Understanding HMOs and Implications in Infant

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Understanding HMOs and Implications in Infant

Health

A/Professor Daniel Avi Lemberg Head of Gastroenterology Sydney Children’s Hospital

Conjoint University of New South Wales

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My town

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My Place

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Thank you for having me

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Conflict

• Many and varied but currently these talks are sponsored by Nestle

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What are HMOs?

BREAST IS BEST

Human breast milk ^1,2

•Is the most suitable nutrition for infants

•Has a unique composition

•Offers various health benefits to infants

•Including reduced risk of:

•gastrointestinal infections

•respiratory infections

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So Breast is best !

What are HMOs?

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Human milk oligosaccharides (HMOs) are specific bioactive compounds present in human milk. ¹

The amount and variety of oligosaccharides in human milk is

unique and is not found in the milk of cows or other animals. ^2,3,4

1 Kunz, Adv Nutr 2012; 2 Urashima et al. Adv Nutr 2012; 3 Ninonuevo et al. Pediatr Res 2008; 4 Ruhaak et al. Anal Bioanal Chem 2014

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Human milk contains two major types of carbohydrates:

Lactose is a major nutrient source and the most abundant solid component of human milk. ¹

Oligosaccharides are the third most abundant solid component of human milk, generally

regarded to be non-digestible by the infant due to lack of the necessary enzymes in humans. ^2,3

1 Pacheco et al. Annu Rev Abim Biosci 2015; 2 Jantscher-Krenn et al. Minerva Pediatr 2012; 3 Ruhaak et al. Anal Bioanal Chem 2014

Monosaccharides

e.g. glucose 1 monosaccharide

molecule

Disaccharides

e.g. lactose 2 monosaccharide

molecules linked

Oligosaccharides

e.g. HMO

3-10 monosaccharide molecules linked

Polysaccharides

e.g. starch

>10 monosaccharide molecules linked

Carbohydrates (also called saccharides) can be classified into mono-, di-, oligo- and

polysaccharides:

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HMOs ARE THE 3 ^RD LARGEST SOLID COMPONENT IN HUMAN MILK

The HMO fraction is quantitatively larger than that of protein (which is typically around 8g/L) ^1,2 ; HMOs and are thus considered a key component of breast milk. ¹

HMO concentrations range between 20–25 g/L for colostrum ² and 5–15 g/L for mature milk. ¹

1 Zivkovic et al. PNAS 2011; 2 Bode, Glycobiology 2012; fig. adapted from Zivkovic et al. Functional Food Reviews 2013

HMOs

…

Sialyllacto-N-tetraose 3’-and 6’-Sialyllactose

3-Fucosyllactose

Lacto-N-(Neo)tetraose

Lacto-N-fucopentaose

Lactose (70 g/L)

2’-Fucosyllactose Water

Human milk Macro- &

Micronutrients and HMOs

Proteins (8 g/L) HMOs (5-15 g/L)

Lipids (40 g/L)

Solid

components

¹

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HMOs commonly contain a lactose core (= a disaccharide consisting of glucose Glc and galactose Gal) ^2,3 which is elongated by one or more of the following 4 monosaccharides: ¹

N-acetylglucosamine (GlcNac) Fucose (Fuc)

Sialic acid (N-acetylneuraminic acid (NeuAc)) Galactose

1 Bode, Glycobiology 2012; 2 Bode et al. Adv Nutr 2012; 3 Jantscher-Krenn et al. Minerva Pediatr 2012

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HMO CATEGORIES

1 Ninonuevo et al. J Agric Food Chem 2006; 2 Urashima et al. Adv Nutr 2012; 3 Bode, Nutr Rev 2009; 4 Hennet et al. Swiss Med Wkly 2014; 5 Bode et. al. Early Hum Dev 2015; 6 Adapted from

More than 200 HMOs ^1,2,3,4 have been identified so far and classified into three different categories ⁵ :

Fucosylated (= neutral) HMOs (e.g. 2’FL)

Non-fucosylated neutral HMOs (e.g. LNnT, LNT)

Sialylated or “with sialic acid” (= acidic) HMOs (e.g. 3’SL)

2’FL

The proportion of fucosylated, non-fucosylated neutral and sialylated HMOs in term breast milk was recently reported as 35–50%, 42–55%, and 12–14%, respectively.

