobacillus spp, Streptococcus spp., Enterococcus spp., Bifidobacterium spp. and Saccharomyces spp.

[0367] In some particular embodiments, the probiotic is a probiotic bacterial strain. In some specific embodiments, it is particularly Bifidobacteria and / or Lactobacilli.

[0368] Suitable probiotic bacterial strains include Lactobacillus rhamnosus ATCC 53103 available from Valio Oy of Finland under the trademark LGG, Lactobacillus rhamnosus CGMCC 1 .3724, Lactobacillus paracasei CNCM 1-2116, Lactobacillus johnsonii CNCM 1-1225, Streptococcus salivarius DSM 13084 sold by BLIS Technologies Limited of New Zealand under the designation KI2, B. longum CNCM 1-2618 (B. longum NCC2705), Bifidobacterium breve sold by Danisco under the trademark Bb-03, Bifidobacterium breve sold by Morinaga under the trade mark M-16V, Bifidobacterium infantis sold for example by Procter & Gamble Co. under the trademark Bifantis, and Bifidobacterium breve sold by Institut Rosell (Lallemand) under the trademark R0070. The composition or combination according to the invention may contain from 10e3 to 10e12 cfu of the at least one (further) probiotic strain, more preferably between 10e7 and 10e12 cfu such as between 10e8 and 10e10 cfu of probiotic strain per g of composition or combination on a dry weight basis.

[0369] In one embodiment, the probiotics are viable. In another embodiment, the probiotics are nonreplicating or inactivated. There may be both viable probiotics and inactivated probiotics in some other embodiments. Probiotic components and metabolites can also be added.

[0370] The nutritional composition according to the invention generally contains a protein source. The protein can be in an amount of from 1 .6 to 3 g per 100 kcal. In some embodiments, especially when the composition is intended for premature infants, the protein amount can be between 2.4 and 4 g / 100kcal or more than 3.6 g / 100kcal. In some other embodiments the protein amount can be below 2.0 g per 100 kcal, e.g. between 1.8 to 2 g / 100 kcal, or in an amount below 1.8 g per 100 kcal.

[0371] Protein sources based on whey, casein and mixtures thereof may be used as well as protein sources based on soy. As far as whey proteins are concerned, the protein source may be based on acid whey or sweet whey or mixtures thereof and may include alpha-lactalbumin and beta-lactoglobulin in any desired proportions.

[0372] In some advantageous embodiments the protein source is whey predominant (i.e. more than 50% of proteins are coming from whey proteins, such as 60% or 70%).

[0373] The proteins may be intact or hydrolysed or a mixture of intact and hydrolysed proteins. By the term “intact” is meant that the main part of the proteins are intact, i.e. the molecular structure is not altered, for example at least 80% of the proteins are not altered, such as at least 85% of the proteins are not altered, preferably at least 90% of the proteins are not altered, even more preferably at least 95% of the proteins are not altered, such as at least 98% of the proteins are not altered. In a particular embodiment, 100% of the proteins are not altered.

[0374] The term “hydrolysed” means in the context of the present invention a protein which has been hydrolysed or broken down into its component amino acids. The proteins may be either fully or partially hydrolysed. It may be desirable to supply partially hydrolysed proteins (degree of hydrolysis between 2 and 20%), for example for infants or young children believed to be at risk of developing cow’s milk allergy. If hydrolysed proteins are required, the hydrolysis process may be carried out as desired and as is known in the art. For example, whey protein hydrolysates may be prepared by enzymatically hydrolysing the whey fraction in one or more steps. If the whey fraction used as the starting material is substantially lactose free, it is found that the protein suffers much less lysine blockage during the hydrolysis process. This enables the extent 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 greatly improves the nutritional quality of the protein source.

[0375] In an embodiment of the invention at least 70% of the proteins are hydrolysed, preferably at least 80% of the proteins are hydrolysed, such as at least 85% of the proteins are hydrolysed, even more preferably at least 90% of the proteins are hydrolysed, such as at least 95% of the proteins are hydrolysed, particularly at least 98% of the proteins are hydrolysed. In a particular embodiment, 100% of the proteins are hydrolysed.

[0376] In one particular embodiment the proteins of the nutritional composition are hydrolyzed, fully hydrolyzed or partially hydrolyzed. The degree of hydrolysis (DH) of the protein can be between 8 and 40, or between 20 and 60 or between 20 and 80 or more than 10, 20, 40, 60, 80 or 90.

[0377] The protein component can alternatively be replaced by a mixture or synthetic amino acid, for example for preterm or low birth weight infants.

[0378] In a particular embodiment, the nutritional composition or the growing-up milk according to the invention is a hypoallergenic composition. In another particular embodiment, the composition according to the invention is a hypoallergenic nutritional composition or growing-up milk.

[0379] The nutritional composition according to the present invention generally contains a carbohydrate source. This is particularly preferable in the case where the nutritional composition of the invention is an infant formula. In this case, any carbohydrate source conventionally found in infant formulae such as lactose, sucrose, saccharose, maltodextrin, starch and mixtures thereof may be used although one of the preferred sources of carbohydrates is lactose.

[0380] The nutritional composition according to the present invention generally contains a source of lipids. This is particularly relevant if the nutritional composition of the invention is an infant formula. In this case, the lipid source may be any lipid or fat which is suitable for use in infant formulae. Some suitable fat sources include palm oil, structured triglyceride oil, high oleic sunflower oil and high oleic safflower oil, medium-chain-triglyceride oil. The essential fatty acids linoleic and a-linolenic acid may also be added, as well small amounts of oils containing high quantities of preformed arachidonic acid and docosahexaenoic acid such as fish oils or microbial oils. The fat source may have a ratio of n-6 to n-3 fatty acids of about 5:1 to about 15: 1 ; for example about 8: 1 to about 10: 1. The nutritional composition of the invention may also contain all vitamins and minerals understood to be essential in the daily diet and in nutritionally significant amounts. Minimum requirements have been established for certain vitamins and minerals. Examples of minerals, vitamins and other nutrients optionally present in the composition of the 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, phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form. The presence and amounts of specific minerals and other vitamins will vary depending on the intended population.

[0381] If necessary, the nutritional composition of the invention may contain emulsifiers and stabilisers such as soy, lecithin, citric acid esters of mono- and di-glycerides, and the like.

[0382] The nutritional composition of the invention may also contain other substances which may have a beneficial effect such as lactoferrin, nucleotides, nucleosides, and the like.

[0383] The nutritional composition of the invention may also contain carotenoid(s). In some particular embodiments of the invention, the nutritional composition of the invention does not comprise any carotenoid.

[0384] Manufacture of a nutritional composition

[0385] The nutritional composition according to the invention may be prepared in any suitable manner. A composition will now be described by way of example.

