CA2852589A1 - Immunomodulatory compositions and uses thereof - Google Patents

Abstract

Polynucleotides comprising immunosuppressive motifs and their use to supplement food products in order to improve immune system function in a subject, is provided.
The food products may be infant formula, breast milk, nutritional supplements or other food products. The use of the polynucleotides in cosmeceutical compositions is also provided.
Such cosmeceutical compositions may be particularly useful for subjects suffering from an immune-related skin condition.

Description

IMMUNOMODULATORY COMPOSITIONS AND USES THEREOF
FIELD OF THE INVENTION
[00011 The present invention relates to the field of immunology and, in particular, to immunomodulatory compositions comprising polynueleotides including immunosuppressive motifs.
BACKGROUND OF THE INVENTION

[0002] The benefits of human milk compared to the use of commercial infant formulas are largely realized because of its bioactive components, including prebiotics, immune proteins and the microbiome of human milk itself. Breastfeeding is associated with a decreased incidence of gastrointestinal (GI) tract infections [1,2], which is corroborated by several studies that have correlated breastfeeding with a lower incidence of necrotizing enterocolitis in humans and animal models [3-5]. Breastfeeding is also associated with an altered fecal microbiome; two studies showed at two weeks of age over 90% of the total fecal bacteria of a breast-fed (BF) infant is Bifidobacteria, whereas in most formula-fed (FF) infants Bifidobacteria is non-detectable [6,7]. Because the community of gut-colonizing bacteria prevents adhesion and colonization of pathogenic bacteria whilst stimulating mueosal cell proliferation and enhancing immune development, the types of predominant bacteria in the fecal microbiome of the developing infant can affect the health outcomes of the individual, as has been discussed in a recent review article [8].
10003] Human milk bacteria have been analyzed by culture-dependent and -independent mechanisms, confirming the presence of a magnitude of bacterial phylotypes [13-20]. In one study, Staphylococcus and Streptococcus dominated the milk microbiome of most mothers, whereas commercially well known bovine milk associated genera, Lactobacillus and Bilidobacterium, contributed as minor milk microbiota members (2-3% of genera) [17]. Another study showed that the human milk microbiome changes over time, and may be dependent on the mother's weight and the baby's mode of delivery [20]. Most recent methods for determining the milk microbiome have included amplification of 16S ribosomal RNA genes (rRNA) followed by pyrosequencing [17,20]. Although this technique is widely accepted as a means to determine microbial diversity, it does present limitations such as a lack of information on the functional capacity of the microbes within the milk matrix and also prevents data accumulation on the types of DNA motifs to which an infant is exposed.
[0004] Unmethylated cytosine phosphate guanine (CpG) dinucleotides within bacterial DNA
are known as potent immune stimulators, acting through toll-like receptor 9 [9]. Conversely, immune suppressive motifs including polyguanosine or guanosine cytosine-rich sequences, such as those on the telomere region of mammalian DNA, that can block immune activation induced by CpGs [10]. Recently, immune suppressive motifs (TTAGGG and TCAAGCTTGA) that are able to counter the effects of CpGs have been identified in Lactobacillus [11].
[0005] U.S. Patent Application Publication No. 2006/0089326 describes nucleic acids containing unmethylated CpG dinucleotides and their use to stimulate an immune response, and to redirect a Th2 response to a Th 1 response. Methods for treating atopic diseases are also described. U.S. Patent Application Publication No. 2011/0201672 describes specific C-type semi-soft immunostimulatory oligonucleotides that are useful for stimulating an immune response, in particular, for treating allergy, cancer and infectious diseases.
[0006] U.S. Patent Application Publication No. 2011/0201676 describes oligodeoxynucleotides comprising multiple guanosine-rich regions or TTAGGG motifs and their use to suppress immune activation. Methods of treating arthropathies, in particular by intra-articular injection, are also described. U.S. Patent Application Publication No. 2012/0258144 also describes oligodeoxynucleotides comprising multiple guanosine-rich regions or TTAGGG
motifs, and further describes the use of these oligodeoxynucleotides to treat or prevent autoimmune diseases.
[0007] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION
[0008] The present invention relates to immunomodulatory compositions and uses thereof. In one aspect, the invention relates to a composition formulated for oral or topical administration comprising an isolated polynucleotide, the isolated polynucleotide comprising deoxyribonucleotides and one or more immunosuppressive motifs.
[0009] In an aspect, the invention relates to an infant formula comprising the composition described above, and one or more of a protein source, a fat source and a carbohydrate source.
[0010] In another aspect, the invention relates to a nutritional supplement comprising the composition described above, and one or more of a protein source, a fat source and a carbohydrate source.
[0011] Additionally, the invention relates to a cosmetic formulation comprising the composition described above and one or more of a cosmetically acceptable carrier, diluent and excipient.
100121 The invention provides a method for enhancing immune system development in an infant comprising orally administering to the infant the composition described above.
[0013] The invention also relates to a method of improving immune system function in a subject comprising orally administering to the subject the composition described above.
[0014] In an aspect, the invention relates to a method of ameliorating a skin condition in a subject comprising topically administering to the subject the composition described above.
[0015] In another aspect, the invention relates to a use of a composition comprising an isolated polynucleotide as a nutritional supplement, the isolated polynucleotide comprising deoxynucleotides and one or more immunosuppressive motifs.
[0016] The invention also relates to a use of a composition comprising an isolated polynucleotide to supplement human breast milk, the isolated polynucleotide comprising deoxyribonucleotides and one or more immunosuppressive motifs.

3 BRIEF DESCRIPTION OF THE DRAWINGS
[0017] These and other features of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings.
[0018] Figure 1: Best hit analysis of 51 bp DNA sequences from human milk. DNA
from human milk was sequenced using 11lumina sequencing followed by alignment to known prokaryotic genomes. Sequences (75,521,216) were BLATed against 1,731 known prokaryotic genomes imported from NCBI (min 95% identity), with 1,331,996 sequences aligning to 370 prokaryotic genera. Other refers to genera each representing <0.1% of all sequences.
[0019] Figure 2: Pair-wise comparison of categorized open reading frames from human milk versus infants' and mothers' feces. Pair-wise comparisons for the human milk metagenome versus (A) breast-fed infants' feces, (B) formula-fed infants' feces and (C) mothers feces are shown. For comparison, a plot of breast-fed infants' feces and formula-fed infants' feces (D) is also shown. Each point represents a different SEED subsystem and its relative abundance within the human milk metagenome compared to the fecal metagenomes. Points lying on or near the dotted line have equal or similar abundance in both metagenomes. Points closer to the x-axis are more abundant in the feces metagenome, whereas points closer to the y-axis are more abundant in the human milk metagenome. Red dots signify those with significantly different proportions between the two metagenomes (Student's t-test, P < 0.05). Breast-fed and formula-fed infants' feces values are an average of five individuals, and mothers' feces values are an average of three individuals. All subjects are unrelated.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention is based on the finding that human breast milk comprises both CpG immunostimulatory DNA sequences and immunosuppressive DNA sequence motifs.
This finding suggests that, while ingestion of viable bacteria in breast milk may lead to effective colonization of the infant gastrointestinal tract, it is the DNA complement of breast milk that contributes to development of the infant immune system. Accordingly, in certain embodiments, the invention relates to supplementation of breast milk or infant formula with polynucleotides comprising immunosuppressive motifs, such as those found naturally in breast milk, in order to