⁶

The neutral oligosaccharides, to which 2´FL and

LNnT belong, account for more than 75% of the total HMOs.

⁶

Proportion of HMO categories in human milk:

2’-Fucosyllactose

ß1-4 α1-2

LNnT

^ß1-4 _ß1-3

Lacto-N-neotetraose

ß1-4

LNT Lacto-N-tetraose

ß1-4 ß1-3

3’SL

^ß2-3

3-Sialyllactose

ß1-4 ß1-3

Smilowitz et al. Annu Rev Nutr 2014

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FUCOSYLATED HMOs DETERMINED BY GENETIC FACTORS

The ability to secrete the enzyme fucosyltransferase is genetically determined. ^1,3 Women with active Secretor gene = Secretors (Se+), 80% of all women. ⁵

Oligosaccharide amount and composition in human milk vary between individuals and

diurnally, by infant gestational age, over the course of lactation as well as with the mother’s nutritional status. ^1,2,4

The most extreme interpersonal variations are based on women’s secretor status. ^1,2

1 Bode et al. Adv Nutr 2012; 2 Goehring et al. Plos One 2014; 3 Blank et al. Adv Nutr 2012; 4 Morrow et al. J Nutr 2005; 5 Urashima et al. Adv. Nutr. 2012; 6 Jantscher-Krenn et al. Minerva Pediatr 2012

Levels of HMO also fluctuate over time with less being produced with time

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THE MOST COMMON HMOs IN NATURE AND RESEARCH

2´FL and LNnT belong to the neutral oligosaccharide groups that account for a total of more than 75% of the total HMOs. ¹

2’FL is by far the most abundant HMO in the milk of Se+ women (80% of women worldwide): average 3 g/L (20% of total HMOs). ^1,2

LNnT constitutes ~ 2-3 % of the total HMO fraction, which qualifies it as one of the 10 most important HMOs by abundancy out of a pool of more than 200. ¹

Individually or together, 2’FL and LNnT have so far been the main focus of pre-clinical and clinical research on the effects of HMOs in infants.

2’FL

ß1-4

2-Fucosyl-Lactose (trisaccharides)

LNnT

Lacto-N-neotetraose (tetrasaccharides)

LNT

ß1-3

Lacto-N-tetraose (tetrasaccharides)

α1-2

ß1-4

ß1-3 ß1-4

ß1-4

ß1-3

1 Roehrig, Glycom A/S, Denmark, Kgs. Lyngby 3rd of June 2015; 2 Urashima et al. Adv Nutr 2012

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MAJORITY OF HMO REMAIN IN GUT LUMEN AND PASS INTO STOOL

1 Ruhaak et al. Anal Bioanal Chem 2014; 2 Rudloff et al. Acta Paediatr 1996; 3 Rudloff et al. Glycobiology 2006; 4 Rudloff et al. Br J Nutr 2012; 5 Obermeier et al. Environ Health Stud 1999; 6 Rudloff et al.

HMOs are generally regarded to be non-

digestible by the infant’s gut due to lack of human production of the

necessary enzymes. ¹

Approximately 1%- 2% of the HMOs are absorbed in the small intestine or colon and reach the systemic circulation. ^1-6

From Rudloff and Kunz, Adv Nutrition, 2012

Anal Bioanal Chem 2014

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MAIN EFFECTS OF HMOS THAT WE CURRENTLY KNOW ABOUT

Luminal & mucosal effects

Microbiota establishment

& function

Protection from infection Mucosal barrier function

Systemic effects

Immunity - Allergy Brain

Metabolic health

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**HMOs shape the early-life gut microbiota by promoting the growth of Bifidobacteria, which may be involved in the programming of a healthy immune system.** ^1,2,3

HMOs nurse beneficial Bifidobacteria ¹

HMOs serve as “food” for specific Bifidobacteria. 1,2,3,4,5 ,6,7

enhance growth of Bifidobacteria that occupy attachment sites on the gut mucosa

➔ prevents adhesion of pathogens.

