New generation of HMOs: From latest Research to Clinical Practice

Generation Next of HMOs: the breakthrough pre-clinical research

Ryan Carvalho, Chief Medical Officer & Global Head of Nutrition Product Development Center (NPTC) – Nestlé Nutrition

Key Messages
→ Various studies have shown that HMOs impact immune, gut and brain health. Mechanisms behind some of these benefits include the impact on microbiome, epithelial barrier functions and immune & cellular pathways.
→ New avenues of potential future benefits of HMOs seen in pre-clinical models are the impact of early life HMOs exposure on later bone health and influence on allergic outcomes.
→ Together, these studies reveal that diversity is important, and the specific structure of HMOs determines their function.

Abstract

Improved rates of exclusive breastfeeding contribute to reduced rates of respiratory and gastrointestinal infections, a reduction in overweight and diabetes later in life, probable protection from certain atopic conditions, and improved cognitive development.¹ Human milk oligosaccharides (HMOs) are an abundant group of diverse bioactives that may be generally categorized as: Nonfucosylated (core), fucosylated, sialylateds. Some of the established areas of HMO benefits include immune health, gut health and the impact on neurocognitive development. The mechanisms behind some of these benefits include impact on the microbiome, epithelial barrier function, and immune and cellular pathways. Various studies have demonstrated the mechanisms by which HMOs may impact immune outcomes. We know from preclinical models that specific HMOs can activate specific G-protein coupled receptors (GPCRs), which may in turn impact inflammation and lead to pain reduction. Lacto-N-tetraose (LNT) and 6’-O-sialyllactose (6’SL) have been shown to individually activate GPR35.2 Other studies have shown that 2-O-fucosyllactose (2’FL) and/or 6’-SL may attenuate the progression of inflammation and protect from allergy in a mouse model, and modulate TLR-4 signalling, which may lead to protection from Necrotizing Enterocolitis (NEC).³ Studies have revealed that HMO usage by infant gut bacteria seems strain specific and HMO structure specific. A seminal publication showed how certain HMOs may help to protect from Group B Streptococcus in preclinical models, while other studies have shown that HMOs may inhibit growth of pathogenic E. coli by dampening inflammation and strengthening barrier function. Certain HMOs may also modulate the immune response in certain viral exposures.^4-14

Further experimental and pre-clinical evidence support the role of HMO diversity on gut health. 6’SL and 3’-O-sialyllactose (3’SL), single and in combination, increase the adhesion of beneficial gut bacteria to human epithelial cells in vitro. The reduction of visceral pain, induced by chronic stress, was shown for a combination of 2’FL and
difucosyllactose (diFL), in an animal model. 2’FL seems to regulate gut contractibility in vitro and there is new evidence that 2’FL and LNT strengthen gut barrier function in vitro.^15-20 Specific HMOs are shown in ex vivo and animal models to increase brain sialylation, attenuate stressor induced anxiety, increase spatial and recognition memory, as well as attention.^17,18,21-28 There has also been newer evidence to suggest the beneficial impact of early life HMOs exposure on bone health.²⁹ Also gaining in promise is the immunomodulatory effects of HMOs, which suggest that HMOs may have some therapeutic potential in allergic diseases.³⁰ In conclusion, we can confidently say that diversity is important, and the structure of specific structure of HMOs influence their function in a very structure specific way

References
1. Victora CG, Bahl R, Barros AJ, França GV, Horton S, Krasevec J, Murch S, Sankar MJ, Walker N, Rollins NC; Lancet Breastfeeding Series Group. Breastfeeding in
the 21st century: epidemiology, mechanisms, and lifelong effect. Lancet. 2016. 30;387(10017):475-90. 10.1016/S0140-6736(15)01024-7.
2. Foata F, Sprenger N, Rochat F. et al. Activation of the G-protein coupled receptor GPR35 by human milk oligosaccharides through different pathways. Sci Rep 10, 16117. 2020. 10.1038/s41598-020-73008
3. Sodhi C.P, Wipf P, Yamaguchi Y. et al. Insights image for “The human milk oligosaccharides 2’-fucosyllactose and 6’-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling.”. Pediatr Res 89, 248. 2021. 10.1038/s41390-020-01184-w
4. Xiao L, Leusink-Muis T, Kettelarij N, van Ark I, Blijenberg B, Hesen NA, Stahl B, Overbeek SA, Garssen J, Folkerts G, van’t Land B. Human Milk Oligosaccharide 2’-Fucosyllactose Improves Innate and Adaptive Immunity in an Influenza-Specific Murine Vaccination Model. Frontiers in Immunology. 2018. 10.3389/fimmu.2018.00452
5. Ruiz-Palacios GM, Cervantes LE, Ramos P, Chavez- Munguia B, Newburg DS. Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1,
4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection. J Biol Chem. 2003 Apr 18;278(16):14112-20. 10.1074/jbc.M207744200.
6. Yu ZT, Nanthakumar NN, Newburg DS. The Human Milk Oligosaccharide 2’-Fucosyllactose Quenches Campylobacter jejuni-Induced Inflammation in Human Epithelial Cells HEp-2 and HT-29 and in Mouse Intestinal Mucosa. J Nutr. 2016. 10.3945/ jn.116.230706.
7. He Y, Liu S, Kling DE, et al The human milk oligosaccharide 2’fucosyllactose modulates CD14 expression in human enterocytes, thereby attenuating LPS-induced inflammation Gut. 2016. 65:33-46.