4 aid proper immune development in infants. In certain embodiments, supplementation of breast milk or infant formula with polynucleotides comprising immunosuppressive motifs together with polynucleotides comprising immunostimulatory motifs is also contemplated.
[0021] Additional embodiments of the invention relate to the use of polynucleotides comprising immunosuppressive motifs to supplement other food products in order to improve immune system function in a subject consuming the food product. Further embodiments contemplate the use of the polynucleotides in cosmeceutical compositions. Such cosmeceutical compositions may be particularly useful for subjects suffering from immune-related skin conditions such as inflammation, psoriasis and eczema.
Definitions [0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[0023] As used herein, the term "about" refers to an approximately +/-10%
variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
[0024] The term "plurality" as used herein means more than one, for example, two or more, three or more, four or more, and the like.
[0025] "Naturally occurring," as used herein, as applied to an object, refers to the fact that an object can be found in nature. For example, an organism, or a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
[0026] The term "isolated," as used herein with reference to a material, means that the material is removed from its original environment (for example, the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide separated from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such

5 polynucleotides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
[0027] The use of the word "a" or "an" when used herein in conjunction with the term "comprising" may mean "one," but it is also consistent with the meaning of "one or more," "at least one" and "one or more than one."
[0028] As used herein, the terms "comprising," "having," "including" and "containing," and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps. The term "consisting essentially of' when used herein in connection with a composition, use or method, denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term "consisting of' when used herein in connection with a composition, use or method, excludes the presence of additional elements and/or method steps. A composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
[0029] It is contemplated that any embodiment discussed herein can be implemented with respect to any disclosed method or composition, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve the disclosed methods.
POLYNUCLEOTIDES
[0030] The invention relates to polynucleotides comprising one or more immunosuppressive motifs, such as those found in human breast milk. Preferably, the polynucleotides are polydeoxynucleotides. As shown herein, DNA extracted from human breast milk contains the immunosuppressive motifs TTAGGG and TCAAGCTTGA. Accordingly, in certain embodiments, the polynucleotide may comprise one or more immunosuppressive motifs selected from TTAGGG and TCAAGCTTGA. In some embodiments, the polynucleotide may include a plurality of immunosuppressive motifs, for example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 motifs. In some embodiments, the polynucleotide may include a plurality of immunosuppressive motifs,

6 the plurality comprising one or more of TTAGGG and one or more of TCAAGCTTGA.
In some embodiments, the polynucleotide may include a plurality of immunosuppressive motifs, the plurality comprising two or more TTAGGG motifs, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 motifs. In some embodiments, the polynucleotide may include a plurality of immunosuppressive motifs, the plurality comprising two or more TCAAGCTTGA motifs, for example, 2, 3, 4, 5, 6,

7, 8, 9 or 10 motifs.
[0031] The polynucleotides are typically between about 10 and about 500 base pairs in length, or any amount therebetween, for example, between about 10 and about 400, or any amount therebetween, between about 10 and about 300, or any amount therebetween, between about 10 and about 200, or any amount therebetween, or between about 10 and about 100, or any range therebetween. In certain embodiments, the polynucleotides are between about 10 and 50 base pairs, for example, between about 12 and 50 base pairs, between about 15 and 50 base pairs, or between about 20 and about 50 base pairs, or any range therebetween.
[0032] The polynucleotides may be synthetic or they may be isolated from a natural source, for example, from breast milk. In certain embodiments, the polynucleotides are synthetic. In some embodiments, the polynucleotides are isolated from breast milk, for example, from human breast milk.
[0033] Polynucleotides isolated from breast milk may be purified or partially purified. Certain embodiments of the invention contemplate polynucleotide preparations that comprise a mixture of DNA sequences isolated from breast milk. Methods for isolating and purifying DNA
sequences from natural sources are well-known in the art (see, for example, Ausubel et al. (1994 & updates) Current Protocols in Molecular Biology, John Wiley & Sons, New York). The isolated DNA may be screened for the presence of one or more immunosuppressive motifs by techniques well-known in the art, for example, PCR-based techniques and/or hybridization-based techniques, including Southern blotting and the use of oligonucleotide arrays.
The DNA may optionally be fractionated to obtain polynucleotides within a certain size range, and may further optionally be enriched by selection of certain sequences. For example, the DNA
may be enriched for polynucleotides comprising immunosuppressive motifs. In certain embodiments, the DNA
may be enriched for polynucleotides comprising immunosuppressive motifs, and also contains polynucleotides comprising immunostimulatory motifs. In certain embodiments, the polynucleotides comprising immunosuppressive motifs are purified such that the final polynucleotide preparation is substantially free of other polynucleotides that do not comprise immunosuppressive motifs.
[0034] In certain embodiments, the polynucleotides are synthetic sequences. In accordance with these embodiments, the polynucleotides may optionally be modified, for example, to improve stability or bioavailability. Methods of synthesizing polynucleotides are well-known in the art (see, for example, Ausubel et al., ibid.). Various modifications to improve stability, half-life, bioavailability, and the like are also well-known in the art. Examples include, but are not limited to, phosphate backbone modifications and/or nucleotide modifications (for example, modifications to the sugar moiety or the nucleoside moiety).
[0035] Examples of modified backbones include, but are not limited to inclusion of one or more phosphorothioate linkages (for example, at least two phosphorothioate linkages at the 5' end of the polynucleotide and multiple (for example, five) phosphorothioate linkages at the 3' end), phosphodiester-modified nucleic acids, arylphosphonates, alkylphosphorothioates, arylphosphorothioate, methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, morpholino, and combinations thereof.
[0036] Examples of modified backbone sugars include those which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Other examples include 2'-0-alkylated ribose groups such as 2'-0-methylribonucleosides (2.-0Me) (Zhao et al. (1999) Bioorg Med Chem Lett 9:24:3453-8). Combinations of 2*-0Me ribose modifications and phosphorothioates may also be used. Other sugar modifications include the use of sugars such as arabinose instead of ribose.
[0037] Nucleoside modifications include replacement of a natural nucleoside by a modified nucleoside, for example hypoxanthine; dihydrouracil; pseudouracil; 2-thiouracil; 4-thiouracil; 5-aminouracil; 5-(C_{1-C 6})-alkyluracil; 5-( C2-C6)-alkenyluracil; 5-(C2-C6)-alkynyluracil;
5-(hydroxymethyl)uracil; 5-chlorouracil; 5-fluorouraci1; 5-bromouracil; 5-hydroxycytosine; 5-(C1-C6)-alkyleytosine; 5-(C2-C6)-alkenylcytosine; 5-(C2-C6)-alkynylcytosine; 5-chlorocytosine;
5-fluorocytosine; 5-bromocytosine; N2-dimethylguanine; 2,4-diamino-purine; 8-azapurine

8 (including, in particular, 8-azaguanine); a substituted 7-deazapurine (including, 7-deazaguanine, as well as 7-deaza-7-substituted and/or 7-deaza-8-substituted purine); and the like.
[0038] In certain embodiments, the polynucleotides may be associated with a delivery vehicle, for example, to stabilize the polynucleotides, to improve in vivo delivery of the polynucleotides, or to target the polynucleotides to specific cells or tissues. Examples of delivery vehicles include, but are not limited to, lipids, sterols, liposomes, nanoparticles, and the like, as is known in the art.
COMPOSITIONS
[0039] In one aspect, the invention relates to compositions comprising one or a plurality of the polynucleotides comprising one or more immunosuppressive motifs. In particular, the invention relates to compositions formulated for oral or topical administration.
Compositions formulated for tube feeding are also contemplated in some embodiments.
[0040] In its simplest form, the composition may comprise one or a plurality of the polynucleotides comprising one or more immunosuppressive motifs and a physiologically acceptable carrier. In certain embodiments, the composition may further comprise one or more polynucleotides comprising immunostimulatory motifs, such a CpG motifs.
[0041] In certain embodiments, the composition comprises a single polynucleotide, which may comprise one or a plurality of immunosuppressive motifs. In some embodiments, the polynucleotide comprised by the composition comprises at least two immunosuppressive motifs, for example between about 2 and about 10, between about 2 and about 6, or about 4 immunosuppressive motifs.
[0042] In certain embodiments, the composition comprises a plurality of isolated polynucleotides, at least one of which comprises one or more immunosuppressive motifs. The plurality of polynucleotides may be synthetic polynucleotides or they may have been isolated from a natural source. In some embodiments, the plurality of polynucleotides in the composition is provided by a DNA preparation extracted from breast milk, such as human breast milk, which preparation may be partially or substantially pure. Using a DNA preparation from breast milk may provide an additional advantage in that the composition will then also include any further