⁷

HMOs do not “feed” growth of numerous potentially pathogenic bacteria, like Clostridium. ^7,8,9

fig. adapted from Bode, Glycobiology 2012

HMOs Beneficial bacteria Potential pathogens

HMO ^S HELP ESTABLISH A PROTECTIVE GUT MICROBIOTA

1. Sela et al. Trends in Microbiology 2010; 2 Zivkovic et al. PNAS 2011; 3 Oozer et al. Am J Clin Nutr 2013; 4 Smilovotz et al. Annu Rev Nutr 2014; 5 Zivkovic et al. Functional Food Reviews 2013; 6 Yatsunenko et al. Nature 2012 ; 7 Sela et al. PNAS 2008; 8 Yu Glycobiology 2013; 9 Hoeflinger J. Agric. Food Chem. 2015;

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HMOs function as soluble receptor analogs (decoys) that compete for bacterial binding against the intestinal mucosa ¹

Many pathogens need to attach to the mucosal surface to cause infectious disease. ¹

HMOs (especially fucosylated structures) resemble the host epithelial cell surface glycans (= receptors) to which pathogens adhere. ^2,3

Pathogens (bacteria, viruses and their toxins) are deceived by the structural similarity and attach to the HMOs. ⁴ HMOs thus serve as soluble decoy receptors to prevent pathogen binding to the host cell and thereby reduce the risk of infectious disease. ¹

HMOs serve as decoys for pathogens. ¹

1 Bode et al. Adv Nutr 2012; 2 Smilovic et al. Annu Rev Nutr 2014; 3 Sprenger et al. Eur J Nutr 2016; 4 Jantscher-Krenn et al. Minerva Pediatr 2012

HMO ^S BLOCK PATHOGENS FROM DOING HARM

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The surface of intestinal epithelial cells, like that of any living cell, is covered in glycans. They

represent the first and crucial interface to the intestinal epithelial cell’s environment. ¹

HMOs can directly interact with intestinal epithelial cells and modulate their glycan expression. ^2,3

Modulating the glycans of the host cell can be an alternative mechanism to prevent pathogen

attachment to the host cell, increasing resistance to infection. ³

HMOs improve host defense by strengthening intestinal barrier function. ¹

1 Angeloni et al. Glycobiology 2005; 2 Bode, Glycobiology 2012; 3 Jantscher-Krenn et al. Minerva Pediatr 2012

HMO ^S STRENGTHEN THE GUT BARRIER FUNCTION

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SUMMARY : MAIN KNOWN IMMUNE FUNCTIONS OF HMO

HMOs help establish a potentially protective gut microbiota with predominance of Bifidobacteria.

HMOs block pathogens from doing harm by functioning as soluble receptor analogs to which pathogens adhere.

HMOs strengthen the gut barrier function.

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HMO ^S AND GUT MICROBIOTA – PRECLINICAL MODELS

*Formerly germfree mice that are inoculated with bacteria commonly present in the infant gut microbiota

1 Sela et al. Trends Microbiol 2010; 2 Li et al. J Nutr 2012; Yu et al. Glycobiology 2013; 3 Marcobal et al. Cell Host Microbe 2011; 4 Sprenger et al. unpublished; 5 Yu et al. 2013; 6 Hoeflinger et al. JAFC 2015

**HMOs promote specific Bifidobacteria growth and metabolic activity** in vitro and establishment in humanized gnotobiotic mice.* ^1-4

HMOs do not allow for growth of numerous potentially pathogenic bacteria, like

Clostridium, Enterobacter, Klebsiella, Escherichia. ^5-6

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HMO ^S AND GUT MICROBIOTA – PRECLINICAL DATA

**LNnT stimulates Bifidobacterium spp. growth and promotes short-chain fatty acids in vitro.**

**LNnT provides selective advantage to B. infantis over Bacteroides species in vivo.**

**2´FL boost growth of specific Bifidobacteria, and production of short-chain fatty acids in vitro.**

Enterobacteriaceae strains do not grow on 2´FL, 6´SL, or LNnT in vitro.

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HMO ^S AND PROTECTION FROM INFECTIONS

What do we know ?

