Human Milk Oligosaccharides in preterm nutrition

1. Human Milk Oligosaccharides
(HMOs): 2’FL & LNnT
NARROWING THE DIVERSITY GAP
1
Dr Gaurav Mandhan
Consultant and Director Neonatology and Pediatrics
Miracles Apollo Cradle, Gurugram

2. • Basics of HMOs and Physiological Significance
• What are HMOs
• Role of HMOs
• Narrowing the Diversity Gap with 2 HMOs: 2’FL & LNnT
• HMOs functions in Real Life scenarios – Clinical Evidence
2

3. Breast Milk is the Best Feeding for every newborn and Infants

4. Basics of HMOs and Physiological Significance
5

5. The Cornerstones of Lifelong Health are Dependent on
Early-life Nutritional Exposures
1.Georgieff MK et al. Acta Paediatr. 2018;107(8):1310-1321. 2. Maggini S et al. Nutrients. 2018;10(10):1531. 3. Koletzko B et al. Am J Clin Nutr. 2009 Jun;89(6):1836-45. 4. Rees L. SA Fam Pract 2005;47(7):
31-32). 6
HMOs are key
nutrients that shape
gut microbiome &
immune health in
early life

6. Human Milk Protects Infants by Providing Components that Benefit Immune
Health
Donovan et al. Ann Nutr Metab 2016 67
Among the bioactive
components in human
milk that are modulators
of these processes, human
milk oligosaccharides are a
key component.

7. This Imbalance of Gut Microbiota* in Early Life has Reported to Impact Both Current and
Future Health
Up to 59% increased
likelihood for childhood
obesity1
20%-50% increased
risk for respiratory
infection1,2
3Xincreased likelihood for
food allergy3
Upto 2.6Xin likelihood
for metabolic disorders4
9
Associated with caesarean mode of delivery

8. What are Human Milk Oligosaccharides (HMOs)?
7

9. 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; 1 adapted from Kunz C. Adv Nutr 2012.
11
HMOs are specific bioactive compounds present in human milk the amount and variety of
oligosaccharides in human milk is unique and not to be found in the milk of cows or other
animals.1,2,3,4
History of HMO Discovery
• Difference is linked to milk
carbohydrate composition
• First description of
microorganisms
(Lactobacilli, Bifidobacteria)
Characterization of
the first individual
HMOs
1930
1950-1980s
Intense work on HMOs as
growth factors for
microorganisms
(Bifidobacteria) as well as on
their anti-adhesive and anti-
inflammatory properties
Fecal microbiota
composition different in
breast-vs-bottle-fed
Identification of further
HMOs and functional
studies
Scientists recognized that these compounds in
human milk contribute to the protection of
breastfed infants‘ health.1
1900
20th century
7-fold increased
mortality of non-
breastfed infants in a
time of an
alarmingly high
overall infant
mortality of 20-30%.1

10. 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
Smilowitz et al. Annu Rev Nutr 2014
More than 200 HMOs1,2,3,4 have been identified so far and classified into three different categories5:
The neutral oligosaccharides, to which 2´FL and LNnT belong, account for more than 75% of the total HMOs.6
12
HMO Categories
2’FL​ - 2’-Fucosyllactose​
Fucosylated HMOs
35%–50%
LNnT - Lacto-N-neotetraose
Non-Fucosylated neutral HMOs
42%–55%
3’SL - 3-Sialyllactose​
Sialylated HMOs
12%–14%

11. Factors affecting HMO composition
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; 7 Newburg et al. Glycobiology 2004
• 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
13

12. Protein(8g/L)
HMOs(5-15g/L)
Lactose(70 g/L)
Lipids(40g/L)
Human milk Solid components1 HMOs
3’-and 6’-Sialyllactose
3-Fucosyllactose
Lacto-N-(Neo)tetraose
Lacto-N-fucopentaose
2’-Fucosyllactose
Sialyllacto-N-tetraose
Water
Macro &
Micronutrients
and HMOs
HMOs are the 3rd Largest Solid Component in Human Milk
1 Zivkovic et al. PNAS 2011; 2 Bode, Glycobiology 2012; fig. adapted from Zivkovic et al. Functional Food Reviews 2013
2'FL & LNnT are
among the most
abundant HMOs
known so far
18