[0386] For example, a formula such as an infant formula may be prepared by blending together the protein source, the carbohydrate source and the fat source in appropriate proportions. If used, the emulsifiers may be included at this point. The vitamins and minerals may be added at this point but they are usually added later to avoid thermal degradation. Any lipophilic vitamins, emulsifiers and the like may be dissolved into the fat source prior to blending. Water, preferably water which has been subjected to reverse osmosis, may then be mixed in to form a liquid mixture. The temperature of the water is conveniently in the range between about 50°C and about 80°C to aid dispersal of the ingredients. Commercially available liquefiers may be used to form the liquid mixture.

[0387] The oligosaccharide(s) may be added at this stage, especially if the final product is to have a liquid form. If the final product is to be a powder, they may likewise be added at this stage if desired.

[0388] The liquid mixture is then homogenised, for example in two stages. The liquid mixture may then be thermally treated to reduce bacterial loads, by rapidly heating the liquid mixture to a temperature in the range between about 80°C and about 150°C for a duration between about 5 seconds and about 5 minutes, for example. This may be carried out by means of steam injection, an autoclave or a heat exchanger, for example a plate heat exchanger.

[0389] Then, the liquid mixture may be cooled to between about 60°C and about 85°C for example by flash cooling. The liquid mixture may then be again homogenised, for example in two stages between about 10 MPa and about 30 MPa in the first stage and between about 2 MPa and about 10 MPa in the second stage. The homogenised mixture may then be further cooled to add any heat sensitive components, such as vitamins and minerals. The pH and solids content of the homogenised mixture are conveniently adjusted at this point.

[0390] If the final product is to be a powder, the homogenised mixture is transferred to a suitable drying apparatus such as a spray dryer or freeze dryer and converted to powder. The powder should have a moisture content of less than about 5% by weight. The oligosaccharide(s) may also or alternatively be added at this stage by dry-mixing or by blending them in a syrup form of crystals, along with the probiotic strain(s), and the mixture is spray-dried or freeze-dried.

[0391] If a liquid composition is preferred, the homogenised mixture may be sterilised then aseptically filled into suitable containers or may be first filled into the containers and then retorted.

[0392] In another embodiment, the composition of the invention may be a supplement. The supplement may be in the form of tablets, capsules, pastilles or a liquid for example. The supplement may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellifying agents and gel forming agents. The supplement may also contain conventional pharmaceutical additives and adjuvants, excipients and diluents, including, but not limited to, water, gelatine of any origin, vegetable gums, lignin-sulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like.

[0393] Further, the supplement may contain an organic or inorganic carrier material suitable for oral or parenteral administration as well as vitamins, minerals trace elements and other micronutrients in accordance with the recommendations of Government bodies such as the USRDA. Embodiments

[0394] The present invention provides the embodiments according to the following numbered clauses:

[0395] 1 . A composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0396] 2. The composition according to clause 1 , wherein the wherein the composition further comprises Bifidobacterium longum subsp. infantis and / or Bifidobacterium lactis.

[0397] 3. A composition comprising a Bifidobacterium longum transitional microorganism, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N- neotetraose (LNnT).

[0398] 4. A combination consisting of a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0399] 5. A combination consisting of a Bifidobacterium longum transitional microorganism, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N- neotetraose (LNnT).

[0400] 6. The composition or combination according to any one of clauses 2-5, wherein the Bifidobacterium longum subsp. infantis is Bifidobacterium longum subsp. infantis LMG 11588 or has an Average Nucleotide Identity (ANI) of at least 99.9% to Bifidobacterium longum subsp. infantis LMG 11588.

[0401] 7. The composition or combination according to any one of clauses 2-6, wherein the Bifidobacterium lactis is Bifidobacterium lactis CNCM 1-3446 or has an Average Nucleotide Identity (ANI) of at least 99.9% ANI to Bifidobacterium lactis CNCM 1-3446. 8. The composition or combination according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism is capable of metabolizing one or more of the HMO(s), preferably all of the HMOs.

[0402] 9. The composition or combination according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism preferentially utilizes 3-FL over 2’-FL.

[0403] 10. The composition or combination according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism is capable of metabolizing a glycan substrate selected from the group recited in any of Tables 1 to 3.

[0404] 11. The composition or combination according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism encodes one or more CAZymes selected from the group recited in Table 1 , preferably wherein the Bifidobacterium longum transitional microorganism further encodes one or more CAZymes selected from Table 2 and 3.

[0405] 12. The composition or combination according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism has an Average Nucleotide Identity (AN I) of at least 98% with at least one Bifidobacterium longum strain selected in the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687, CMCC-P0001 (ATCC BAA-2753), and any combination thereof.

[0406] 13. The composition or combination according to any one of clauses 1-11 , wherein the Bifidobacterium longum transitional microorganism has an Average Nucleotide Identity (ANI) of at least 98% with at least one Bifidobacterium longum strain selected in the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687, and any combination thereof.

[0407] 14. The composition or combination according to any one of the preceding clauses, wherein the HMO mixture consists of 2’-FL, DFL, LNT, 6SL and 3SL.

[0408] 15. The composition or combination according to any one clauses 1-13, wherein the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL, and 3-FL.

[0409] 16. The composition or combination according to any one of clauses 1-13, wherein the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL, and LNnT.

[0410] 17. The composition or combination according to any one of clauses 1-13, wherein the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL, 3-FL and LNnT. 18. The composition or combination according to any one of clauses 1-14, wherein the HMO mixture consists essentially of: i. 31 wt% to 82 wt% of 2FL, preferably 41wt% to 70 wt%; ii. 10 wt% to 27 wt% of LNT, preferably 14 wt% to 23 wt%; iii. 4 wt% to 11 wt% of DFL, preferably 6 wt% to 10 wt%; and iv. 9 wt% to 34 wt% of 6SL and 3SL combined, preferably 11 wt% to 29 wt%.

[0411] 19. The composition or combination according to any one of clauses 1-13 or 15, wherein the HMO mixture consists essentially of: i. 16 wt% to 69 wt% of 2’-FL, preferably 22 wt% to 59 wt%; ii. 9 wt% to 24 wt% of LNT, preferably 12 wt% to 21 wt%; iii. 2 wt% to 10 wt% of DFL, preferably 3 wt% to 8 wt%; iv. 8 wt% to 26 wt% of 6SL and 3SL combined, preferably 11 wt% to 22 wt%; and v. 18 wt% to 50 wt% of 3-FL, preferably 11 wt% to 43 wt%.