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HMO ^S AND PROTECTION FROM INFECTIONS – PRECLINICAL MODELS

1 Brassart et al. Infect Immun 1991; 2 Cravioto et al. J Infect Dis 1991;3 Weichert et al. Nutr Res 2013; 4 Barthelson et al. Infect Immun 1998; 5 Duska-McEwan et al. FNS 2014; 6 Idänpään-Heikkilä et

HMOs block numerous pathogens, like Campylobacter jejuni, Candida, E. coli, Salmonella, Streptococcus

pneumonia, Pseudomonas aeruginosa, Influenza in vitro. ^1,2,3,4,5

HMOs reduce respiratory and gastrointestinal

pathogen load in animal models and, in some cases, protect from weight loss. ^6,7,8,9,10

al. JID 1997; 7 Palacios et al. J Biol Chem 2003; 8 Hester et al. Br J Nutr 2013; 9 Li et al. The IMSE Journal 2014; 10 He et al. Gut 2016

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Does it protect against diarrhoea

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PROTECTION FROM GASTROINTESTINAL INFECTION – IN VIVO

Method

One of the most common causes of bacterial diarrhea is Campylobacter jejuni.

Suckling mice from transgenic dams (=

transfected with a plasmid containing the human FUT1 gene) and control pups from nontransgenetic dams were challenged with inocula of Campylobacter jejuni.

Every third day, one pup from each litter was tested for campylobacter intestinal

colonization.

Results

The neutral, fucosylated oligosaccharide fraction in milk of transgenic mice protects their nursing pups from intestinal

**colonization by Campylobacter.**

Fucosylated HMOs (2’FL) inhibit Campylobacter binding to intestinal host cells.

Shaded areas indicate the period during which an appreciable number of mice are infected.

Campylobacter

Ruiz-Palacios et al. J Biol Chem 2003

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**2´FL prevent adherent-invasive E. coli (AIEC) infection and reduce intestinal inflammation in a mouse model.**

2´-FL pretreatment

prevented body weight loss due to prevention of AIEC infection

reduced colon inflammation.

PROTECTION FROM GASTROINTESTINAL INFECTION – IN VIVO

Method

Mice were inoculated via gavage with adherent-invasive E. coli (AIEC) which are able to invade epithelial cells of the colon. Half of them received 2´FL for 4 days before they were infected.

Results

AIEC infection manifested as body weight loss (∼10%).

He et al. Gut 2016

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HMO ^S AND INFECTIOUS DIARRHEA – OBSERVATIONAL STUDY

2’Fucosyl-HMOs related to protection from infectious diarrhea

Campylobacter diarrhea occurred less often in infants whose mother’s milk contained high levels of 2´FL

(measured as % of HMOs). ¹

Moderate-to-severe diarrhea of all causes occurred less often in infants whose milk contained high levels of total 2-linked fucosyl-oligosaccharide (measured as % of HMOs). ¹

This study provides evidence suggesting that HMOs are clinically relevant to protection against infant diarrhea, with high 2‘FL levels associated with greater protection. ¹

1 Morrow et al. J Pediatrics 2004

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HMO ^S AND INFECTIOUS DIARRHEA – OBSERVATIONAL STUDY

2’Fucosyl-HMOs related to protection from infectious diarrhea

A high ratio of 2-linked (FUT2-dependent) to non-2-linked fucosyloligosaccharides (FUT3-dependent) in human milk was associated with lower risk of symptomatic infection **with heat stable toxin (ST) of E. coli in breast-fed infants.**

Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of diarrhea in the developing world.

FUT2 = α1-2-fucoslyltransferase FUT3 = α1-3/4-fucosyltransferase

Milk with higher 2´-linked fucosyloligosaccharide ratios affords greater protection against infant diarrhea.

Newburg et al. Glycobiology 2004

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For Nestlé Internal Use Only

HMO ^S AND IMMUNE COMPETENCE /

ALLERGY

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IMMUNE EDUCATION / COMPETENCE – IN VIVO

Method

Mice were sensitized with ovalbumin (OVA).

Sensitized mice were orally challenged with OVA and 2´FL, 6´SL or lactose.

Results

Treatment with either 2´FL or 6´SL led to a significant attenuation of oral OVA-

challenge-induced intestinal allergy as assessed by diarrhea score.

2´FL and 6´SL reduced the severity of food allergy-induced diarrhea.