8. Angeloni S, Ridet JL, Kusy N, Gao H, Crevoisier F, Guinchard S, Kochhar S, Sigrist H, Sprenger N. Glycoprofiling with micro-arrays of glycoconjugates and lectins. Glycobiology. 2005 Jan;15(1):31-41. 10.1093/glycob/cwh143.
9. Duska-McEwen G, Senft, A, Ruetschilling, Barett E, Buck R. Human Milk Oligosaccharides Enhance Innate Immunity to Respiratory Syncytial Virus and Influenza in Vitro. Food and Nutrition Sciences. 2014. 5. 1383-1395.

10.4236/fns.2014.514151. 10. Sprenger, et al. NRC, unpublished results;
11. Kim J, Kim YJ, Kim JW. Bacterial Clearance Is Enhanced by α2,3- and α2,6-Sialyllactose via Receptor-Mediated Endocytosis and Phagocytosis.
Infect Immun. 2018 Dec 19;87(1):e00694-18. 10.1128/ IAI.00694-18.
12. Lin AE, Autran CA, Szyszka A, Escajadillo T, Huang M, Godula K, Prudden AR, Boons GJ, Lewis AL, Doran KS, Nizet V, Bode L. Human milk oligosaccharides inhibit growth of group B Streptococcus. J Biol Chem. 2017 Jul 7;292(27):11243-11249. 10.1074/jbc.M117.789974.
13. Ackerman DL, Doster RS, Weitkamp JH, Aronoff DM, Gaddy JA, Townsend SD. Human Milk Oligosaccharides Exhibit Antimicrobial and Antibiofilm Properties against Group B Streptococcus. ACS Infect Dis. 2017 Aug 11;3(8):595-605. 10.1021/ acsinfecdis.7b00064.
14. Moore RE., Xu LL., Townsend SD. Prospecting Human Milk Oligosaccharides as a Defense Against Viral Infections. ACS infectious diseases. 2021. 7(2), 254–263. 10.1021/acsinfecdis.0c00807
15. Kavanaugh DW, O’Callaghan J, Buttó LF, Slattery H, Lane J, Clyne M, Kane M, Joshi L, Hickey RM. Exposure of Bifidobacterium longum subsp. infantis to Milk Oligosaccharides Increases Adhesion to Epithelial Cells and Induces a Substantial Transcriptional Response. PLoS One. 2013 Jun 21;8(6):e67224. 10.1371/journal.pone.0067224.
16. Eutamène H, Ferrier L, Tondereau V, et al. Ingestion of human milk oligosaccharides alleviates visceral hypersensitivity induced by chronic stress. Abstract at the 6th WCPGHAN, June 2020, Copenhagen,
Denmark.
17. Bienenstock J, Buck RH, Linke H, Forsythe P, Stanisz AM, Kunze WA. Fucosylated but not sialylated milk oligosaccharides diminish colon motor contractions.
PLoS One. 2013 Oct 2;8(10):e76236. 10.1371/journal. pone.0076236.
18. Farhin S, Wong A, Delungahawatta T, Amin JY, Bienenstock J, Buck R, Kunze WA. Restraint stress induced gut dysmotility is diminished by a milk oligosaccharide (2’-fucosyllactose) in vitro. PLoS One. 2019 Apr 24;14(4):e0215151. 10.1371/journal. pone.0215151.
19. Natividad JM, Rytz A, Bergonzelli G, Garcia-Rodenas CL. Human milk oligosaccharides confer resistance against inflammation-mediated intestinal epithelial
barrier dysfunction in-vitro. Abstract at the 6th WCPGHAN, June 2020, Copenhagen, Denmark.
20. Natividad JM, Rytz A, Rochat F, Garcia-Rodenas CL. Microbial products derived from human milk oligosaccharides fermentation by infant microbiota
protect against inflammation-mediated intestinal epithelial barrier dysfunction in-vitro. 2020. Abstract at the 6th WCPGHAN, June 2020, Copenhagen, Denmark.
21. Henry M, Sabine S, Manfred K. Fucose and fucosyllactose enhance in-vitro hippocampal longtermmpotentiation, Brain Research, 1996. 276-280.1016/0006-8993(96)00406-4.