9 beneficial immunomodulatory sequences that may be found in breast milk. In certain embodiments, the DNA preparation may further comprise at least one or more polynucleotides comprising immunostimulatory motifs.
[0043] In certain embodiments, the composition is formulated for oral administration and may optionally include other actives or nutrients. For example, in some embodiments, the composition may be formulated as a food product or nutritional supplement and the composition may optionally include a protein source, a fat source and/or a carbohydrate source.
[0044] In certain embodiments, the composition is an infant formula, a follow-on formula, or a toddler beverage and comprises one or a plurality of the polynucleotides comprising one or more immunosuppressive motifs and one or more of a protein source, a fat source and/or a carbohydrate source. One or more of the protein source, fat source and carbohydrate source may be provided by human breast milk. Alternatively or in addition, the protein source may be whey protein, bovine milk or soy protein or any of their derivative products, for example cheese. The infant formula, a follow-on formula, or a toddler beverage may further optionally include other nutritional or active components, for example, one or more of a probiotic, a prebiotic, fatty acids, oligosaccharides, and the like. The infant formula, a follow-on formula, or a toddler beverage may be formulated as a liquid or it may be in solid form, for example as a powder or granules, for reconstitution in a liquid.
[0045] In some embodiments, the composition is an infant formula. In some embodiments, the composition is an infant formula for neonates.
[0046] In certain embodiments, the composition is a nutritional supplement product. In certain embodiments, the composition is a nutritional supplement product that comprises one or a plurality of polynucleotides comprising one or more immunosuppressive motifs and optionally one or more of a protein source, a fat source and/or a carbohydrate source.
[0047] Non-limiting examples of protein sources include dairy based proteins, plant based proteins, animal based proteins and artificial proteins. Dairy based proteins may include, but are not limited to, casein, caseinates, casein hydrolysate, whey, whey hydrolysates, whey concentrates, whey isolates, milk protein concentrate, milk protein isolate, or combinations thereof. Examples of plant based proteins include, for example, soy protein (including soy protein concentrate and soy protein isolate), pea protein (including concentrate and isolate), canola protein (including concentrate and isolate), wheat and fractionated wheat proteins, corn protein and its fractions (including zein), rice protein, oat protein, potato protein, peanut protein, and combinations thereof. Other examples include proteins derived from beans, buckwheat, lentils, pulses, chenopods (such as quinoa), single cell proteins, or combinations thereof. Animal based proteins may include, for example, beef, poultry, fish, lamb, seafood, or combinations thereof.
[0048] Examples of carbohydrates that may be included in the nutritional supplements include, but are not limited to, sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, modified starch, amylose starch, tapioca starch, corn starch, gums, agar, karageenans, or combinations thereof.
[0049] Examples of fats that may be included in the nutritional supplements include, but are not limited to, vegetable fat (such as olive oil, corn oil, sunflower oil, high-oleic sunflower, rapeseed oil, canola oil, hazelnut oil, soy oil, palm oil, coconut oil, blackcurrant seed oil, walnut oil, borage oil, lecithins, and the like), animal fats (such as milk fat), and combinations thereof.
[0050] In certain embodiments, the nutritional supplement product may optionally comprise one or more of: grains (for example, whole grains); prebiotics; probiotics;
fibre; amino acids;
antioxidants; vitamins; minerals, and/or a source of omega-3 and/or omega-6 fatty acids.
[0051] Prebiotics are food substances that promote the growth of beneficial bacteria in the gut.
Non-limiting examples of prebiotics include acacia gum, alpha glucan, arabinogalactans, beta glucan, dextrans, fructooligosaccharides, fucosyl lactose, galactooligosaccharides, galactomannans, gentiooligosaccharides, glucooligosaccharides, guar gum, inulin, isomaltooligosaccharides, lactoneotetraose, lactosucrose, lactulose, levan, maltodextrins, milk oligosaccharides, partially hydrolyzed guar gum, pecticoligosaccharides, resistant starches, retrograded starch, sialooligosaccharides, sialyllactose, soy oligosaccharides, sugar alcohols, xylooligosaccharides, or their hydrolysates, or combinations thereof.

[0052] Probiotics are food-grade live microorganisms (including semi-viable or weakened, and/or non-replicating) intended to beneficially affect a host by improving the intestinal microbial balance. Non-limiting examples of probiotics include Aerococcus, Akkertnansia, Aspergillus, Bacteroides, Bifidobacterium, Candida, Clostridium, Debaromyces, Enterococcus, Fusobacterium, Lactobacillus, Lactococcus, Leuconostoc, Melissococcus, Micrococcus, Mucor, Oenococcus, Pediococcus, Penicillium, Peptostreptococcus, Pichia, Propionibacterium, Pseudocatenulatum, Rhizopus, Saccharomyces, Staphylococcus, Streptococcus, Torulopsis, Weissella, and combinations thereof. Most commonly used probiotics include strains of Bacillus, Bifidobacterium, Lactobacillus and Streptococcus, including, but not limited to, Bacillus coagulans, Bifidobacterium lactis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus bulgaricus and Streptococcus thermophilus. In certain embodiments, when the nutritional supplement products comprise probiotics, the probiotics are selected from strains of Bacillus, strains of Bifidobacterium, strains of Lactobacillus, strains of Streptococcus, and combinations thereof.
[0053] The nutritional supplement products may further optionally comprise one or more conventional food additives (synthetic or natural), for example one or more acidulants, additional thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipients, flavor agents, osmotic agents, preservatives, stabilizers, sweeteners, texturizers, and the like, which may be added in conventional amounts.
[0054] The nutritional supplements may be a source of either incomplete or complete nutrition.
For example, in some embodiments the nutritional supplements may be intended to supplement a regular diet that provides all or the majority of essential nutrients. In some embodiments, the nutritional supplements are intended to supplement an inadequate diet. In some embodiments, the nutritional supplements are intended to provide complete nutrition, for example, as total parenteral nutrition (TPN) preparations.
[0055] The nutritional supplements may be formulated for administration orally or by tube feeding. In certain embodiments, the nutritional supplements may be formulated as a liquid or in solid form, for example as a powder or granules, for reconstitution in a liquid, or as a bar.