[0412] 20. The composition or combination according to any one of clauses 1-13 or 16, wherein the HMO mixture consists essentially of: i. 34 wt% to 85 wt% of 2’-FL, preferably 40 wt% to 71 wt%; ii. 10 wt% to 40 wt% of LNT, preferably 12 wt% to 26 wt%; iii. 4 wt% to 14 wt% of DFL, preferably 5 wt% to 10 wt%; iv. 9 wt% to 31 wt% of 6SL and 3SL combined, preferably 10 wt% to 28 wt%; and v. 6 wt% to 30 wt% of LNnT, preferably 7 wt% to 22 wt%.

[0413] 21. The composition or combination according to any one of clauses 1-13 or 17, wherein the HMO mixture consists essentially of: i. 20 wt% to 60 wt% of 2’-FL, preferably 22 wt% to 55 wt%; ii. 4 wt% to 30 wt% of LNT, preferably 6 wt% to 20 wt%; iii. 1 wt% to 12 wt % of DFL, preferably 2 wt% to 8 wt%; iv. 7 wt% to 23 wt% of 6SL and 3SL combined, preferably 8 wt% to 22 wt%; v. 10 wt% to 50 wt% of 3-FL, preferably 13 wt% to 46 wt%; and vi. 3 wt% to 25 wt% of LNnT, preferably 5 wt% to 20 wt%.

[0414] 22. The composition according to any one clauses 1-3 or 6-21 , wherein the composition further comprises a glycan substrate selected from the group recited in any of Tables 1 to 3. 23. The composition according to any one of clauses 1-3 or 6-22, wherein the composition is a nutritional composition selected from an infant formula, a starter infant formula, a follow- on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement.

[0415] 24. A composition as defined in any one of clauses 1-3 or 6-23 for use in preventing, reducing the risk of and / or treating an infection in a subject.

[0416] 25. A combination as defined in any one of clauses 4-23 for use in preventing, reducing the risk of and / or treating an infection in a subject.

[0417] 26. Use of a composition as defined in any one of clauses 1-3 or 6-23 for the manufacture of a medicament for preventing, reducing the risk of and / or treating an infection in a subject.

[0418] 27. Use of a combination as defined in any one of clauses 4-23 for the manufacture of a medicament for preventing, reducing the risk of and / or treating an infection in a subject.

[0419] 28. A method of preventing, reducing the risk of and / or treating an infection in subject, comprising administering a composition as defined in any one of clauses 1-3 or 6-23 to the subject.

[0420] 29. A method of preventing, reducing the risk of and / or treating an infection in subject, comprising administering a combination as defined in any one of clauses 4-23 to the subject.

[0421] 30. A prebiotic for use in preventing, reducing the risk of and / or treating an infection in a subject by promoting the growth and / or survival of a Bifidobacterium longum transitional microorganism in the gut of the subject, wherein the prebiotic is a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N- neotetraose (LNnT).

[0422] 31. Use of a prebiotic for the manufacture of a medicament for preventing, reducing the risk of and / or treating an infection in a subject by promoting the growth and / or survival of a Bifidobacterium longum transitional microorganism in the gut of the subject, wherein the prebiotic is a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3- fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0423] 32. A method of preventing, reducing the risk of and / or treating an infection in a subject by promoting the growth and / or survival of a Bifidobacterium longum transitional microorganism in the gut of the subject, the method comprising administering a prebiotic to the subject, wherein the prebiotic is a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

[0424] 33. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to any one of clauses 24-32, wherein the composition, combination or prebiotic is for preventing and / or reducing the risk of an infection in the subject.

[0425] 34. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to any one of clauses 24-33, wherein the infection is a viral, bacterial or fungal infection.

[0426] 35. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to any one of clauses 24-34, wherein the infection is an airway infection.

[0427] 36. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to any one of clauses 24-35, wherein the infection is a viral airway infection

[0428] 37. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to clause 36, wherein the viral airway infection is selected from influenza virus, respiratory syncytial virus, rhinovirus, parainfluenza viruses, metapneumovirus, coronavirus, adenovirus, and bocavirus.

[0429] 38. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to clause 36 or clause 37, wherein the viral airway infection is influenza virus, respiratory syncytial virus or rhinovirus.

[0430] 39. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to any one of clauses 34-38, wherein the viral infection causes a disease selected from the group consisting of common cold, influenza (flu), bronchitis, bronchiolitis and pneumonia, preferably bronchiolitis or pneumonia, more preferably bronchiolitis.

[0431] 40. A composition as defined in any one of clauses 1-3 or 6-23 for use in promoting a longterm immune benefit in a subject.

[0432] 41. A combination as defined in any one of clauses 4-23 for use in promoting a long-term immune benefit in a subject.

[0433] 42. Use of a composition as defined in any one of clauses 1-3 or 6-23 for the manufacture of a medicament for promoting a long-term immune benefit in a subject. 43. Use of a combination as defined in any one of clauses 4-23 for the manufacture of a medicament for promoting a long-term immune benefit in a subject.

[0434] 44. A method of promoting a long-term immune benefit in a subject comprising administering a composition as defined in any one of clauses 1-3 or 6-23 to the subject.

[0435] 45. A method of promoting a long-term immune benefit in a subject comprising administering a combination as defined in any one of clauses 4-23 to the subject.

[0436] 46. The composition for use, the combination for use, the use, or the method according to any one of clauses 40-45, wherein promoting a long-term immune benefit in a subject comprises: i. promoting long-term respiratory health; ii. preventing and / or reducing the risk of allergen sensitisation; and / or iii. preventing and / or reducing the risk of developing a respiratory condition.

[0437] 47. A composition as defined in any one of clauses 1-3 or 6-23 for use in i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0438] 48. A combination as defined in any one of clauses 4-23 for use in i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0439] 49. Use of a composition as defined in any one of clauses 1-3 or 6-23 for the manufacture of a medicament for i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0440] 50. Use of a combination as defined in any one of clauses 4-23 for the manufacture of a medicament for i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject.

[0441] 51. A method of i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject, the method comprising administering a composition as defined in any one of clauses 1-3 or 6-23 to the subject.

[0442] 52. A method of i) preventing and / or reducing the risk of allergen sensitisation and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject, the method comprising administering a combination as defined in any one of clauses 4-23 to the subject.

[0443] 53. The composition for use, the combination for use, the use, or the method according to any one of clauses 46-53, wherein the respiratory condition is a chronic inflammatory disease of the respiratory tract or an allergic respiratory tract disease.

[0444] 54. The composition for use, the combination for use, the use, or the method according to clause 53, wherein the chronic inflammatory disease of the respiratory tract is asthma or chronic obstructive pulmonary disease (COPD).

[0445] 55. The composition for use, the combination for use, the use, or the method according to clause 53, wherein the allergic respiratory tract disease is recurrent wheeze or asthma, preferably allergic asthma.