22. Oliveros E, Vázquez E, Barranco A, Ramírez M, Gruart A, Delgado-García JM, Buck R, Rueda R,
Martín MJ. Sialic Acid and Sialylated Oligosaccharide Supplementation during Lactation Improves  Learning and Memory in Rats. Nutrients. 2018 Oct 16;10(10):1519. 10.3390/nu10101519.
23. Tarr AJ, Galley JD, Fisher SE, Chichlowski M, Berg BM, Bailey MT. The prebiotics 3’Sialyllactose and 6’Sialyllactose diminish stressor-induced anxiety-like
behavior and colonic microbiota alterations: Evidence for effects on the gut-brain axis. Brain Behav Immun. 2015 Nov;50:166-177. 10.1016/j.bbi.2015.06.025.
24. Sakai Y, Kumano H, Nishikawa M, Sakano Y, Kaiya H, Imabayashi E, Ohnishi T, Matsuda H, Yasuda A, Sato A, Diksic M, Kuboki T. Changes in cerebral glucose
utilization in patients with panic disorder treated with cognitive-behavioral therapy. Neuroimage. 2006 Oct 15;33(1):218-26. 10.1016/j.neuroimage.2006.06.017
25. Jacobi SK, Yatsunenko T, Li D, Dasgupta S, Yu RK, Berg BM, Chichlowski M, Odle J. Dietary Isomers of Sialyllactose Increase Ganglioside Sialic Acid Concentrations in the Corpus Callosum and Cerebellum and Modulate the Colonic Microbiota of Formula-Fed Piglets. J Nutr. 2016 Feb;146(2):200-8. doi: 10.3945/jn.115.220152.
26. Vázquez E, Barranco A, Ramírez M, Gruart A, Delgado-García JM, Martínez-Lara E, Blanco S, Martín MJ, Castanys E, Buck R, Prieto P, Rueda R. Effects of a human milk oligosaccharide, 2’-fucosyllactose, on hippocampal long-term potentiation and learning capabilities in rodents. J Nutr Biochem. 2015 May;26(5):455-65. 10.1016/j.jnutbio.2014.11.016.
27. Pisa, E, Tomasi, F, Simone, M, Hauser, J. Modulation of 3’SL level in pre-weaning milk impact attention, learning and memory in the adult offspring. Nutrition. 2019
28. Hauser J, Pisa E, Arias Vásquez A et al. Sialylated human milk oligosaccharides program cognitive development through a non-genomic transmission
mode. Mol Psychiatry. 2021. 10.1038/s41380-021- 01054-9
29. Bonnet N, Mletzko K, Baruchet M, Ramos-Nieves M, Favre L, Brassart D, Schmidt FN, Busse B, Koopmans JS, Horcajada MN. Preclinical supplementation of
Human Milk Oligosaccharides (HMOs) during early life positively impacts long-term bone quality. ASBMR Annual Meeting, 2020.
30. Castillo-Courtade L, Han S, Lee S, Mian FM, Buck R, Forsythe P. Attenuation of food allergy symptoms following treatment with human milk oligosaccharides in a mouse model. Allergy. 2015 Sep;70(9):1091-102. 10.1111/all.12650.

Do more HMOs translate into increased benefits

Yvan Vandenplas, Professor Emeritus, Department of Pediatrics, University Hospital Brussels

Key Messages
→ The more we learn about HMOs, the greater the evidence becomes that they are essential for balanced nutrition during infancy.
→ Since approximately 5 years ago it became possible to industrially produce 2’fucosyllactose (2’FL), in an identical structure to the 2’FL present in mother’s milk.
→ Recently, synthesization of more HMOs has become possible. The first clinical trial, with a blend of 5 HMOs (2’FL, difucosyllactose (DFL), Lacto-N-tetraose (LNT), 3’-Sialyllactose (3’SL), has shown them to be safe and well tolerated by healthy infants.