[0056] In certain embodiments, the composition is a cosmetic formulation comprising one or more of the polynucleotides comprising one or more immunosuppressive motifs and one or more of a cosmetically acceptable carrier, diluent and excipient. The cosmeceutical formulation may be, for example, an exfolient, mask, ointment, lotion, gel, cream, pomade, skincream, skinmilk, skinserum, or the like. The cosmeceutical formulation may optionally contain materials normally present in personal care or healthcare formulations, such as emollients, inorganics, humectants, moisturizers, preservatives, pH adjusters, proteins, natural peptides, synthetic peptides, herbal extracts, carriers/solvents, soothing/cooling agents, antioxidants, perfumes, emulsifiers, viscosity modifiers, and/or lipids such as triglycerides, phospholipids and sphingolipids. In certain embodiments, the cosmeceutical composition may optionally further comprise one or more other active ingredients, for example, anti-inflammatory compounds, sunscreen, vitamins, essential fatty acids, sphingolipids, self-tanning compounds, whitening agents such as kojic acid and arbutin, and/or antimicrobials.
USES
[0057] The polynucleotides comprising one or more immunosuppressive motifs and compositions comprising same may be used to supplement food products, including breast milk and infant formulas, in order to improve immune system function in a subject consuming the food product. The polynucleotides may also be used in cosmeceutical compositions to alleviate immune-related skin conditions such as psoriasis, inflammation, and eczema.
[0058] Certain embodiments relate to methods and uses of the polynucleotides comprising one or more immunosuppressive motifs or compositions comprising same to enhance immune system development in an infant. The polynucleotides may optionally be administered in combination with one or more of probiotics, oligosaccharides, prebiotics and/or fatty acids. In some embodiments, the polynucleotides may be administered orally as part of an infant formula or enriched breast milk. In certain embodiments, the infant is a neonate.
[0059] Certain embodiments of the invention relate to the use of the polynucleotides comprising one or more immunosuppressive motifs or compositions comprising same to supplement human breast milk. The breast milk may be, for example, banked, stored or freshly pumped breast milk.

[0060] Certain embodiments of the invention relate to methods and uses of the polynucleotides comprising one or more immunosuppressive motifs or compositions comprising same to improve immune system function in a subject. The subject may be an infant, a child or an adult. The polynucleotides may be administered orally or topically. In certain embodiments, the polynucleotides are administered orally as part of a nutritional supplement product, for example, a beverage, a shake, a smoothie, a sports drink or a bar. In certain embodiments, the polynucleotides are administered as a component of an enriched food, for example, a yoghurt, a yoghurt- or milk-based product.
[0061] The amount of polynucleotide(s) administered to a subject will be an effective amount, that is, an amount of the polynucleotide(s) that when administered to the subject, either as a single dose or as part of a series of doses, is effective to improve immune system development or function. It will be understood that in vivo administration of the polynucleotides is dependent upon a variety of factors, including for example, the duration, dose and frequency of administration, and the general condition of the subject. A suitable dose is typically approximated from animal model studies.
[0062] In certain embodiments, an effective dose of the polynucleotides for oral administration is from about 0.1 mg to about 250 mg, for example, from about 0.1 mg to about 200 mg, 150 mg, 100 mg, 50 mg, 25 mg, 10 mg, or any amount therebetween. In certain embodiments, the dose of polynucleotides is from about 0.5 mg to about 250 mg, 200 mg, 150 mg, 100 mg, 50 mg, 25 mg,

10 mg, or any amount therebetween, or from about 1 mg to about 250 mg, 200 mg, 150 mg, 100 mg, 50 mg, 25 mg, 10 mg, or any amount therebetween. In certain embodiments, an effective dose of the polynucleotides for oral administration is about 25 mg, 20 mg, 15 mg, 10 mg, or 5mg, or any amount therebetween, per day.
[0063] In some embodiments, the polynucleotides are administered topically.
The polynucleotides may be administered in the form of a cosmetic formulation. In certain embodiments, the invention relates to methods and uses of the polynucleotides or compositions comprising same to ameliorate a skin condition in a subject by topically administering the polynucleotides. The skin condition may be one associated with immune dysfunction, for example, baby rash, psoriasis or eczema.

[0064] To gain a better understanding of the invention described herein, the following examples are set forth. It will be understood that these examples are intended to describe illustrative embodiments of the invention and are not intended to limit the scope of the invention in any way.
EXAMPLES
EXAMPLE 1: A Functional Capacity Analysis of the Human Milk Metagenome [0065] In this study a metagenomic analysis of the bacteria in human milk was performed using Illumina sequencing and the MG-RAST pipeline [21]. The aims were to determine the genera of bacteria in human milk, search for immune-modulatory DNA motifs, and determine the types of bacterial open reading frames (ORFs) in human milk that may influence bacterial presence and stability in this complex yet foundational and immunomodulatory food matrix.
Results Phyla and genera within human milk [0066] Metagenome sequencing of a pooled human milk sample resulted in 261,532,204 sequenced reads of 51 bp, which were binned into those aligning to the human genome (186,010,988, 72.01 3.06%), known prokaryotic genomes (1,331,996, 0.53 0.16%) or those not aligning to either category (74,189,220, 27.46 3.72%). Using a best hit analysis of the 1,331,996 51-bp sequences, 75% aligned to Staphylococcus, 15% to Pseudomonas, 2% to Edwardsiella, and 1% to Pantoea, Treponema, Streptococcus and Campylobacter, respectively (Figure 1). The remaining 3% of the known prokaryotic sequences mapped to 361 bacterial genera, demonstrating the diversity of the human milk metagenome while confirming the presence of key genera like Akkermansia.
[0067] Sequences not aligning to prokaryotic or human genomes with a <2 bp mismatch were re-aligned to the human genome with decreased stringency (<10 bp mismatch), leaving 32,991,450 sequences for contig assembly (Table 1). Using Ray v1.7 [22], 56,712 contigs were assembled and submitted to the MG-RAST pipeline [21]. Post quality control, 53,785 sequences (94.8%), with a mean length of 160 55 bp, were used for further analysis (Table 1). When the contigs were analyzed using a best hit approach through MG-RAST, they aligned predominantly to the phyla of Proteobacteria (65.1%) and Firmicutes (34.6%). The contigs aligned to 194 known genomes at the genus level, predominantly Pseudomonas (61.1%), Staphylococcus (33.4%) and Streptococcus (0.5%), with the highest level of diversity at the genus level within the Proteobacteria phylum (125 different genera). These results are similar to the best hit analysis performed with the non-assembled sequences in that the majority of sequences are from Staphylococcus and Pseudomonas, but differ in their proportion (Figure 1).
Contigs matching viral genomes were observed (<0.04%), including phages derived from Pseudomonas and Staphylococcus. Contigs also aligned to the genomes of humans, gorillas, chimps and orangutans, likely due to the 60% identity criteria used. The observation of some of the genera, including Staphylococcus, Pseudomonas and Pantoea, was further validated through the presence of their rRNA ORFs.
Open reading frames within human milk [0068] A total of 41,352 ORFs were predicted using MG-RAST, of which 82% were annotated (33,793 ORFs), and 18% were unrecognized (7,559 sequences, Table I). A total of 30,128 ORFs corresponded to a functional category. For example, many ORFs encoded proteins for basic cellular function, including those for respiration (4.2%), cell signaling (4.8%), RNA (7.0%), DNA (2.6%), and amino acid metabolism (5.3%). ORFs encoding proteins for carbohydrate metabolism (5.7% of all ORFs) included those for lactose metabolism (oligosaccharide 6.7%), but none for human milk oligosaccharide metabolism, likely due to the lack of sequences aligning to the genome of Bifidobacteria. Virulence related ORFs (4.5% of all ORFs) included those for antibiotic resistance (60.2%), adhesion (17%), bacteriocins (2.7%), as well as others.
Stress-related ORFs (4.0% of all ORFs) included those for oxidative stress (40.3%), osmotic stress (20.2%), heat and cold shock (12.0% and 4.0%, respectively) and many others.
Human milk metagenome compared to mothers' and infants' feces [0069] The metagenome of human milk was compared to that of feces from 10 unrelated infants (five BF and five FF) and three unrelated mothers. Using a best hit analysis at the phylum level, contigs from human milk were dissimilar from contigs from feces in regards to the lack of diversity within the human milk metagenome, as over 99% of the contigs were from just two phyla, Proteobacteria and Firmicutes (65.1% and 34.6%, respectively). BF-infants' feces had a high proportion of Actinobacteria (70.4%), followed by FF-infants' feces (27.3%), mothers' feces (12.6%), and human milk (0.15%). The proportion of Proteobacteria in the human milk metagenome (65.1%) was most similar to that of BF-infants' feces (10.8%), but was significantly different from FF-infants' feces and mothers' feces (7.5% and 4.3%, respectively, P <0.05). The metagenomes of FF-infants' feces and mothers' feces were most similar in regards to their high proportion of Bacteroidetes (17.6% and 20.6%, respectively). Conversely, when using a lowest common ancestor approach at the phylum level in comparison to the best hit analysis, human milk appeared more similar to the fecal metagenomes in terms of an increase in diversity, but was still dominated by Proteobacteria (38.5%). Also, using the lowest common ancestor analysis increased the proportion of contigs aligning to Actinobacteria in human milk (0.15% to 11.58%), as well as in mothers' feces (12.6% to 30.6%).
100701 The metagenomes of human milk and feces were also compared at the functional level.
The functional ORF profile of the human milk metagenome is similar to that of each fecal metagenome, but two fecal profiles were even more similar, for example BF-versus FF-infants' feces, as seen using pair-wise comparison plots (Figure 2). The human milk metagenome is most dissimilar from that of FF-infants' feces as 17 out of the 26 functional categories contain a significantly different proportion of the ORFs (Figure 2). The three fecal metagenomes had a significantly higher proportion of ORFs encoding genes for dormancy and sporulation (2.3%, 2.3% and 2.7%, for BF-infants', FF-infants' and mothers' feces, respectively) than did the human milk metagenome (no associated ORFs, Figure 2).
[0071] Both BF- and FF-infants' fecal metagenomes had significantly higher proportions of cell division (3.5% each, respectively) and phosphorus metabolism related ORFs (3.1% and 3.0%, respectively) than did the human milk metagenome (2.3% and 2.1%, Figure 2). The human milk metagenome, in comparison to BF- and FF-infants' feces, did, however, have significantly higher proportions of membrane transport (5.0% compared to 4.0%
and 4.0%), nitrogen (3.5% compared to 3.1% and 3.0%) and RNA metabolism (4.9% compared to 4.1% and 4.3%), cell regulation (4.4% compared to 3.5% and 3.3%), respiration (4.3%
compared to 3.4%
and 3.4%), stress response (4.2% compared to 3.7% and 3.5%) and virulence-related ORFs (4.4% compared to 3.7% and 3.7%, Figure 2).

