WO2014028416A1 - Milk glycans, arrays, compositions and uses related thereto - Google Patents

Description

MILK GLYCANS, ARRAYS, COMPOSITIONS AND USES RELATED THERETO

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant number GM62116 and GM085448 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application number 61/682,912 filed August 14, 2012 hereby incorporated by reference in its entirety.

BACKGROUND

As the natural food source for newborns, human milk provides not only all of the nutrients necessary for infants to grow and develop but also provides health benefits in early childhood. In addition to antibodies that greatly enhance defense of the infant against various diseases, human milk possesses a rich pool of oligosaccharides (glycans) that generally are unique in composition to humans and differ from those in the milk of other mammals. The mature milk is estimated to contain 5-15 g/liter free human milk glycans (HMGs) depending on individual lactation time and blood group type. See Thurl et al, Br. J. Nutr., 2010, 104, 1261-1271, Thurl et al, Glycoconj. J., 1997, 14, 795-799, and Blank et al, Anal. Bioanal, 2011, Chem. 401, 2495-2510.

Unique HMGs have been detected and characterized. The remarkable structural diversity of these glycans suggests that they possess multiple biological effects. The prebiotic and anti-adhesive effects are the most common functions attributed to HMGs. Sela & Mills, Trends Microbiol., 2010, 18, 298-307. Furthermore, HMGs are associated with neonatal intestinal development and limiting risks of necrotizing enterocolitis. Bode, Nutr. Rev., 2009, 67, S183-S191.

Although the multiple functions of HMGs are poorly understood, numerous studies have shown that their functional activities are dependent on their structures. For example, sialylated HMGs inhibit cholera toxin binding and leukocyte adhesion to cultured human umbilical vein endothelial cells. Specific fucosylated HMGs are recognized by

enteropathogens, including Helicobacter pylori, rabbit calicivirus, and Norwalk virus.

Neutral HMGs, especially H type 2 glycans, inhibit Campylobacter jejuni adherence to Hep-2 cells and intestinal mucosa. Although the reported in vitro and in vivo data provide important information for understanding the effect of HMGs, typical experiments with HMG utilize either a small number of defined glycans or mixtures of HMG fractions. Such limitations represent challenges in studying HMGs, where the goal is to determine the roles of specific glycans in the milk glycome and to establish the relationships between glycan structures and their biological effects. Many HMGs are comprised of linear and branched polymers of type 1 and type 2 lactosamine, Gaipi-3GlcNAc and Gaipi-4GlcNAc, respectively, substituted with a- linked Neu5Ac and Fuc. It is not possible to assign complete structures by mass spectrometry alone because of isobaric and isomeric structures, and a wide variety of approaches are often required. Thus, there is a need for improved methods of evaluating glycan structures.

Shotgun glycomics utilizes free glycans derived from glycoproteins and glycolipids. They are derivatized with a bifunctional fluorescent tag and separated by multidimensional HPLC, and individual glycans are printed as a shotgun glycan microarray (SGM). In this approach glycan structures are defined after they are identified through their recognition by a glycan binding protein (GBP) or pathogen and, therefore, are potentially functionally important. The combined the use of mass spectrometry, recognition by defined GBPs, and exoglycosidase treatments has helped to provide more detailed information about specific glycan structures in an approach termed metadata-assisted glycan sequencing (MAGS). See Song et al, Nat. Methods, 2011, 8, 85-90, Song et al, J Biol Chem., 2011, 286(36):31610- 22, Stowell et al, Nat. Med., 2010,16, 295-301; Bohnsack et al, J. Biol. Chem., 2009, 284, 35215-35226, Song et al, J. Biol. Chem., 2009, 284, 35201-35214, Song et al, Glycoconj. J., 2008, 25, 15-25, and Yu et al, J Biol Chem., 2012 287, 44784-44799.

SUMMARY

This disclosure relates to milk glycans, nutritional supplements, pharmaceutical compositions, and uses related thereto. In certain embodiments, the disclosure relates to an array, e.g., on a solid substrate, comprising a plurality of zones or wells wherein each zone contains a conjugate comprising a glycan conjugated to a fluorescent tag, wherein the glycan is derived from milk of a mammal. In certain embodiments, the mammal is a human.