13. 1 Bode, Glycobiology 2012; 2 Urashima et al. Biosci Biotechnol Biochem 2013; 3 Tao et al. J Proteome Res 2011; 4 Oozeer et al. Am J Clin Nutr 2013.
HMOs are more complex, varied, and in higher concentration and different structures
than milk oligosaccharides of other species.
Human milk Bovine milk
Oligosaccharides (g/l) 10-15  0.05
Number of identified
oligosaccharides
>2001  40
% neutral fucosylated 50-80%2  1%
% sialylated acidic 10-20%  70%
Main differences between human milk and bovine milk
oligosaccharides: 1
21
HMOs compared to Cow’s Milk Oligosaccharides
HMOs in Human Milk

14. 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
• Non-digestible 1
• Approximately 1%- 2% of
the HMOs are absorbed.1-5
From Rudloff and Kunz, Adv Nutrition, 2012
19

15. What Do HMOs Do?
23

16. Main Effects of HMOs
Luminal & mucosal effects
• Microbiota establishment &
function
• Protection from infection
• Mucosal barrier function
Systemic effects
• Immunity - Allergy
• Brain function
• Metabolic health
25

17. The Key Role and Best Researched Effect Of HMOs
Impact on The Infant’s Immune System
24

18. 25
HMOs Help the Immune Defenses in 4 Main Ways
Nurture
bifidobacteria
Act as trap for
pathogens
Strengthen
gut barrier
function
Educate the
developing
immune system

19. HMOs promote growth of beneficial Bifidobacteria
HMOs Help Establish a Protective Gut Microbiota and Act as Trap for
Pathogens
Bode, Glycobiology 2012.
A
HMOs serve as decoys for pathogens
26

20. Pre-clinical
Clinical observation
Clinical intervention
Microbiota growth, function & establishment
Protection from Infection
Immune competence - Allergy
Brain - ENS
• Growth and function of specific Bifidobacteria
• No growth of Enterobacteriaceae
• Protection from gastrointestinal pathogens
• Protection from respiratory pathogens
• Modulation of immune competence – Allergy
• Brain function
Functional Data on HMOs
2’FL
LNnT ß1-4
α1-2
ß1-4
ß1-3
ß1-4
29
Note: Specific effects of 2’FL & LNnT are discussed in later sections

21. Narrowing the Diversity Gap with 2 HMOs: 2’FL & LNnT
31

22. Why are 2’FL and LNnT Important?
• Are among the most abundant HMOs
• Belong to the 2 most abundant categories of HMOs
• Are among the most well researched HMOs
• Since HMO functions are unique to their structure, different structures confer
potentially wider benefits
32

23. • Promotes the growth of bifidobacterial strains and to suppress potentially pathogenic bacteria
• Bifidobacterial strains multiplied abundantly and produced ample SCFA
2´FL Promotes the Growth of Specific Bifidobacteria
Yu ZT et al. Glycobiology 2013 34
B. longum ATCC15697, B. infantis; FL, fucosylated; HMO, human milk oligosaccharide; SCFA, short-chain fatty acid.
Growth of 4 selected bacterial species after 48 h
incubation with 2’FL
Changes in SCFA production

24. 2’FL & LNnT Help Protect From GI &
Respiratory Infections
Antiadhesive and Antimicrobial effects
GI: Gastrointestinal
1.Ruiz-Palacios G et al, J Biol Chem 2003; 2. Idänpään‐Heikkilä I et al, J Infect Dis 1997. 37

25. 2´FL Inhibits Binding of Enteric Pathogens to Human Intestinal cell Lines –
In Vitro
1. Ruiz-Palacios G et al, J BiolChem 2003 . 2.Weichert et al. Nutr Res 2013
BM = human milk Caco-2 cells = human intestinal cell line Lac=lactose
EPEC (Enteropathogenic E. coli), and C. (Campylobacter) jejuni
38
Adhesion of EPEC to Caco-2 cells. Effects on
pathogen adhesion of 2´FL and BM in %
(relative adhesion) compared to lactose
Adhesion of C. jejuni to Caco-2 cells. Effects of 2´FL
and BM on pathogen adhesion in % (relative
adhesion) compared to lactose
EPEC
Percentage colonization
With 2’FL Without 2’FL