Immune-modulatory DNA motifs in human milk and feces [0072] When contigs were searched for the presence of immune suppressive motifs, TCAAGCTTGA was found in 0.02% of the human-milk assembled contigs (11 sites, Table 2) with an occurrence 1.5 times that of the human genome alone (once per 844,000 bp compared to once per 1,276,500 bp in the human genome, Z-score ¨1.6). The contigs positive for TCAAGCTTGA aligned to the genomes of Pseudomonas (45%), Nocardia (9%), Staphylococcus (9%) and contigs of unknown origin (36%, Tables 3 and 3B). When the contigs from BF-infants' feces, FF-infants' feces and mothers' feces were scanned for TCAAGCTTGA, it was found at a relative occurrence of 1.19, 1.64, and 1.64 times that in the human genome, respectively (Table 2). Another immune suppressive site, TTAGGG was observed 1,684 times in the human milk metagenome (3.2% of contigs), and at a relative occurrence 0.48 times that of the human genome (once per 5,600 bp compared to once per 2,670 bp, Z-score 22.54, Table 2).
Contigs containing TTAGGG corresponded to genomes of Staphylococcus (59%), Pseudomonas (10%), Lactobacillus (0.5%), 21 other known prokaryotic genomes (2.7%), and contigs from unknown genomes (27%, Table 3). When the contigs from BF-infants' feces, FF-infants' feces and mothers' feces were scanned for TTAGGG, this sequence was observed at a relative occurrence of 0.33, 0.18 and 0.26 times that in the human genome, respectively (Table 2).
Assembled contigs were also searched for the presence of synthetically-assembled immune suppressive or immune stimulatory DNA motifs (7 and 5 motifs, respectively), such as those used in vaccine production [23-27]. No synthetically-assembled sequences were observed in the human-milk contigs, whereas three motifs were found in less than 5 x 10-4% of contigs from the fecal metagenomes (maximum of 4 hits per 834,774 contigs).
Discussion Genera within human milk [0073] Determining the human milk metagenome, a bodily fluid notably absent from the human microbiome project [28], is crucial for enabling better insight on the process of infant GI
colonization and immune development. By pooling DNA from ten human milk samples and subjecting it to Illumina sequencing we have demonstrated the large diversity of the human milk metagenome with over 56,000 contigs aligning to 177 bacterial genera. Previous studies investigating the microbiome of human milk have used both culture-dependent and -independent approaches. Using 16S rRNA sequencing, Hunt et al. have reported several predominant species in human milk including a core of genera found in 15 human milk samples across time:
Streptococcus, Staphylococcus, Serratia, Pseudomonas, Corynebacteria, Ralstonia, Propionibacteria, Sphingomonas, and Bradyrhizobiaceae [17]. Other studies showed colostrum was populated mostly by Weisella and Leuconostoc, followed by Staphylococcus, Streptococcus, and Lactococcus, and that Akkermansia were more prevalent in overweight mothers [20,29].
Using a best hit analysis of the Si bp Illumina reads, alignments for Akkermansia, Propionibacteria, Sphingomonas and Weisella were observed in our milk samples.
Using PCR-denaturing gradient gel electrophoresis and quantitative PCR, two studies from Martin et al.
reported the presence of Bifidobacterium breve, B. adolescentis, B. bifidum and B. dentium in human milk, which differs from our findings [15,16], potentially due to differences in DNA
extraction methods [30]. The differences between the previously reported microbial communities and our analysis may also be due, in part, to the geographic location of the mothers, which has been shown to greatly impact the microbiome of individuals [31]. Furthermore, other differences between our characterization of the milk microbiome and those previously reported may be attributed to the means of milk collection. In comparison to previous studies where human milk was expressed from an aseptic breast [13-20], the current method determines the total microbiome (i.e. metagenome) ingested by the infant (from a non-sterilized breast), indicative of what an infant would receive from its mother during suckling.
[0074] Because our samples were collected from a nonsterilized breast, it could be hypothesized the human milk metagenome reported here would be similar to that of the skin microbiome. Although no reference database was freely available within MG-RAST
for comparison, the metagenome of human milk is similar to previously reported skin profiles in that there is a large proportion of Staphylococcus, which is found in moist areas of skin.
[0075] These moist areas, such as the antecubital fossa (inner fold of the elbow), also contain Betaproteobacteria, such as Burkholderia and Bordetella, which are present in the milk metagenome [32,33]. The human milk metagenome is also similar to drier areas of the skin such as the plantar heel, which contains Gamaproteobacteria such as Pseudomonas [32]. The human milk metagenome is, however, more similar to fecal microbiomes (as described in 16S rRNA