Castillo-Courtade et al. Allergy 2015

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HMOs may reduce the risk of IgE-associated ^Eczema

• Higher levels of 2′FL in human breast milk were associated with a lower incidence of IgE-

associated eczema (P=.0065) among

C-section-born infants, with a family risk for allergies, at 2 years of age

Association between 2′FL in

human breast milk and IgE

eczema in C-section–born infants

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CLINICAL INTERVENTION TRIAL WITH HMO 2´FL + GOS (Abbott)

ß1-4

α1-2 ß1-3

ß1-6 ß1-4

n n

GOS

galactose glucose fucose

Marriage et al. JPGN 2015

2’FL

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RCT WITH 2´FL (ABBOTT): STUDY OBJECTIVES AND METHODS

Objective

To assess growth and tolerance in healthy term infants who received infant formulae with a caloric density closer to human milk supplemented with 2´FL + GOS from 0-4 months and to study uptake of 2´FL.

¹

To investigate effects of feeding formulae supplemented with 2´FL on biomarkers of immune function.

²

Methods

Randomized, placebo-controlled, double-blinded, multicenter intervention trial at 28 sites throughout the United States

Feeding groups (from ≤ 5 day to 119 days of life)

Exclusively formula-fed infants randomly assigned to 1 of 3 low-caloric (64.3 kcal/100 ml) formulae:

enrolled in blood collection subgroup (immune markers)²

1 Marriage et al. JPGN 2015; 2 Goehring et al. J Nutr 2016

Exclusively breastfed infants

• HM n = 107 n = 51

• Experimental Formula 1 (EF1) : test formula 1 (0.2 g/L 2´FL + 2.2 g/L GOS) n = 105 n = 54

• Experimental Formula 2 (EF 2): test formula 2 (1 g/L 2´FL + 1.4 g/L GOS)

• CF: control formula (2.4 g/L GOS)

n = 111 n = 101

n = 48

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RCT WITH 2´FL (ABBOTT):

GROWTH AND 2’FL ABSORPTION/ EXCRETION

Methods

Randomized, placebo-controlled, double-blinded, multicenter intervention trial at 28 sites

throughout the United States

Results

No significant differences among any groups for growth parameters (weight, length or head circumference) over the 4-month study period.

All formulae were well tolerated.

Infants fed 2´FL-supplemented formulae with a caloric density similar to human milk had a growth from birth to 4 months of age similar to that of human milk-fed infants.

Weight growth plotted on WHO growth charts

Female infants Male infants

CF=control formula; EF=experimental formula (EF1=0.2g/L, EF2=1 g/L 2´FL

HM= human milk

Marriage et al. JPGN 2015

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RSV

stimulated

n = 48 n = 51 n = 54 n = 48

In ex vivo RSV-stimulated cell (PBMC*) cultures, breastfed infants were not different than either of the groups fed the formulae with 2´FL+GOS, but they had lower concentrations of inflammatory cytokines TNF-α and IFN-Ƴ (p ≤0.05) than infants fed the control formula. Similar trends were observed for IL-1ra, IL-6, and IL-1β. (RSV stimulated. Fig.

A+B).

*Peripheral blood mononuclear cells = immune cells including lymphocytes, monocytes, and dendritic cells

Labeled means withou a common letter differ (p ≤ 0.05)

RCT WITH 2´FL (ABBOTT):

RESULTS - BLOOD IMMUNE MARKERS

Plasma cytokine concentrations (A) in 6-wk-old infants and ex vivo cytokine production by RSV-stimulated PBMCs*

Infants fed formulae with 2´FL + GOS have blood immune markers (baseline and RSV stimulated) similar to breastfed infants, infants fed formula with GOS do not.

Goehring J Nutr. 2016

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For Nestlé Internal Use Only

FIRST CLINICAL INTERVENTION TRIAL WITH 2 HMO ^S :

2´FL + LNnT (NESTLÉ)

ß1-4 α1-2

ß1-4 ß1-3

ß1-4

2’FL LNnT

Puccio et al. JPGN 2017

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STUDY OBJECTIVES

Overall purpose:

Assess growth, tolerance, morbidity and stool microbiota in healthy term infants who received infant formula supplemented with HMOs from 0-6 months

Primary objective:

Weight gain of infants fed an intact protein experimental formula (EF) with two human milk identical

oligosaccharides – 2´FL (1.0 g/L) and LNnT (0.5 g/L) - compared to infants fed an identical control formula without HMOs (CF).