[0446] 56. The composition for use, the combination for use, the use, or the method according to any one of clauses 47-55, wherein the composition and / or combination is for i) preventing and / or reducing the risk of allergen sensitisation in the subject later in life and / or ii) preventing and / or reducing the risk of developing a respiratory condition in the subject later in life.

[0447] 57. A composition as defined in any one of clauses 1 -3 or 6-23 for use in preventing and / or reducing the risk of developing asthma in a subject.

[0448] 58. A combination as defined in any one of clauses 4-23 for use in preventing and / or reducing the risk of developing asthma in a subject.

[0449] 59. Use of a composition as defined in any one of clauses 1-3 or 6-23 for the manufacture of a medicament for preventing and / or reducing the risk of developing asthma in a subject.

[0450] 60. Use of a combination as defined in any one of clauses 4-23 for the manufacture of a medicament for preventing and / or reducing the risk of developing asthma in a subject.

[0451] 61. A method of preventing and / or reducing the risk of developing asthma in a subject comprising administering a composition as defined in any one of clauses 1-3 or 6-23 to the subject.

[0452] 62. A method of preventing and / or reducing the risk of developing asthma in a subject comprising administering a combination as defined in any one of clauses 4-23 to the subject.

[0453] 63. The composition for use, the combination for use, the use, or the method according to any one of clauses 57-62, wherein the asthma is allergic asthma. 64. The composition for use, the combination for use, the use, or the method according to any one of clauses 47-63, wherein the composition is for use in preventing and / or reducing the risk of developing asthma in the subject later in life.

[0454] 65. The composition for use, the combination for use, the use, or the method according to clause 56 or clause 64, wherein later in life is from 12 months to 12 years after termination of the treatment.

[0455] 66. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to any one of clauses 24-65, wherein the subject is an infant, a young child or a child.

[0456] 67. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to clause 66, wherein the subject is an infant or a young child.

[0457] 68. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to any one of clauses 24-67, wherein the composition, combination and / or prebiotic increases the levels of IL-6 in the subject.

[0458] 69. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to any one of clauses 24-68, wherein the composition, combination and / or prebiotic increases the levels of short-chain fatty acids (SCFA) in the subject.

[0459] 70. The composition for use, the combination for use, the use, the method, or the prebiotic for use according to clause 69, wherein the SCFA is selected from acetate, butyrate and / or propionate.

[0460] 71 . The composition for use, the combination for use, the use, the method, or the prebiotic for use according to any one of clauses 24-70, wherein the composition, combination and / or prebiotic modulates the permeability of the gut epithelial barrier; preferably wherein the composition, combination and / or prebiotic decreases the permeability of the gut epithelial barrier.

[0461] Those skilled in the art will understand that they can freely combine all features of the present invention disclosed herein. In particular, features described for the product of the present invention may be combined with the method of the present invention and vice versa. Further, features described for different embodiments of the present invention may be combined. Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification. Further advantages and features of the present invention are apparent from the figures and non-limiting examples.

[0462] Examples

[0463] Example 1 : Transitional B. longum increase short-chain fatty acid production

[0464] 3-fucosylactose (3-FL), short-chain fatty acid (SCFA), tricarboxylic acid (TCA) intermediates and SCFA intermediates were measured by 1 H-NMR technique. Results are shown in Figures 2 and 3. The heatmaps highlight the dynamic of consumption and production of key metabolites in SCFAs pathways by showing the Z score of each metabolite abundance at TO, T24 and T48.

[0465] Total SCFAs corresponds to the sum of the peak integrals of acetate, butyrate, and propionate. Significant difference in metabolite Z score between Bifidobacterium longum transitional or Bifidobacterium longum spp infantis and no supplementation is calculated with ANOVA and highlighted with a star symbol (* p-value<0.05, ** p-value<0.01 , *** p- value<0.001). Significant difference in metabolite Z score between Bifidobacterium longum transitional and Bifidobacterium longum spp infantis is calculated with ANOVA and highlighted with a round symbol (°p-value<0.05, ° ° p-value<0.01 , ° ° ° p-value<0.001). The box plots indicate the strain abundance (i.e. the strain specificity gene copy measured by qPCR) of Bifidobacterium longum transitional or Bifidobacterium longum spp infantis over 48h or fermentation. Figure 2 and Figure 3 are proofs of concept that Bifidobacterium longum transitional is well implanted in the microbial community, is metabolically active on 3-FL or Pea fiber and produces more SCFAs than Bifidobacterium longum subsp. Infantis.

[0466] Figure 2 shows SCFAs production (i.e acetate, butyrate and propionate) over 48h of batch fermentation with 3-fucosylactose (3-FL).

[0467] Figure 3 shows SCFAs production (i.e acetate, butyrate and propionate) over 48h of batch fermentation with pea fiber (rich in arabinan).

[0468] Example 2: Transitional B. longum increases the anti-infection cytokine, IL-6

[0469] Monocytes were isolated from the buffy coat of healthy donors. One hundred thousand monocytes were seeded in each well of a 96-well plate and incubated with 1e6 CFU of B. longum transitional for 24 hours for immune training. Cells were washed by centrifugation and allowed to rest for 6 days. Monocytes were stimulated with LPS for 24hrs. IL-6 was thereafter measured in the cell culture supernatants to assess immune training (see Figure 4). Bars indicate the median IL-6 production from 3 donors with dotted line indicating the IL-6 level by untrained monocytes.

[0470] Method

[0471] Immunoprofiling with PBMC cells

[0472] Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats obtained from healthy adults by density gradient. PBMC were then seeded at 1.5x106cells / ml in a 48-well bottom plate in complete Isocove’s modified Dulbecco’s medium (cIMDM) containing 10% fetal bovine serum, 1% glutamine, 1% penicillin / streptomycin and 0.1 % gentamycin. PBMC were stimulated for 36 hours in the presence of different bacterial strains including all transitional B longum isolates at 107CFU / ml and probiotic strains. Cell culture supernatants were collected to assess cytokine expression for IL-10 and IL-12p40 by ELISA. Standard curve for each cytokine was used to calculate absolute amount (picogram / ml) from optical density readouts.

[0473] Example 3: Transitional B. longum increases gut epithelial barrier resistance

[0474] In vitro experiments using a human colorectal adenocarcinoma cell line (Caco-2) have shown that the transitional B. longum strains were able to increase the transepithelial electrical resistance (TEER) when incubated with the epithelial cells.

[0475] Caco-2 cells were seeded on Transwell and grown for 3 weeks. Caco-2 monolayers were preincubated with transitional B. longum NCC5002 (black line) at 4.106CFU / well, B lactis NCC2818 (grey line) at 4.106CFU / well or vehicle (dotted line) in the presence of 10ng / mL IFNy for 24 hours (0-24). After that period, cells were challenged with 50ng / mL TNFa proinflammatory cytokine for another 24 hours (24-48) followed by a recovery phase of 24 hours (48-72). Transepithelial electrical resistance was measured at O, 24, 48 and 72 hours. Data is represented as mean ± SD. For each time point, statistical difference was assessed using two-way ANOVA with Dunnett test for multiple comparison and represented by asterisks or hash marks for NCC5002 and NCC2818, respectively. * / #= p<0.05; ** / ##=P<0.01 compared to control group (Figure 5).