In certain embodiments, the glycan does not contain mannose, terminal GalNAc, or terminal Neu5Aca2-3Gaipi-4GlcNAc. In certain embodiments, the number of zones is greater than 20, 50, 100, 200, or 300. In certain embodiments, the glycan is selected from one or more glycans disclosed herein. In certain embodiments, the disclosure relates to methods comprising mixing an array disclosed herein, with a biological molecule or pathogen and detecting the biological molecule bind a glycan or pathogen bind a glycan in a zone. In certain embodiments, the biological molecule is a peptide, protein, receptor, ligand, antibody, lectin, or virus particle. In certain embodiments, the pathogen is a virus, bacteria, fungus, or parasite. In certain embodiments, the antibody is a monoclonal antibody for a blood group epitope. In certain embodiments, the antibody is anti-Le^a, anti-sialyl Le^a, anti-Le^b, and anti-blood group H type- 1.

In certain embodiments, the disclosure relate to computer readable mediums, such as a hard drives or transportable memory comprising data on the sequence of the glycan in each zone of an array disclosed herein.

In certain embodiments, the disclosure relates to devices comprising an array disclosed herein, a source of electromagnetic irradiation, e.g. visible or ultraviolet light, and a fluorescence detector.

In certain embodiments, the disclosure relates to nutritional supplements comprising one or more of the human milk glycans selected from those disclosed herein or combinations thereof. In certain embodiments, the nutritional supplement comprises or consists of twenty five, thirty, forty, fifty, sixty, seventy, eighty, ninety, or more of the human milk glycans disclosed herein.

In certain embodiments, the nutritional supplement comprises or consists of H-71, H-

99, H-108, H-111, H-125, H-126, or H-127, and combinations thereof. In certain

embodiments, the nutritional supplement comprises or consists of H-28, H-30, H-31, H-36, H-43, or H-60, and combinations thereof. In certain embodiments, the nutritional supplement comprises or consists of H-1, H-5, H-6, H-12, H-14, H-15, H-16, H-17, or H-56, and combinations thereof.

In certain embodiments, the disclosure relates to methods comprising feeding a subject younger than one or two years old a nutritional supplement of disclosed herein.

In certain embodiments, the disclosure relates to pharmaceutical composition comprising a pharmaceutically acceptable excipient and a glycan disclosed herein, e.g., with a terminal Neu5Aca2-6Gaipi-4GlcNAc. In certain embodiments, the glycan is H-28, H-30, H-31, H-36, H-43, or H-60, and combinations thereof. In certain embodiments, the glycan is H-1, H-5, H-6, H-12, H-14, H-15, H-16, H-17, or H-56, and combinations thereof. In certain embodiments, the pharmaceutical composition of further comprises a second anti-viral or anti-bacterial agent. In certain embodiments, the disclosure relates to methods of treating or preventing a pathogenic infection comprising administering a pharmaceutical composition disclosed herein to a subject at risk or, exhibiting symptoms of, or diagnosed with a pathogenic infection, e.g., a viral infection.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows glycan determinants defined by GBPs. The determinants defined by six commercially available lectins and five monoclonal antibodies were determined by analysis on the defined glycan microarray provided by the Consortium for Functional Glycomics. The determinants, whose presence on an array can be defined by positive signals in an array analysis, are outlined in each structure. Con A, concanavalin A.

Figure 2 shows predicted and unique lectin/antibody binding patterns to hypothetical isobaric N-glycans immobilized on a hypothetical glycan microarray used for structural analysis. Ten hypothetical N-glycans (1-10) are listed, and the predicted binding patterns for the hypothetical microarray of the lectins and anti-glycan antibodies described in Fig. 1 are shown. The + or - indicate positive or negative binding, and each data point is divided to provide the results of binding with no treatment (above the slash) and the results of binding after treatment with nonspecific neuraminidase (below the slash).

Figure 3 shows metadata-assisted glycan sequencing is an extension of the shotgun glycan microarray concept. Beginning with the generation of the TGL, each glycan is assigned an accession number and printed on the array, and the metadata are collected for each glycan and stored in a database. Pre -printing information can include the following: number of negative charges based on ion-exchange chromatography; location of the glycan in the two-dimensional HPLC separation profiles; percentage of total glycomes that each glycan represents; MALDI-TOF data to provide information on purity, composition, additional MS data as obtained; defined GBP binding before and after exoglycosidase digestion; and any other information deemed useful regarding the nature of the glycan.