Hypothesis: Weight gain (g/d) from enrollment to age 4 mo in infants fed EF will be non inferior to that of infants CF (non-inferiority margin ∆= -3g/day)

Secondary objectives:

Growth

Digestive tolerance

Morbidity [parental reported adverse events (AEs) / medication use]

Stool microbiota profile and metabolic signature

Puccio et al. Abstract at Nutr Growth Conference 2016; Steenhout et al. Abstract at Exp Biol Conference 2016; Puccio et al. Publication in progress

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STUDY DESIGN AND STUDY POPULATION

Prospective, randomized, controlled, multi-center interventional clinical trial of 2 parallel formula-fed groups conducted in Italy and Belgium

Study population

Healthy, full-term (≥ 37 weeks, ≤ 42 weeks) infants, birth weight 2500-4500g, age ≤ 14 days at enrollment, exclusively formula-fed at the time of enrollment.

Reference group: exclusively breastfed infants, enrolled at 3 months of age.

Feeding groups

Control Formula (whey-predominant, with LC-PUFAs) (Control/CF)

Test Formula (control formula + 2 HMOs ; 1 g 2’FL/L and 0.5 g LNnT/L) (Test/EF) Breastfed group (BF)

n = 87 n = 88 n = 38

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Mean weight in girls and boys from 0-12 months of age, compared with the WHO growth curve (ITT population)

GROWTH

Mean weight from enrollment to age 12 months tracked closely with the WHO growth standard and was not significantly different between groups at any study visit.

Weight development between birth and the age of 4 months was not significantly

different between infants receiving formula with HMOs (Test) and those receiving

formula without HMOs (Control).

Mean weight gain in the test group was non-inferior to the mean weight gain in the control group.

Puccio et al. Abstract at Nutr Growth Conference 2016; Puccio et al. Publication in progress

Mean weight gain (g/d)

Control Test Difference between groups p-value

ITT population 29.52 29.39 -0.13 0.864

PP population 30.15 29.84 -0.30 0.715

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DIGESTIVE TOLERANCE

1Stooling and other GI symptoms, as well as behavioral pattern results were analyzed by Cochran-Mantel-Haenszel test, *p<0.05. ²Bristol scale score from 1 (hardest stool) to 7 (most liquid stool).

3Infant flatulence, spitting-up and vomiting.⁴Infant restlessness/irritability, colic and waking-up at night

No significant difference in stool consistency between the groups, except at 2 months (softer in the formula with HMOs (EF) vs. control formula (CF))

No significant difference in stool frequency between the groups

Gastrointestinal symptoms ³ and behavioral pattern ⁴ were similar between the groups

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MORBIDITY

The occurrences of total AEs, GI-related AEs, SAE, and AEs related to study formulae were similar between the formula with HMOs and control formula groups.

AEs: adverse events

SAEs: serious adverse events SOC: system, organ and class

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AE ^S **IDENTIFIED A PRIORI AND MEDICATION USE**

Infants receiving formula with HMOs (vs. CF) had significantly fewer reports of bronchitis through 4, 6 and 12 mo, lower respiratory tract infections (AE cluster) through 12 mo, antipyretics use through 4 mo, and antibiotics use through 6 and 12 mo. The protective

effects continued after the 6 months intervention period.

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CAESAREAN-BORN INFANTS BENEFIT EVEN MORE FROM FORMULA SUPPLEMENTED WITH HMO S

Caesarean-born infants receiving formula with HMOs (vs. CF) had significantly fewer reports of bronchitis and lower respiratory tract infections (AE cluster) through 12 months.

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CONCLUSIONS 1: CLINICAL TRIAL WITH 2’FL AND LNnT

Infant formula supplemented with 2’FL and LNnT is safe, well-tolerated, and supports normal, age- appropriate infant growth.

Formula supplemented with 2’FL and LNnT reduced the likelihood of parental reported morbidity (particularly bronchitis and lower respiratory tract infections) and medication use (particularly antibiotics, antipyretics).

Findings suggest the addition of 2´FL and LNnT may provide immune benefits. However, these findings warrant confirmation in future studies.

Caesarean-born infants benefit even more from formula supplemented with HMOs. However, these findings warrant confirmation in future studies.

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RDA Genus

The 3 groups - BF, Test, Control - significantly separated at genus level (p<0.001);

Centroids of Control & BF clearly separated whereas Test & BF partially overlapped.