26. 2’FL and LNnT Inhibits Pathogenic Colonisation And Protects From
Respiratory Infections
HMO, human milk oligosaccharide
1.Idänpään-Heikkilä et al. JID 1997; 2. Duska-McEwen et al. FNS 2014;
Viable S. pneumonia in bronchoalveolar lavage1
LNnT reduced S. pneumoniae adhesion1
39
In vitro, LNnT and 2´FL2
LNnT decreased Influenza (IAV) viral load and 2’FL decreased Respiratory
Syncytial Virus (RSV) viral load in airway epithelial cells2

27. Immune-Modulatory Effects Attenuate
Inflammatory Pathways
41

28. 2’FL can Directly Attenuate Inflammation Stimulated By Pathogens
*P≤.05; **P≤.01
*P≤.05; **P≤.01
2’FL significantly diminished AIEC-induced stimulation of
inflammatory cytokines in vivo
2’FL significantly decreased LPS-induced
stimulation of IL-8 in vitro
2’FL, 2’-fucosyllactose; AIEC, adherent-invasive E. coli; HMO, human milk oligosaccharide; LPS, lipopolysaccharide; TNF, Tumor necrosis factor.
He Y et al, Gut 2014. 42

29. 2´FL Reduced the Severity of Food Allergy-induced Diarrhoea and
Hypothermia
OVA, oral ovalbumin.
Castillo-Courtade L et al, Allergy 2015.
43

30. Clinical study : 2’FL Added to Infant Milk Diets is Associated with Immune
Biomarkers Similar to Breastfeeding
• Infants fed with 2’FL-supplemented infant milk diets had similar levels of plasma inflammatory
cytokines to that of breastfed infants
Labelled means without a common letter are statistically significant (P≤.05)
Plasma Cytokine Levels in 6-Week-Old Infants
BF, breastfed; IL, interleukin; TNF, tumour necrosis factor.
Goehring K et al, J Nutr 2016.
45

31. • 2′FL affects cognitive performance and improves learning
Vázquez E et al., J Nutr Biochem, 20151
• 2′FL significantly improves the long-term induction and maintenance of the
hippocampus (brain area involved in memory function)
Matthies H et al., Brain Res, 19962
ENS: Enteric Nervous System
1. Vázquez E et al, J Nutr Biochem 2015; 2. Matthies H et al, Brain Res 1996; 3. Tarr A et al, Brain Behav Immun 2015.
47

32. 53
What Can These HMOs
(2’FL & LNnT) add to
Real-life Practice?

33. HMOs and Protection From Infections:
Clinical Studies
49

34. V1
1 mo
V2
2 mo
V3
3 mo
V4
4 mo
V5
6 mo
V6
12 mo
V0
≤14 d
Control infant milk diet without HMOs (Control)
Experimental infant milk diet with HMOs (Test)
Follow-up infant milk diet without HMOs
Follow-up infant milk diet without HMOs
Enrollment End of treatment End of study
V0
Informed consent
Randomization
Medical history
Clinical examination
Anthropometry
Reported AEs / medication use
V1-V6
Clinical examination
Digestive tolerance (3-day diary)
Daily record of infant milk diet intake
Anthropometry
Reported AEs / medication use
Breastfed reference group (BF)1
1A non-randomized reference group of exclusively breastfed infants was enrolled at 3 months of age for measurements of stool microbiota and fecal markers of intestinal health
Puccio et al. JPGN 2017
Exclusive feeding Complementary foods
51

35. Puccio et al. JPGN 2017
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.
Mean weight in girls and boys from 0-12 months of age,
compared with the WHO growth curve (ITT population)
52
Mean head circumference from 0 to 12 months of age,
compared with the WHO growth curve (ITT population)