[0476] Caco-2 cells were seeded on Transwell and grown for 3 weeks. Caco-2 monolayers were preincubated with transitional B. longum NCC5002 (black line) at 4.106CFU / well, B lactis NCC2818 (grey line) at 4.106CFU / well or vehicle (dotted line) in the presence of 10ng / mL IFNy for 24 hours (0-24). After that period, cells were challenged with 50ng / mL TNFa proinflammatory cytokine for another 24 hours (24-48) followed by a recovery phase of 24 hours (48-72). At the 72-hour timepoint, permeability of the caco-2 monolayers was assessed by measuring the flux of fluorescein sulfonic acid (478 Daltons) across the epithelium for 180 minutes. Data is represented as mean ± SD. For each time point, statistical difference was assessed using two-way ANOVA with Dunnett test for multiple comparison and represented by asterisks. *= p<0.05 compared to control group (Figure 6).

[0477] Method

[0478] CACO-2 cells culture and transepithelial electrical resistance measurement

[0479] Caco-2 cells (HTB-37; American Type Culture Collection) were seeded in 24-well semi- permeable inserts. Caco-2 monolayers were cultured for 14 days, with three medium changes / week, until a functional cell monolayer with a transepithelial electrical resistance (TEER) was obtained. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing glucose and glutamine and supplemented with HEPES and 20% (v / v) heat- inactivated fetal bovine serum. Before addition of bacteria to the apical compartment, the TEER of the Caco-2 monolayers was measured (= Oh time point). The TEER of an empty insert was subtracted from all readings to account for the residual electrical resistance of an insert. Then, probiotic strains (directly taken from a glycerol stock) were diluted in Caco-2 complete medium and apically added to the Caco-2-bearing inserts at 2x10E6 colony-forming unit. Cells were also exposed to Caco-2 complete medium (CM) in both chambers as control and to 0.75% glycerol in the apical compartment as vehicle control. Cells were treated for 24h and TEER was measured at several time points (2h, 4h, 6h and 24h). After subtracting the TEER of the empty insert, all timepoint values were normalized to its own Oh value (to account for the differences in initial TEER of the different inserts) and are presented as percentage of initial value.

[0480] Example 4: Analysis of Carbohydrate Active Enzyme (CAZyme) genes of Bifidobacterium longum transitional microorganism

[0481] Genomes of Bifidobacterium longum subspecies listed in Figure 7 were annotated to CAZymes combining dbCAN2 (Zhang et al., Nucleic Acids Res. 46(W1):W95-W101 (2018)) tools and databases HMMdb (v9) and Diamond (v2.0.8). Query sequences with > 0.50 coverage and e-value < 1e-15 were annotated with HMMER according to the dbCAN CAZyme domain HMM database. Diamond was also used to annotate query sequences with hits in the CAZy database (Drula et al., Nucleic Acids Res. 50(D1):D571-D577 (2022)) (http: / / www.cazy.org / ) with > 0.90 identity, and e value < 1e-102. HMMER annotation was prioritized and used in instances of mismatched CAZyme annotations of query sequences between HMMER and DIAMOND tools. Only CAZyme families and subfamilies encoding Glycoside Hydrolases (GHs) and Polysaccharide Lyases (PLs) were used for comparative analyses of B. longum subspecies (see Figure 7).

[0482] Example 5: Utilization of glycan substrates

[0483] Pulverized or homogenized stool samples were mixed 10-fold by adding PBS / glycerol (1 / 10) (w / v) before centrifugation at 2000g for 2 minutes. The slurry and pellet were then stored at - 80°C. Frozen fecal samples were thawed from storage at -80°C before centrifugation at 2000g for 2 minutes. The resulting supernatant was inoculated with media based on that disclosed in Daguet et al. (Journal of Functional Foods; 2016; 20; 369-379). This media was supplemented with specific fibers to be tested at 5 g / L and a Bifidobacteria supplement of 5E07 CFU / ml.

[0484] The culture was set up at 37°C, N2 gas flow to ensure anaerobic conditions and gentle stirring. Aliquots were taken and analyzed at the time points indicated.

[0485] B. longum transitional strain NCC5001 growth is promoted by pectin (sugar beet) and arabinogalactan (larch wood) (Figure 8).

[0486] B. longum transitional strain NCC5002 growth is promoted by arabinogalactan (larch wood) and starch (potato) (Figure 9).

[0487] Example 6: Characterization of B. longum transitional microorganism

[0488] B. longum transitional strains were isolated from the feces of breast-fed infants using Eugon Tomato Agar (ETA). Obtained isolates were sequenced using PacBio to obtain a fully closed assembled genome for each of the strain. Each strain was deposited in the internal Nestle Culture Collection (NCC, Lausanne, Switzerland) and at the Collection Nationale de Microorganisms (CNCM) at the Pasteur Institute (Paris, France) together with their genome sequence data. The genome of the strains was compared by Average Nucleotide Identity (AN I) using OrthoAni (htps: / / www.ezbiocloud.net / tools / orthoani) to other publicly available genomes representing the overall diversity of the B. longum species (Table 5), and to the Metagenomic Assembled Genomes (MAG) obtained from metagenomic sequences issued from infant feces of the same cohort.

[0489] Table 5 - list of genomes used for ANI analysis and their publicly available references. (T) stands for typestrain.

[0490] The analysis demonstrates that the newly described strains group together with the MAGs obtained from the same cohort, defining a well delineated clade belonging to the B. longum species. Two previously isolated strains BSM11-5 and 3_mod are found to be grouped within this newly described clade. The clade is genetically different from B. longum subspecies longum (96.40 % ANI) subspecies. The clade is related, while still clearly distinct, to B. longum subspecies, suis / suillum (98.207%), and to the group of strains (JDM301 , CMCC_P0001 and BXY01) previously suggested to be a new B. longum subspecies (O’Callaghan et al. 2015), sharing an identity of 98.260 % to this group of strains. Figure 1 shows ANI LIPGMA based phylogenetic tree. The scale represents the percentage (%) of identity at each branch point.