Abstract

Human milk oligosaccharides (HMOs) are the third most important component in mother’s milk, they are virtually absent in cow’s milk and thus also absent in cow’s milk
based infant formula. HMOs are known to stimulate the growth of the health promoting species bifidobacteria and lactobacilli in the gastrointestinal (GI) microbiome. In comparison to breastfed infants, the GI microbiome of unsupplemented formula fed infants has low numbers of bifidobacteria and lactobacilli.  The addition of prebiotic oligosaccharides such as fructooligosaccharides, galacto-olgosaccharides and inulin were shown to stimulate the growth of these health promoting species, and brought the composition of the GI microbiome of formula fed infants closer to that of breastfed infants. Approximately 5 years ago it became possible to industrially produce 2’fucosyllactose (2’FL), identical in structure to the 2’FL present in mother’s milk. The first studies showed the benefit of the addition of 2’FL to infant formula on the GI microbiota and on the immune response. Soon after, a second oligosaccharide, Lacto-N-neotetraose (LNnT), could be produced as well, and the first clinical trials with the addition of this specific mixture were started.

The results of this trial showed a reduction of bronchitis, lower respiratory tract infection and use of antibiotics and antipyretics. This beneficial effect persisted for at least
six months longer (up to the age of 12 months) than the intervention period (up to the age of 6 months).¹ This trial was followed by trials in infants with cow’s milk allergy, treated with a whey based extensively hydrolyzed formula (w-eHF) showing comparable benefit. The addition of both HMOs (2’FL and LNnT) to a w-eHF for at least four months was shown to be well tolerated, to result in normal growth,² and appeared to better reduce infective episodes (lower incidence of gastroenteritis (-40%) and lower and upper respiratory tract (-33.6%; -5.2%) infections, otitis media (-70%)).2 According to the safety analysis set, including 190 infants, the estimated incidence of upper respiratory tract infections was reduced with 42 %, and the decrease for lower respiratory tract infections was -23%.³ Use of antibiotics was reduced by 20%. The same mixture of two HMOs was also shown to be safe and well tolerated in preterm infants.4 Recently, synthesization of more HMOs has become possible, resulting in a blend of 5 HMOs (2’FL, difucosyllactose (DFL), Lacto-N-tetraose (LNT), 3’-Sialyllactose (3’SL) and 6’SL). This new blend was tested in infant formula and was shown to be very safe and well tolerated, resulting in normal growth (5). These 5 specific HMOs were chosen because they are amongst the most abundant HMOs in breast milk, and represent together the 3 HMO families in breast milk.

References
1. Puccio G, et al., Effects of infant formula with human milk oligosaccharides on growth and morbidity: a randomized multicenter trial. J Pediatr
Gastroenterol Nutr, 2017. 64: p. 624-631.JPGN 2017;64: 624–631
2. Vandenplas Y et al. Growth tolerance and safety of an extensively hydrolyzed formula containing two human milk oligosaccharides in infants with cow’s
milk protein allergy. Oral abstract presentation. EAACI PAAM, 17-19 October, 2019.
3. Vandenplas Y et. al. Extensively hydrolysed formula with two human milk oligosaccharides reduces the rate of upper respiratory tract infections in infants
with cow’s milk allergy. Oral abstract presentation. EAACI Congress. 2020
4. Hascoet JM et al. Effect of a liquid supplement containing 2 human milk oligosaccharides (hmos) in preterm infants: a multi-centered, double-blind,
randomized, controlled trial. Abstract presentation. 6th WCPGHAN 2021
5. Bauer V et. Al. Term infant formula supplemented with a unique blend of five human milk oligosaccharides supports age-appropriate growth, is safe and well-tolerated: A double-blind, randomized controlled trial. Abstract presentation. Pediatric Academic Societies Conference 2021 (EP-218.2048)

Advancing the management of cow’s milk protein allergy: From research to clinical practice

Anna Nowak-Wegrzyn, Professor of Pediatrics at the New York University School of Medicine

Key Messages
→ Initial studies in healthy infants established that formulas with HMO (2’FL and LNnT) were well tolerated and supported normal growth.
→ In a multi-center study, a whey-based extensively hydrolyzed formula (w-eHF) containing 2’FL and LNnT, was shown to be tolerated by more than 90% of the infants and young children with IgE-mediated CMPA.
→ These studies demonstrated the safety and efficacy of the novel hypoallergenic w-eHF with 2’FL and LNnT in infants with CMPA and showed an important benefit of reducing the frequency of upper respiratory tract infections and ear infections.