36. 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
1. Puccio et al. Oral Presentation at Nutrition & Growth Conference 2016; 2. Puccio et al. JPGN 2017
• No significant difference in stool consistency between the groups, except at 2 months (softer in the infant milk diet
with HMOs (EF) vs. control infant milk diet (CF))
• No significant difference in stool frequency between the groups
• Gastrointestinal symptoms3 and behavioural patterns4 were similar between the groups
97
53
Digestive Tolerance with 2’FL & LNnT supplemented diet

37. 1. Puccio et al. Abstract at Nutr Growth Conference 2016; 2. Puccio et al. JPGN 2017
%
Infants
Infants
(%)
Bronchitis
(0-12 months)
Lower respiratory tract
infection* (0-12 months)
-55% RR* -70 % RR* -53 % RR*
*RR = risk reduction
infant milk diet with 2’FL and LnNT
infant milk diet without 2’FL and LnNT
*AE(adverse events)-cluster
Antibiotic use
(0-12 months)
%
Infants
-56 % RR*
15.9
29.9
0
5
10
15
20
25
30
35 P=0,032
Antipyretic use
(0-4 months)
%
Infants
99
54
Reduced risk of Morbidities and Medication Use

38. Caesarean-born Infants Benefit Even More from Infant Milk Diet Supplemented with 2’FL
& LNnT
1. Puccio et al. Abstract at Nutr Growth Conference 2016; 2.Puccio et al. JPGN 2017
Caesarean-born infants receiving infant milk diet with HMOs (vs. CF) had significantly fewer reports of
bronchitis and lower respiratory tract infections) through 12 months.
0
5
10
15
20
25
30
35
40
45
Bronchitis (0-12 months) Lower respiratory tract
infection (0-12 months)
34.4%
-99 % RR* -79 % RR*
%
Infants
P=0.003 P=0.022
infant milk diet with 2’FL and LnNT
infant milk diet without 2’FL and LnNT
3.1%
12.5%
40.6%
*RR = risk reduction
55

39. Caesarean-section-related Dysbiosis may Play a Role in Long-term Health Outcomes
Microbial Exposure
Immune Response
Host Genetics
Allergies
Infections
Metabolic
disorders
Autoimmune
conditions
Caesarean-section led
microbial dysbiosis
Long-term health
outcome
Salas Garcia MC, et al. Ann Nutr Metab 2018.
57

40. Conclusions: Clinical Trial with 2’FL and LNnT
1. Puccio et al. Abstract at Nutr Growth Conference 2016;2. Puccio et al. JPGN 2017
• Infant milk diet supplemented with 2’FL and LNnT supports normal age-appropriate
infant growth.
• Digestive tolerance with 2’FL and LNnT supplemented infant milk diet
• Infant milk diet supplemented with 2’FL and LNnT reduced the likelihood of parent
reported morbidity (particularly bronchitis and lower respiratory tract infections) and
medication use (particularly antibiotics, and antipyretics). These findings suggest the
addition of 2´FL and LNnT may provide immune benefits.
• Caesarean-born infants benefit even more from infant milk diet supplemented with
HMOs.
58

41. Conclusions: Clinical Trial with 2’FL and LNnT
• Infant milk diet supplemented with 2’FL and LNnT shifts global gut microbiota
composition (bacterial diversity and abundance) closer to that observed in the breastfed
reference group at 3 months.
• Infant milk diets supplemented with 2’FL and LNnT increased potentially beneficial
Bifidobacterium and decreased potentially pathogenic Escherichia and
Peptostreptococcaceae at 3 months.
• Infant milk diet supplemented with 2’FL and LNnT shifts stool metabolic signature
towards the profile of BF and,
• Shift in gut ecology of infant milk diet-fed infants towards the breastfed standard
potentially leads to clinical benefits.
108
1. Puccio et al. Abstract at Nutr Growth Conference 2016; 2. Steenhout et al. Abstract at Exp Biol Conference/ FASEB J 2016; 3. Puccio et al. JPGN 2017; 4.Berger et al. Abstract at WCPGHAN &
EAPS Congress 2016 62

42. THANK YOU
63