[0491] A selection of the above mentioned genomes, representing the diversity of the B. longum subspecies, were annotated for Carbohydrate-Active enZYmes(CAZY) using the dbCAN annotation pipeline (http: / / bcb.unl.edu / dbCAN / ). Results showed that B. longum subsp. longum, B. longum subsp. Suis and B. longum subsp.suillum strains contained a GH20 (lacto- N-biosidase) enzyme, implicated in the degradation and metabolization of Lacto-N-tetraose (LNT). Similarly to B. longum subsp. infantis strains, B. longum transitional strains also possessed a similar enzyme, and in addition harbored GH29 (fucosidase) encoding genes which are implicated in the degradation and metabolization of fucosylated human milk oligosaccharides, such as 2’-FL, 3-FL or diFL. Additionally, three of the strains (CNCM 1-5684, BSM1-15& 3_mod) also harbor a GH 33 (sialidase) encoding gene implicated in the degradation and metabolization of sialilated HMO such as 3’SL or 6’SL (Table 6). Table 6 - Number of genes encoding for GH20 (lacto-N-biosidase), GH29 (a-fucosidase), GH95 ((a -fucosidase / ( a -galactosidase) and GH33 (sialidase) glucohydrosylhydrase family enzymes in each of the represented genomes.

[0492] CAZYmes_pred Genome N° of N° of N° of N° of reference predicted GH20 predicted GH29 predicted predicted encoding genes encoding genes GH95 GH33 encoding encoding genes genes

[0493] All newly obtained genomes were compared and aligned with the genome of two strains (B. longum subsp. infantis ATCC15697 and B. kashiwanohense DSM 21854) belonging to species for which the genes responsible for fucosylated HMOs utilization were elucidated (James et al. 2019).

[0494] Results

[0495] As shown in Figure 11 , all newly described strains contained genes responsible for the utilization of fucosylated HMOs. While NCC 5001 organization reflects the one of 8. longum subsp. infantis ATCC 15697, all other strains (NCC 5000, NCC 5002, NCC 5003, NCC 5004) harbor a gene organization closer to that of 8. kashiwanohense DSM 21854. Overall, the similarity to the well described fucosidases of 8. longum subsp. infantis ATCC 15697 is above 77% (for BLON_2334) and 88% (BLON_2335) in all newly described strains.

[0496] Example 7: Utilization of fucosylated HMOs

[0497] All strains retrieved from the Nestle Culture Collection (Table 7) were reactivated from a freeze-dried stock, using two successive culturing steps (16h, 37°C, anaerobiosis) in MRS supplemented with 0.05 % cysteine (MRSc). Reactivated cultures were then centrifuged, washed and resuspended in 1 volume of PBS. Washed cells were used to inoculate MRS based medium without a carbon source (MRSc-C) (10 g 1-1 of bacto proteose peptone n°3, 5 g 1-1 bacto yeast extract, 1 g 1-1 Tween 80, 2 g 1-1 di-ammonium hydrogen citrate, 5 g 1-1 sodium acetate, 0.1 g 1-1 magnesium sulphate, 0.05 g 1-1 manganese sulfate, 2 g 1-1 di-sodium phosphate, 0.5 g 1-1 cysteine) in which glucose, 2’-FL or 3-FL were added as unique carbon source at a concentration of 0.5%. Growth was then performed in a 96 well microplate, with a volume of 200 pl per well. Incubation was performed in anaerobiosis for 48h, and optical density was measured in a spectrophotometer at 600 nm. As shown in Figure 9, all B. longum transitional strains grew on fucosylated HMOS. Results

[0498] All B. longum transitional strains grew better on 3-FL than 2’-FL and reached a higher cell density on this carbohydrate. This behavior indicates a preference for 3-FL over 2’-FL (ratios from 1 .8 to 2.8- see Figure 12), which is not observed in B. longum subsp. infantis LMG 11588.

[0499] Table 7 - list of the strains (and corresponding numbers) used for the individual fucosylated

[0500] HMO growth studies

[0501] Example 8: Efficacy testing of synbiotic in PVM infection model and pollution-enhanced allergic airway inflammation model

[0502] Detailed trial design

[0503] Fig. 14 shows a schematic of the model of early life viral airway infection and pollution enhanced allergic airway inflammation. The trial design is described in more detail below.

[0504] WT C57BL / 6 breeders were fed either a control fiber or low fiber diet, starting 3 weeks prior to gestation. Offspring of these breeders were fed the corresponding diet of their mothers throughout the experiment, i.e. from post-natal day (PND) 0 to PND66.

[0505] At PND5, litters of low fiber diet fed animals were randomly assigned to the different experimental groups (n=14-16 / group). Mice were fed once daily with different combinations of nutritional ingredients (6HMOs; B. infantis LGM11588; B. infantis LGM11588+6HMOs) via intra-gastric (i.g.) gavage of 50 pl of ingredients in a saline solution ending PND20. Control groups were fed with saline solution only.

[0506] At PND10, all animals (n=13-16 / group) were infected intra-nasally (i.n.) with 10 PFU of PVM (PVM, J3666) in a total volume of 40 pl in a saline solution (20 pl / nostril) to induce bronchiolitis. The solution containing the virus was delivered as droplets on the nostrils using P20 pipettes. Isoflurane anesthetized mice were hold in supine position to make sure the solution was properly inhaled.

[0507] Animals (n=7-8 / group) were sacrificed at PND20 (at peak immunopathology of bronchiolitis) to measure virus induced lung inflammation and associated pathology.

[0508] Starting from PND42, animals that recovered from PVM-infection (n=7-8 / group) were sensitized i.n. with cockroach allergen extracts (CRE, 1mg / application) in combination with particulate matter PM2.5 (10mg) once a week ending PND63 (i.e. on PND42, PND49, PND56, PND63) to induce pollution-enhanced allergic airway inflammation.

[0509] Animals were either sacrificed on PND20 (N=7-8 / group, peak immunopathology bronchiolitis) to test impact of nutritional interventions to promote protective immunity and on PND66 (N=7- 8 / group) to assess susceptibility to pollution-enhanced allergic airway inflammation post viral airway infection.

[0510] Probiotic dose

[0511] 106CFU / day.

[0512] HMO dose

[0513] A concentration of 4mg / day and application of a mix of 6HMOs (2FL / DFL, LNT, 3SL, 6SL, 3FL) has been selected based on most recent recommendations for HMOs in infant formulae (Stage 1 : 1.8-2g / L / day in the first 6 months).

[0514] Calculations were based on 1-week old infant consuming 500 mL of breast milk / day and 10 day old mouse pup consuming up to 2mL of milk / day (Source: contemporary topics in laboratory animal science / American Association for Laboratory Animal Science 43(3):50-3).

[0515] The ratio between HMO ingredients is inspired on breast milk composition: 2FL / DFL ingredient 53%; LNT ingredient 19%; 6SL ingredient 8%, 3SL ingredient 6.2%; 3FL ingredient 13.8%.