studies) due to the large proportion of Firmicutes bacteria within human milk, which is a very minor member of the skin microbiome [32,33].
[0076] Also, the skin of adults tends to contain a high level of Propionibacteria, which notably tends to colonize the skin of cesarean-section birthed babies, whereas this genus is minimally represented in our human milk sample using a best hit analysis of the 51 bp IIlumina reads (0.2%, [34,35]). This observation suggests that mother's milk may prove useful as a skin lotion, to rebalance the skin microbiome of C-section babies.
Phylogenetic differences between human milk and feces [0077] Comparing the metagenome of human milk to that of publicly available infants' and mothers' fecal profiles provides insight as to how human milk may lead to proper colonization of the infant gut. When comparing the human milk metagenome to the infant fecal metagenome, there are numerous differences. For example, the metagenome of BF-infants' feces contains a high proportion of Actinobacteria (70.4%), which correlates with previous studies demonstrating a high abundance of Bifidobacterium in the feces of BF-infants whereas FF-infants had a more varied microbiota [6,31,36]. Contigs from human milk, however, aligned mostly with Proteobacteria and Firmicutes (65.1% and 34.6%, respectively). At the phylum level, the present milk metagenome was less diverse than the fecal metagenomes as over 99% of the contigs were from just two phyla, Proteobacteria and Firmicutes. FF-infants' feces and mothers' feces were similar in that they both contained contigs aligning to the phylum Bacteroidetes (17.6% and 20.6%, respectively), whereas Bacteroidetes was a very minor component of BF-infants' feces and human milk (0.3% and 0.02%, respectively). Also, the similar proportion of Firmicutes in human milk compared to mothers. feces (34.6% and 59.6%, respectively) correlates with the hypothesis that mothers' milk may be inoculated by immune cells carrying bacteria from the GI
tract of the mother to her breast [37-39]. This may be a mechanism by which the human milk microbiome is shaped by the general health of the mother, including her weight [20].
Functionality of the human milk metagenome [0078] Using Illumina sequencing of all DNA within milk samples permits the prediction of ORFs within assembled contigs and allows for determination of the functional capability of the milk metagenome. A total of 41,352 ORFs were predicted, including those for basic cell function, as well as those that may enable the bacteria to remain in human milk, such as ORFs for carbohydrate metabolism (5.7% of ORFs). The predominant carbohydrate in human milk, lactose, is a potential carbon source for human milk bacteria, and therefore the presence of ORFs associated with its metabolism (6.7% of carbohydrate-associated metabolism) is expected.
Another carbon source for bacteria in human milk is human milk oligosaccharides (HMOs), which cannot be digested by the infant [40]. These oligosaccharides, which are heavily fucosylated and readily digested by Bifidobacteria, are thought to be responsible for the colonization of BF-infants with high levels of Bifidobacteria [41]. Recently, HMOs have also been correlated with increased abundance of Staphylococcus within human milk, regardless of their inability to utilize the human milk oligosaccharides as a carbon source [42]. The predominance of Staphylococcus-aligning contigs in our milk samples supports these findings.
Furthermore, there was a significantly higher number of ORFs related to nitrogen metabolism within the human milk metagenome in comparison to BF- and FF-infants' feces (P
< 0.05).
Because human milk contains 1.48-2.47 g of nitrogen per 100 g of milk, the bacteria within human milk may use it as a nutrient source in addition to lactose and HMOs [43].
[0079] Human milk contains an abundance of immune cells, antibodies and antimicrobial proteins (such as lactoferrin, CD14, alpha-lactalbumin, and lysozyme), and therefore the bacteria residing within human milk must harbor mechanisms to combat the milk-endogenous immune system [44-46]. For example, the metagenome of human milk includes ORFs for stress response and defense (4.0% and 4.5% of all ORFs, respectively) including those for oxidative stress (40.3% of stress-related ORFs) and toxic compound resistance (60.2% of defense ORFs). The human milk metagenome also contains ORFs for both heat and cold shock (12% and 4% of stress-related ORFs), which may enable the bacteria to persist in milk post-breast pumping, refrigeration and reheating. This may be of particular importance as human milk banks gain more popularity over time. For example, as described in a review by Urbaniak et al., some milk banks deem pasteurization of breast milk unnecessary, while others have an upper limit of 105 organisms per ml [47]. In unpasteurized banked milk and in-home stored milk, if some organisms are able to survive the storage and re-heating process better than others, the bacterial profile of human milk may change to favor better surviving (and not necessarily more beneficial) bacteria. Furthermore, ORFs encoding genes related to virulence and disease (4.5% of all ORFs), are also observed in the human milk metagenome. These ORFs could allow some of the human milk microbes, such as Staphylococcus aureus, to cause mastitis in humans when the balance of human milk-antimicrobials to microbes is tilted towards microbial growth [48].
For example, some bacteria within human milk harbor antibiotic resistance genes (60.2% of virulence associated ORFs) allowing them to proliferate regardless of the mother's potential antibiotic use, and some bacteria are able to produce bacteriocins (2.7% of virulence associated ORFs), which could impact the growth of other, less virulent, microbes within the community.
Immune-modulatory landscape of the human milk metagenome 100801 Because human milk contains a broad spectrum of microbes at the genus level, it likely contributes significantly towards effective colonization of the infant GI
tract. In the case of banked human milk, which is Holder pasteurized (65 C for 5-30 min), most bacteria are destroyed, but their proteins and DNA remain [49]. The presence of non-viable bacteria and bacterial DNA in human milk, which are indistinguishable from live bacteria using our approach of DNA isolation and sequencing, is a logical way to prime the infant immune system and lead to tolerance of the trillions of bacteria that will inhabit the gut following birth. For example, the immune suppressive motifs, TTAGGG and TCAAGCTTGA [11], are present in 3.0% and 0.02%
of the 56,950 human milk-contigs, respectively (1,684 sites, and 11 sites, Table 2). The occurrence of the immune suppressive motifs is similar to that in the metagenomes of BF- and FF-infants' feces, as well as mothers' feces. This suggests that having a diverse community of microbes may lead to a similar abundance of immune suppressive motifs, regardless of the genera present in the sample. Interestingly, the immune suppressive motif TTAGGG was found in higher abundance in the human genome than in bacterial contigs (one per 2,670 bp in the human genome compared to one per 5,600 bp in the bacterial contigs, Table 2).
Colostrum and mature human milk contain between 5 x 108 to 4 x 109 leukocytes/L and between 1 x 108 to 4 x 109 leukocytes/L, respectively, which are mostly macrophages (55%-60%) and neutrophils (30%-40%), with natural killer cells representing up to 12% of the population [50,51]. This suggests that ingestion of the mothers' DNA, through ingestion of her immune cells and any free circulating DNA may also lead to proper immune development through a balance of concomitant exposure to immune stimulatory bacterial CpGs and immune suppressive DNA in the mothers' genome and bacterial genomes.