Abstract

Cow’s milk proteins (CMP) are among the most common food allergens in infancy.¹ Dietary avoidance of CMP is the mainstay of management of CMP allergy (CMPA). When breastfeeding is not possible, hypoallergenic formulas are indicated for CMPA. The new generation of hypoallergenic infant formulas (whey based extensively hydrolysed formula (w-eHF) and amino acid based formula (AAF) contain human milk oligosaccharides (HMO). HMO are non-digestible oligosaccharides abundant in human milk. HMO act as prebiotics, serving as nutrients for non-pathogenic bacteria (e.g. bifidobacteria) that metabolize them to tolerogenic short chain fatty acids. HMO also acts as decoys for pathogenic bacteria restricting their access to intestinal epithelium. Initial studies in healthy infants established that formulas with two
HMO, 2’fucosyl-lactose (2’FL) and lacto-N-neotetraose (LNnT) were well tolerated and supported normal growth.² In addition, in secondary analyses, feeding formula with 2’FL and LNnT since birth was associated with significant reduction in frequency of infections, the use of antibiotics and antipyretics in the first year of life, compared to formula without HMO.²

Following this important evidence generated in healthy infants, studies were initiated in infants with CMPA. In a multi-center study, a w-eHF containing 2’FL and LNnT was shown to be tolerated by more than 90% of the infants and young children with IgE-mediated CMPA, thus meeting the American Academy of Pediatrics criteria for formula hypoallergenicity.³ A recent international multi-center, controlled, double-blinded and randomized clinical trial showed that the w-eHF with 2’FL and LNnT supported normal growth in infants with CMPA.⁴ It was also associated with a significant reduction in the frequency of upper respiratory tract infections and otitis media.^4,5 Furthermore, w-eHF with 2F’L and LNnT was associated with positive modulation of the gut microbiota. A lower alpha diversity of stool microbiota at 12 months was observed, with delayed colonization with bacteria such as Firmicutes and Bacteroidetes. It was, however, associated with more persistent colonization with Actinobacteria (phylum including Bifidobacteriales) and Proteobacteria.⁶ These studies demonstrated the safety and effcacy of the novel hypoallergenic w-eHF with 2’FL and LNnT in infants with CMPA and showed an important benefit of reducing the frequency of upper respiratory tract infections in the first year of life as well as positively modulating gut microbiota composition.

References
1. Renz H, Allen K, Scherer S et al. Food Allergy, Nature Reviews Disease Primers 2018 4 17098 doi:10.1038/nrdp.2017.98
2. Puccio G, Alliet P, Cajozzo C et al. Effects of Infant Formula With Human Milk Oligosaccharides on Growth and Morbidity: A Randomized Multicenter Trial. J Ped Gastro Nutr. 2017;64: 624–631 doi: 10.1097/MPG.0000000000001520
3. Nowak-Wegrzyn A, Czerkies L, Reyes K, Collins B, Heine RG. Confirmed Hypoallergenicity of a Novel Whey-Based Extensively Hydrolyzed Infant Formula Containing Two Human Milk Oligosaccharides. Nutrients. 2019 Jun 26;11(7). pii: E1447. doi: 10.3390/nu11071447. PMID: 31248026
4. Vandenplas Y et al. Growth tolerance and safety of an extensively hydrolyzed formula containing two human milk oligosaccharides in infants with cow’s milk protein allergy. Oral abstract presentation. EAACI PAAM, 17-19 October, 2019.
5. Vandenplas Y, Żołnowska M, Berni Canani R, Ludman S Tengelyi Z, Moreno Álvarez A, Goh A, Gosoniu ML, Tadi, Heine RG. Extensively hydrolysed formula with two human milk oligosaccharides reduces rate of upper respiratory tract infections in infants with cow’s milk allergy. EAACI Digital Congress 2020, late breaking oral abstract 1885.
6. Pedersen HK et al. An extensively hydrolysed formula supplemented with two human milk oligosaccharides (HMO) shapes the gut microbiome in infants with cow’s milk protein allergy. Poster abstract presentation EAACI FAAM October 2020.