[0516] In some experiments a concentration of 4mg / day and application of a blend of 5HMOs (2FL, DFL, LNT, 3SL, 6SL) has been selected based on most recent recommendations for HMOs in infant formulae (Stage 1 : 1.8-2g / L / day in the first 6 months). Ratios used for 5HMO blend: 2FL / DFL ingredient 65.70%; LNT ingredient 23.40%; 6SL ingredient 9%, 3SL ingredient 2%.

[0517] Low fiber diet

[0518] WT C57BL / 6 mice efficiently clear the PVM virus. Such mild infections are not associated with altered lung tissue remodeling that in infants predispose to allergic airway inflammation later in life. To increase susceptibility to PVM infection and associated risk of allergic airway inflammation in adulthood, dams and corresponding pups were fed a low fiber diet throughout the experiment (susceptible groups; Trompette, Nat. Med, 2014). Animals fed a normal fiber diet were used as protected controls (protected group).

[0519] Statistical analysis

[0520] Statistical significance was determined using a non-parametric, two-was ANOVA followed by Bonferroni post-hoc testing. Results were considered significant at P < 0.05. *P < 0.05; ** P < 0.01 ; *** P < 0.001. All statistical analyses were performed using Prism GraphPad Software.

[0521] Results

[0522] PVM infection model

[0523] Pneumonia Virus of Mice (PVM) is related to the human respiratory syncytial virus (RSV) pathogen that affects >90% of children by the age of two years. PVM has been used to study respiratory virus replication and the ensuing inflammatory response as a component of a natural host-pathogen relationship. As such, PVM infections in mice reproduce many of the clinical and pathologic features of the more severe forms of RSV infections in infants - that if not properly controlled have long-lasting consequences on lung function and predispose to the onset of allergic airway responses later in life (Dyer KD, Viruses, 2012). The highly pathogenic PVM strain J3666 has been selected for these studies to conform to human pathophysiology of RSV infections in infants.

[0524] Severe bronchiolitis in early life is associated with mucus hypersecretion and airway epithelial cell sloughing. Dead epithelial cells together with a viscous exudate can form dense plugs in the bronchioalveolar lumen that impede breathing. Controlled mucus secretion and epithelial cell death are beneficial defense mechanisms limiting viral spread. These responses need to be tightly regulated upon clearance of virus to avoid chronicity and pathological tissue remodeling.

[0525] Pathological hallmarks of a severe viral lower respiratory infection include airway epithelial cell (AEC) sloughing, mucus hypersecretion and airway smooth muscle (ASM) remodelling. AEC detachment is a feature of viral bronchiolitis and is associated with disease severity and viral load.

[0526] AEC sloughing was quantified by measuring the length of sloughed airway epithelium and expressing this as a percentage as basement membrane length of airway in at least five airways per mouse. Mucus hyper-secretion and pathological lung tissue remodeling as other hallmarks of severe bronchiolitis were assessed by quantification of Muc5ac positive AECs, and ASM remodeling was assessed by area / airway circumference, respectively (Lynch JP, JEM 2018).

[0527] Early life nutritional intervention with synbiotic (8. infantis + 6HMOs) resulted in a significant reduction in infection mediated airway epithelial cell (AEC) sloughing (Fig. 15A), mucus secretion (Fig. 15B) and pathological tissue remodeling (Fig. 15C) compared to placebo treated controls. Strikingly, synbiotic intervention showed superior efficacy over prebiotic (6HMO) or probiotic (B. infantis) interventions alone in reducing infection-mediated AEC sloughing, mucus secretion and pathological airway remodeling.

[0528] To assess whether early life nutritional supplementation with the synbiotic, besides eliciting an effective anti-viral immune response, results in controlled resolution of lung inflammation upon viral clearance, numbers of neutrophils and / or eosinophils in the airways of PVM infected mice were assessed by flow cytometry. Importantly, early life supplementation with the synbiotic resulted in controlled resolution of lung inflammation upon clearance of virus as assessed by significantly reduced numbers of granulocytes (neutrophils and eosinophils) in the airways in response to PVM infection.

[0529] Early life nutritional intervention with a second synbiotic (8. infantis + 5HMOs) resulted in a significant up-regulation of the anti-viral cytokine IFN-A at 5 days post infection (peak of viral infection, Fig. 18A) that was associated with a significant reduction in granulocytic cell infiltrates into inflamed lungs as quantified by numbers of lung neutrophils (Fig. 18B) and numbers of lung eosinophils (Fig. 18C), suggesting that early life intervention with the synbiotic (8. infantis + 5HMOs) reduces virus-induced severe lung inflammation. These data indicate that early life supplementation with the synbiotic controls effective anti-viral immune responses and promotes rapid resolution of lung inflammation and thus recovery from respiratory infection.

[0530] Pollution-enhanced allergic airway inflammation model

[0531] Severe RSV infection in early life represents a significant risk factor to develop allergic airway disease later in life. Outdoor air pollution is a major health problem throughout the world. Exposure to particulate matter (PM) has been associated with the exacerbation of several respiratory diseases, including allergic asthma. To mimic such globally relevant conditions, animals recovered from severe PVM infection were sensitized with cockroach allergens in the presence of the pollutant PM2.5 to induce allergic airway inflammation. Such experimental approach allows to test whether nutritional intervention in early life (window of opportunity) does not only alter susceptibility to viral bronchiolitis but may also have sustained immune benefits into adulthood. Early life respiratory viral infections represent a potential tipping point in the balance between long-term respiratory health and chronic airway disease such as airway allergies. The present study shows that nutritional synbiotic intervention in early life not only reduced severity of airway viral infection but also promoted a sustained immune benefit into adulthood as assessed by reduced susceptibility to pollution-enhanced allergic airway inflammation.

[0532] This is exemplified by reduced lung ILC2 (Fig. 16A) and eosinophil numbers (Fig. 16B) as assessed by flow cytometry. Dampened lung inflammation was further associated with a reduced mucus score (Fig. 17A) and improved tissue remodeling (Fig. 17B). Strikingly, and in line with reduced susceptibility to viral airway infection (see Fig. 15), synbiotic intervention (B. infantis + 6HMOs) showed superior efficacy over prebiotic (6HMO) or probiotic (8. infantis) interventions alone in reducing pollution-enhanced allergic airway inflammation in adulthood.

[0533] Conclusions

[0534] Using a neonatal mouse model of PVM-induced bronchiolitis and early life nutritional interventions, we demonstrate that synbiotic intervention (8. infantis in combination with a mix of human milk oligosaccharides (5HMO or 6HMO)) provides protection from virus-induced bronchiolitis. Importantly, we demonstrate that synbiotic intervention (8. infantis in combination with a mix of human milk oligosaccharides (6HMO)) provides superior efficacy over 6HMOs alone or 8. infantis alone to provide protection from virus-induced bronchiolitis. Specifically, synbiotic interventions resulted in a rapid resolution of virus-induced lung inflammation and appropriate lung tissue remodeling upon clearance of the virus.