Conclusions [0081] Current microbiome studies characterizing the microbial communities of various anatomical niches have revealed vast differences between healthy individuals [28]. These differences can often be attributed to the host's environment and diet. As demonstrated previously by preliminary 16S rRNA sequencing, the human milk microbiome is similar to other areas of the body in that its composition is unique to each individual [17].
Milk has evolved as the first nutrient source for mammals ex utero, with a high level of inter-mother diversity as to the proportions of bacterial genera, immune proteins and nutrients within it [29]. The diversity of sequences of DNA within the milk metagenome is furthermore beneficial for infants, as opposed to any one specific bacterial genus or species. Recent reviews on human milk outline the phylotypes of bacteria within human milk, but only speculate on the function of the human milk microbiome due to a lack of data on the functional capacity of the microbes within human milk [47,52]. The discovery of the abundance of immune suppressive DNA motifs observed within bacterial and human DNA from human milk, as well as ORFs within the human milk metagenome that allow bacteria to persist in the biological fluid supports and enables this discovery of the immunological functionality of the milk metagenome.
Methods Donors and sample collection [0082] Breastfeeding women (n = 10) were recruited from the Children's Hospital of Eastern Ontario (CHEO, Ottawa, Canada) in accordance with the Research Ethics Board of CHEO and the University of Ottawa Research Ethics Board (2007303-01H). Informed consent was given by all participants, all donors were healthy, and milk was donated between 9 and 30 days postpartum. Milk samples were collected by either manual or electric breast pump expression into a sterile milk collection bag (Medela AG, Baar, Switzerland). To better represent a milk sample that would be received by the infant, breasts were not sterilized prior to collection.
Samples were immediately frozen and then stored at ¨70 C.
DNA isolation [0083] Milk samples (1 mL) were centrifuged at 5,000 x g for 10 minutes to pellet eukaryotic cells. Prokaryotic cells were pelleted from milk serum by centrifugation at 13,000 x g for 15 minutes. Pellets were resuspended in 2 mL phosphate buffered saline with 1%
Triton X-100 and incubated for 2 hours at 37 C to lyse any remaining eukaryotic cells. Bacteria were pelleted by centrifugation at 13,000 x g for 15 minutes and pellets were resuspended in 500 [IL TE with 30 pt of 10% sodium dodecyl sulfate and 5 tg proteinase K. Samples were incubated for 2 hours at 37 C, and DNA was isolated using phenol/chloroform as previously described [53]. DNA pellets were resuspended in 50 JIL TE buffer and pooled. A total of'-4 ug of double stranded DNA was isolated as quantified with Quant-iT PicoGreen (Invitrogen, Burlington, ON, Canada) using a Typhoon Trio Imager and Image Quant TL software (GE Healthcare, Waukesha, WI, USA).
DNA integrity was also determined by agarose gel electrophoresis prior to sequencing.
DNA sequencing, filtering and contig assembly [0084] The pooled DNA sample was sequenced seven independent times by StemCore Laboratories (Ottawa, Ontario, Canada). DNA was prepared according to the DNA
sample preparation protocol 1003806 Rev. B for Illumina sequencing (Illumina Inc, San Diego, CA, USA). Sequencing was performed using an Illumina GAIIx Genome Analyzer and Illumina CASAVA analysis pipeline (v 1.7.0). Sequences were aligned to the human genome (hg19/
NCBI37) with a stringency of 2 bp mismatching using ELAND (Illumina Inc).
Prokaryotic genomes (1,731 genomes) were imported from NCBI. Sequences were aligned to the genomes using BLAT (Kent Informatics, Inc.) and sorted via best hit analysis to genera according to "List of Prokaryotic Names with Standing in Nomenclature"
(http://www.bacterio.cict.fr/, accessed February 2012). Unidentified sequences were further filtered by using BLAST
against the human genome with a stringency of <10 mismatches or gaps. Both prokaryotic and remaining unknown sequences were assembled into contigs using Ray v1.7 [22].
Contigs, ORF prediction and characterization [0085] Assembled contigs were uploaded to the MG-RAST pipeline [21]. Organism abundance was analyzed using a lowest common ancestor approach with a maximum evalue of 1 x 10-5, a minimum identity of 60%, and a minimum alignment length of 15 measured in amino acids for protein and base pairs for RNA databases. A functional abundance analysis of ORFs was performed using "Hierarchical Classification" by comparing to subsystems with a maximum e-value of 1 x 10-5, a minimum identity of 60%, and a minimum alignment length of 15 measured in amino acids for protein and base pairs for RNA databases. Previously reported and publicly available metagenomes of feces from five unrelated BF-infants, five FF-infants (metagenome IDs: USinfTW4.1, 6.1, 10.1, 11.1, 12.1, 13.1, 15.1, 19.1, 20.1, and 21.11) and three unrelated mothers (metagenome IDs: USchp [1,3,4,18,33]mom) were compared at the phylum level to the human milk metagenome within MG-RAST using the same lowest common ancestor analysis described above [21]. The mean percent alignments of the individuals or the normalized mean percent of ORFs in each functional category was used. Metagenome comparisons were statistically compared by Student's t-tests (P < 0.05) using SigmaPlot (Systat Software, Inc., San Jose, CA, USA).
Immune-modulatory motif identification [0086] An identity of 100% was used to search for immunomodulatory motifs by alignment with assembled contigs from the human milk metagenome (56,712 contigs) or the fecal metagenomes described above (834,774, 64,662 and 553,391 contigs from BF-infants', FF-infants' and mothers' feces, respectively). The human genome (2,865,822,365 bp) was used as a comparative reference. Z-score was calculated using the formula Z = (0-E)/Stdev, where 0 was the observed number of hits and E was the expected number of hits using the formula E =
(Lcont)(Nh/Lh) where Lcont was length of sequences or assembled contigs, Nh was number of sites found in the human genome (or compiled bacterial genomes); Stdev was the standard deviation of occurrence of each motif in 22 + X + Y human chromosomes.
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Table 1 Contig assembly and open reading frame (ORF) prediction of illumina reads (51 bp) from human milk Sequenced reads (51 bp) 261,53Z 204 Matching human 186,010,988 Matching prokaryotic 1,331,9%
Used in contig assembly' 32,991,450 Conti gs 56,712 Post quality control 53,785 Average length (bp) 160 55 Total length (bp) 8,630,997 Predicted ORFs 41,352 Annotated 33,793 rRNAs 103 Functional category 30,128 Unrecognized 7,559 1 all sequences not matching the human genome (_10 bp mismatch).

Table 2 Occurrence of immune suppressive motifs in various metagenomes Sequence Number of hits Base pairs per hit Relative occurrence (Z-score) TCAAGCTTGA II 844000 (Human Milk) 151 (¨LO
344 1,077,000 (BF Infant) 119 (-0.74) 124 779,000 (FF Infant) 1.64 (-1.84) 268 777,000 (Mother) 1.64 (-1.85) 2,245 1,276,500 (Human Genome) TTAGGG 1,684 5,600 (Human Milk) 0.48 (22.54) 18,118 8,200 (BF Infant) 033 (4254) 16,410 15,01X1 (FF Infant) 0.18 (94.85) 20,612 10,200 (Mother) 0.26 (57.92) 1,082,623 2,670 (Human Genome) DNA motifs TTAGGG and TCAAGCTTGA were searched for in contigs derived from human milk, breast-fed infants' feces (BF infant), formula-fed infants feces (FF
infant) and mothers` feces. Relative occurrence is in comparison to the human genome.

Table 3 Occurrence of immune suppressive motifs TTAGGG and TCAAGCTTGA in contigs from human milk Sequence Genus Number of hits TCAAGCTTGA Pseudomonas Nocardia 1 Staphylococcus 1 Unknown 4 TTAGGG Staphylococcus 1000 Pseudomonas 169 Lactobacillus 8 Bacillus 6 Streptococcus 6 Streptomyces 4 Tetragenococcus 4 Unknown 461 Table 3.B. Sequences obtained from assembled contigs and containing immune suppressor motif TCAACCITGA.
Match/ Genus Distance Flanked sequences Length of from ORE
con tigs acttacataact ecccagacTCAAGCTTGActtaaqcaaacaaa 162 -. . . . . . ...... . . . . . . .gT=GCTTGAaaaaaclacrcaaaaccacaaa 368/369 Staphylococcus Qtqcogccccat tcati.7agaTCAAGCTTGArc;ctggtgct.:gtr.T.Tal:. :t.Q 62/216 Pseudomonas end of ORF(-) . . . . . . . . . . . . . ..... . cTCA_A_GCTTGI-ttoac: zgotcc.cp:1 :a 7,-..
tcg 140 -acgt t coTCARGCTTGAccqcgcgcagt tacccaact 40/109 Pseudomonas -ccgccgccag-: act t t tgaaTCALGOTTG2-'.,gotctgcccaqq-cactgccrt 125 --aatactc771ccactggcgeaTCA:A.GCTTGAcicttgcct teagccaccagt 44/118 Pseudomonas -acgcagtgc.'czgrz.acagagcTCAAGCTTGAtcca . . . . . . . . . ...... . 118 _ cagatccaca.acicaccagcaTCAGC1"1'GAtetgatgaatqcigac.ggcac 97/139 Pseudomonas end of ORF(-) actgqtggcc_c_Taacrcicaac..7cTCAAG'CTTGAtacgcc-c4g1=ggccaagtact 40/154 Pseudomonas -gtgacqccccat. t cat cagaTCAAGOTTGAccrctggtget tgtcqactg 88/117 Pseudomonas end of ORF(-) cactggacqtatagaerc :q :7=LLACCTTGtga...-:-.:tgetc.:(Tacaq-z:cki 164 -tuc_gr'z tatcgaccaa 7 = ccik:',-3C:7_¨_'-ccic.a.7.,ctct ocagcggc 111/120 Pseudomonas ggaaggaagacagccag=tTCA2=_GETTC:Acgar_Ictca..-itc: accagaga 104 --actatagataa tact Eia.:-..-:;TC7-,.?.GCTTGaaaaagaccaaaaccaraaa 162/162 Staphylococcus -[0001] The disclosures of all patents, patent applications, publications and database entries referenced in this specification are hereby specifically incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication and database entry were specifically and individually indicated to be incorporated by reference.
[0002] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention. All such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A composition formulated for oral or topical administration comprising an isolated polynucleotide, the isolated polynucleotide comprising deoxyribnucleotides and one or more immunosuppressive motifs.
2. The composition according to claim 1, wherein each of the one or more immunosuppressive motifs are selected from TCAAGCTTGA [SEQ ID NO:1] and TTAGGG