BF Control Test

BACTERIAL DIVERSITY

Beta diversity: Inter-individual diversity at 3 months of age

Formula with HMOs shifted global gut microbiota composition closer

to that of breastfed infants.

Steenhout et al. Abstract at Exp Biol Conference 2016; Puccio et al. Publication in progress

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SPECIFIC BACTERIAL ABUNDANCE

Median interquartile ranges are depicted. Wilcoxon rank sum test at genus level, significant difference indicated by *p<0.05, **p<0.01, and ***p<0.001;

False Discovery Rate (FDR) adjusted P-values between Test and Control groups are 0.16 for Bifidobacterium & Escherichia, and 0.26 for Peptostreptococcaceae uncl.

Test significantly differed from Control on three bacterial genera, being closer to BF.

**Formula with HMOs promote the colonization of potentially beneficial Bifidobacterium and reduce** potentially pathogenic Escherichia and Peptostreptococcaceae.

Relative composition (% of all sequences) of the 3 groups - BF, Test, Control- for the 3 main taxa that showed significant differences between the Test and Control groups at 3 months of age

Steenhout et al. Abstract at Exp Biol Conference 2016; Puccio et al. Publication in progress

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The Good, The Bad and The Ugly

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CONCLUSIONS 2: CLINICAL TRIAL WITH 2’FL AND LNnT

Infant formula supplemented with 2’FL and LNnT shifts global gut microbiota composition (bacterial diversity and abundance) closer but not that observed in the breastfed reference group at 3 months.

Infant formula supplemented with 2’FL and LNnT increased potentially beneficial Bifidobacterium and decreased potentially pathogenic Escherichia and Peptostreptococcaceae at 3 months.

Infant formula supplemented with 2’FL and LNnT shifts stool metabolic signature towards breastfed profile and, of note increased lactate.

Further studies are warranted to evaluate whether such a shift in gut ecology of formula-fed infants towards the breastfed standard leads to benefits.

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Key Points

• HMOs are the third largest solid components in human breast milk, with more than 200 varieties

• HMOs support immunity in 4 main ways:

― promote the growth of beneficial bacteria

― assist gut barrier function

― prevent pathogen adhesion in the gut

― help modulate the immune system

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Key Points

• The safety of 2′FL and LNnT has been approved by the EFSA and the U.S. FDA; furthermore, infant formula supplemented with 2′FL, alone or in combination with LNnT, support normal growth, are safe and

well tolerated.

• HMOs 2’FL and LNnT when added to infant formulae may offer

immune benefits compared to formulae without HMOs.

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Thanks for your attention today

• Any questions

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CLINICAL INTERVENTION TRIALS

Prospective, randomized, controlled clinical intervention studies (RCTs) with HMOs on:

Safety / growth;

Immune markers (substudy) (2’FL+GOS)

Marriage et al. JPGN 2015 Goehring et al. J Nutr 2016

Safety / growth (2’FL+FOS)

Kajzer et al. The FASEB Journal 2016 (Abstract)

Safety / growth (2’FL+LNnT)

Puccio et al. Abstract at Nutr Growth Conference 2016 Steenhout et al. Abstract at Exp Biol Conference 2016

Puccio et al. JPGN 2017

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PROTECTION FROM RESPIRATORY INFECTION – IN VITRO

In vivo, LNnT ¹

Method

S. pneumoniae without or with LNnT or sialylated LNnT were administered into the trachea of rabbits. Viable S. pneumonia in bronchoalveolar lavage was determined.

Results

LNnT and sialylated LNnT reduced S.

pneumoniae adhesion.

1 Idänpään-Heikkilä et al. JID 1997; 2 Duska-McEwen et al. FNS 2014

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Method

Effects of 2´FL and LNnT on human respiratory epithelial cell lines following viral infection in vitro.

Results

**LNnT decreased Influenza (IAV) viral load and 2’FL decreased Respiratory Syncytial Virus (RSV) viral load in airway**

epithelial cells.

PROTECTION FROM RESPIRATORY INFECTION – IN VITRO

In vitro, LNnT and 2´FL ²

1 Idänpään-Heikkilä et al. JID 1997; 2 Duska-McEwen et al. FNS 2014

Understanding HMOs and Implications in Infant