[0535] With the initial host response to respiratory viruses representing a potential tipping point in the balance between long-term respiratory health and chronic airway disease, these findings indicate that early life nutritional intervention with these synbiotics (8. infantis + 6HMOs or B. infantis + 5HMOs) may represent a preventive strategy to fight severe bronchiolitis in infants and associated risk to develop allergic airway disease at school age. In fact, nutritional synbiotic intervention in early life not only reduced severity of airway viral infection but also promoted a sustained immune benefit into adulthood as assessed by reduced susceptibility to pollution-enhanced allergic airway inflammation. In particular, these results deliver string evidence that nutritional synbiotic intervention in early life attenuates pathological airway remodeling known to increase the risk of chronic inflammatory diseases such as asthma or COPD later in life. Example 9: Preclinical model for efficacy testing of B. I. iuvenis in infection model

[0536] Detailed trial design

[0537] An in-vivo preclinical model of infection was developed, as shown in Figure 19.

[0538] WT C57BL / 6 breeders were fed either a control fiber or low fiber diet, starting 3 weeks prior to gestation. Offspring of these breeders were fed the corresponding diet of their mothers throughout the experiment.

[0539] Control groups were nursed by mothers fed either with a low fiber diet only (susceptible group) or with a high fiber diet only (protected group) before weaning and kept on the same diet after weaning.

[0540] At post natal day (PND) 5, litters of low fiber diet fed animals were randomly assigned to the different experimental groups (n=8 / group). C57BL / 6 WT pups received different combinations of nutritional ingredients (6HMOs + 2 bifidobacterial strains (B. infantis LGM11588 and B. lactis CNCM 1-3446); 6HMOs + 3 bifidobacterial strains (B. infantis LGM11588 and 8. lactis CNCM 1-3446 and 8. / . iuvenis)) via oral gavage while being nursed by mothers fed on low fiber diet. Broad antibiotics were supplied through the drinking water from PND16 to 26. Following weaning on PND21 , a selective fiber mix (adapted to 8. longum transitional strain) was introduced in the diet of these mice coupled with oral gavage of the same nutritional ingredients (reduced dose of HMOs + bifidobacterial strains). The HMO mixtures used in the pre-weaning and post-weaning stages are shown in Table 8.

[0541] Infection with pneumonia virus of mice was performed at PND35.

[0542] Table 8 - HMO mixture used in the study Results

[0543] As shown in Figure 20, the nutritional composition containing the B. longum transitional strain mix (Group 4) confers better protection against airway viral infection post weaning, as shown by the increased weight (%PND35) on the days post infection when compared to the low fibre diet (Group 2), or the low fibre diet mix without the B. longum transitional strain (Group 3).

[0544] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention 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 connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1 . A composition comprising a Bifidobacterium longum transitional microorganism and a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N-neotetraose (LNnT).

2. The composition according to claim 1 , wherein the Bifidobacterium longum transitional microorganism:(i) is capable of metabolizing the HMO(s);(ii) preferentially utilizes 3-FL over 2’-FL;(iii) is capable of metabolizing a glycan substrate selected from the group recited in any of Tables 1 to 3; and / or(iv) encodes one or more CAZymes selected from the group recited in Table 1 , preferably wherein the Bifidobacterium longum transitional microorganism further encodes one or more CAZymes selected from Table 2 and 3.

3. The composition according to claim 1 or claim 2, wherein the Bifidobacterium longum transitional microorganism has an Average Nucleotide Identity (ANI) of at least 98% with at least one Bifidobacterium longum strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687, CMCC-P0001 (ATCC BAA-2753), and any combination thereof.

4. The composition according to any one of the preceding claims, wherein the composition further comprises Bifidobacterium longum subsp. infantis and / or Bifidobacterium lactis.

5. The composition according to claim 4, wherein:(i) the Bifidobacterium longum subsp. infantis is Bifidobacterium longum subsp. infantis LMG 11588 or has an Average Nucleotide Identity (ANI) of at least 99.9% to Bifidobacterium longum subsp. infantis LMG 11588; and(ii) the Bifidobacterium lactis is Bifidobacterium lactis CNCM 1-3446 or has an Average Nucleotide Identity (ANI) of at least 99.9% ANI to Bifidobacterium lactis CNCM 1-3446.

6. The composition according to any one of the preceding claims, wherein the HMO mixture consists of 2’-FL, DFL, LNT, 6SL and 3SL.

7. The composition according to any one claims 1-5, wherein the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL, and 3-FL.

8. The composition according to any one of claims 1-5, wherein the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL, and LNnT.

9. The composition according to any one of claims 1-5, wherein the HMO mixture consists of 2’-FL, DFL, LNT, 6SL, 3SL, 3-FL and LNnT.

10. The composition according to any one of the preceding claims, wherein the composition is a nutritional composition selected from an infant formula, a starter infant formula, a follow- on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement.

11. A composition as defined in any one of claims 1-10 for use in preventing, reducing the risk of and / or treating an infection in a subject.

12. A composition as defined in any one of claims 1-10 for use in promoting a long-term immune benefit in a subject.

13. The composition for use according to claim 12, wherein promoting a long-term immune benefit in a subject comprises: i. promoting long-term respiratory health; ii. preventing and / or reducing the risk of allergen sensitisation; and / or iii. preventing and / or reducing the risk of developing a respiratory condition, preferably wherein the respiratory condition is a chronic inflammatory disease of the respiratory tract, such as asthma or chronic obstructive pulmonary disease (COPD) or is an allergic respiratory tract disease, such as recurrent wheeze or asthma.

14. A composition as defined in any one of claims 1-10 for use in preventing and / or reducing the risk of developing asthma in a subject.

15. A prebiotic for use in preventing, reducing the risk of and / or treating an infection in a subject by promoting the growth and / or survival of a Bifidobacterium longum transitional microorganism in the gut of the subject, wherein the prebiotic is a HMO mixture consisting of 2'-fucosyllactose (2’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lacto-N- neotetraose (LNnT).

16. The composition for use according to claim 11 or the prebiotic for use according to claim 15, wherein the infection is a viral, bacterial or fungal infection, preferably wherein the infection is an airway infection.

17. The composition for use or the prebiotic for use according to any one of claims 11 , 15 or 16, wherein the infection is a viral airway infection, preferably wherein the viral airway infection is selected from influenza virus, respiratory syncytial virus, rhinovirus, parainfluenza viruses, metapneumovirus, coronavirus, adenovirus, and bocavirus.

18. The composition for use or the prebiotic for use according to any one of claims 11-17, wherein the subject is an infant, a young child or a child.