[SEQ ID NO:2].
3. The composition according to claim 1 or 2, wherein the polynucleotide is between about and about 500 nucleotides in length, or between about 20 and about 100 nucleotides in length.
4. The composition according to any one of claims 1 to 3, wherein the isolated polynucleotide is a synthetic polynucleotide.
5. The composition according to any one of claims 1 to 3, wherein the isolated polynucleotide is isolated from a natural source.
6. The composition according to any one of claims 1 to 3, wherein the composition comprises a plurality of isolated polynucleotides, at least one of which comprises the one or more immunosuppressive motifs.
7. The composition according to claim 6, wherein at least one of the plurality of isolated polynucleotides comprises one or more immunostimulatory motifs.
8. The composition according to claim 7, wherein the plurality of isolated polynucleotides are DNA sequences isolated from human breast milk.
9. The composition according to any one of claims 1 to 8, wherein the composition is formulated for oral administration.
10. The composition according to claim 9, further comprising one or more probiotic.

11. The composition according to claim 9 or 10, further comprising standard infant formula.
12. The composition according to claim 9 or 10, wherein the composition is a food product or nutritional supplement product.
13. The composition of claim 12, wherein the food product is an infant formula, a follow-on formula, or a toddler beverage.
14. The composition according to any one of claims 1 to 8, wherein the composition is formulated for topical administration.
15. An infant formula comprising the composition according to claim 9 or 10, and one or more of a protein source, a fat source and a carbohydrate source.
16. The infant formula according to claim 15, wherein the infant formula is formulated for administration to neonates.
17. The infant formula according to claim 15, wherein the protein source, fat source and carbohydrate source is human breast milk.
18. The infant formula according to claim 15, wherein the protein source is whey protein.
19. The infant formula according to claim 15, wherein the protein source is soy protein.
20. The infant formula according to claim 15, further comprising one or more probiotics.
21. The infant formula according to any one of claims 15 to 20, wherein the infant formula is a liquid.
22. The infant formula according to any one of claims 15 to 20, wherein the infant formula is a powder or granules for reconstitution in a liquid.
23. A nutritional supplement comprising the composition according to claim 9 or 10, and one or more of a protein source, a fat source and a carbohydrate source.
24. The nutritional supplement according to claim 23, wherein the protein source is whey protein.

25. The nutritional supplement according to claim 23, wherein the protein source is soy protein.
26. The nutritional supplement according to any one of claims 23 to 25, wherein the nutritional supplement is a liquid.
27. The nutritional supplement according to any one of claims 23 to 25, wherein the nutritional supplement is a powder or granules for reconstitution in a liquid.
28. The nutritional supplement according to any one of claims 23 to 25, wherein the nutritional supplement is a sports drink or a bar.
29. A cosmetic formulation comprising the composition of claim 14 and one or more of a cosmetically acceptable carrier, diluent and excipient.
30. A method for enhancing immune system development in an infant comprising orally administering to the infant the composition of claim 9 or 10.
31. The method according to claim 30, wherein the composition is administered in combination with human breast milk.
32. The method according to claim 30, wherein the composition is administered in combination with infant formula.
33. The method according to any one of claims 30 to 32, wherein the infant is a neonate.
34. A method of improving immune system function in a subject comprising orally administering to the subject the composition according to claim 9 or 10.
35. The method according to claim 34, wherein the composition is administered as a component of a nutritional supplement.
36. The method according to claim 35, wherein the composition is administered as a component of an enriched food.
37. The method according to claim 36, wherein the enriched food is a yoghurt or yoghurt-based product.

37. The method according to claim 36, wherein the yoghurt or yoghurt-based product further comprises probiotics.
38. A method of ameliorating a skin condition in a subject comprising topically administering to the subject the composition according to claim 14.
39. The method according to claim 38, wherein the skin condition is psoriasis or eczema.
40. Use of a composition comprising an isolated polynucleotide as a nutritional supplement, the isolated polynucleotide comprising deoxynucleotides and one or more immunosuppressive motifs.
41. The use according to claim 40, wherein each of the one or more immunosuppressive motifs are selected from TCAAGCTTGA [SEQ ID NO:1] and TTAGGG [SEQ ID NO:2].
42. The use according to claim 40 or 41, wherein the polynucleotide is between about 10 and about 500 nucleotides in length, or between about 20 and about 100 nucleotides in length.
43. The use according to any one of claims 40 to 42, wherein the isolated polynucleotide is a synthetic polynucleotide.
44. The use according to any one of claims 40 to 42, wherein the isolated polynucleotide is isolated from a natural source.
45. The use according to any one of claims 40 to 42, wherein the composition comprises a plurality of isolated polynucleotides, at least one of which comprises the one or more immunosuppressive motifs.
46. The use according to claim 45, wherein at least one of the plurality of isolated polynucleotides comprises one or more immunostimulatory motifs.
47. The use according to claim 46, wherein the plurality of isolated polynucleotides are sequences isolated from human breast milk.
49. The use according to any one of claims 40 to 47, wherein the composition further comprises one or more probiotic.

50. The use according to any one of claims 40 to 49, wherein the nutritional supplement is for supplementing infant formula, a follow-on formula, or a toddler beverage.
51. The use according to any one of claims 40 to 49, wherein the nutritional supplement is a sports drink or bar.
52. Use of a composition comprising an isolated polynucleotide to supplement human breast milk, the isolated polynucleotide comprising deoxyribonucleotides and one or more immunosuppressive motifs.
53. The use according to claim 52, wherein each of the one or more immunosuppressive motifs are selected from TCAAGCTTGA [SEQ ID NO:1] and TTAGGG [SEQ ID NO:2].
54. The use according to claim 52 or 53, wherein the polynucleotide is between about 10 and about 500 nucleotides in length, or between about 20 and about 100 nucleotides in length.
55. The use according to any one of claims 52 to 54, wherein the isolated polynucleotide is a synthetic polynucleotide.
56. The use according to any one of claims 52 to 54, wherein the isolated polynucleotide is isolated from a natural source.
57. The use according to any one of claims 52 to 54, wherein the composition comprises a plurality of isolated polynucleotides, at least one of which comprises the one or more immunosuppressive motifs.
58. The use according to claim 57, wherein at least one of the plurality of isolated polynucleotides comprises one or more immunostimulatory motifs.
59. The use according to claim 58, wherein the plurality of isolated polynucleotides are sequences isolated from human breast milk.