Figures 4A and B show an example of MAGS of a single human milk glycan selected for structural analysis based on its binding function. A, lectin and antibody binding to standards and an unknown disialyl human milk glycan. The predicted structure of the unknown glycan identified as a glycan ligand for MVM from human milk is shown at the upper left above a list of 10 glycan standards obtained from human milk. The binding patterns for four defined lectins (SNA, ECL, AAL, and GSL-II) and three monoclonal antibodies (anti-Le^a, anti-type I glycan (Gaipi-3GlcNAc), and anti-Sialyl Le LSTa) from individual microarray analyses are indicated as either positive (+) or negative (-), indicating the presence or absence of the determinants as defined in Fig. 1. B, lectin and antibody binding to standards and an unknown disialyl human milk glycan before and after exoglycosidase digestion. The predicted structures of the unknown glycan and

exoglycosidase products of the enzyme treatments are shown with the patterns of lectin and antibody binding from individual microarray analyses indicated as either + or -. Glycan Mass = 1818.174 (monoisotopic). Predicted composition Neu5Ac(2)Hex(4)HexNAc(2).

Figure 5 schematically illustrates the generation of HMG SGM. Human milk glycans were extracted, fractionated, AEAB-conjugated (labeled with a tag), and separated. The purified fractions were quantified and printed to create a human milk glycan SGM available for studies with GBPs and microorganisms.

Figure 6 shows data on plant lectins binding to human milk SGM. The human milk SGM microarray was characterized with biotinylated lectins AAL (0.1 μg/ml; A), UEA-I (10 μ^ιηΐ; B), LTL (10 μ^πιΐ; C), SNA (5 μ^ιηΐ; D), RCA-I (10 μg/ml; E), and ECL (10 μ^ιηΐ; F). A total of 140 glycans was printed on the microarray. Glycans 1-73 are sialylated glycans (tan), 74-127 are neutral glycans (violet), and 128-140 are controls of structurally defined glycans (light green). The structures in symbols indicate the binding specificity of each lectin identified by defined glycan microarray (CFG v5.0). Considering the variation of binding affinity, the histogram shows the data at the concentration that yielded the best

signal/background ratio instead of the data at the same concentration.

Figure 7 shows data on antibodies binding to human milk SGM. The human milk SGM was interrogated with antibodies: anti-Lea antibody (10 μ^ιηΐ; A), anti-SLea/LSTa antibody (10 μ^ιηΐ; B), anti-blood group HI antibody (1 :10 dilution; C), and anti-CD15 antibody (10 μ^ιηΐ; D). The microarray was also used to test the binding specificity of anti- TRA-1-60 antibody (50 μ^πιΐ; E) and anti-TRA-1-81 antibody (50 μ^πιΐ; F). Glycans 1-73 are sialylated glycans (tan), 74-127 are neutral glycans (violet), 128-140 are controls of structurally defined glycans (light green).

Figure 8 shows data on MVM viruses binding to human milk SGM. The binding preferences of several strains of MVM, MVMp-WT (prototype strain, empty capsid; A), MVMp-VLP (prototype strain, virus-like particles (B)), MVMi-ggA (capsid protein mutant of immunosuppressive strain, empty capsid; C), and MVMi-agD (Non-structural protein mutant of immunosuppressive strain, empty capsid; D) were evaluated on the human milk SGM. Each virus was tested at 200 μ^ιηΐ and detected by anti-MVM capsid antibody. Panel E shows that there was no significant background binding from the anti-MVM capsid antibody to HMG microarray. Glycans 1-73 are sialylated glycans (tan), 74-127 are neutral glycans (violet), and 128-140 are controls of structurally defined glycans (light green).

Figure 9 shows data on influenza viruses binding to human milk SGM. The binding preferences of various influenza virus isolates, A/Brisbane/59/2007 H1N1 (A),

A/Oklahoma/447/2008 H1N1 (B), A/Pennsylvania/08/2008 (C), A/California/04/2009 H1N1 (D), A/Oklahoma/483/2008 H3N2 (E), and hPIV2 were evaluated with HMG microarray (F). Glycans 1-73 are sialylated glycans (tan), 74-127 are neutral glycans (violet), and 128-140 are controls of structurally defined glycans (light green).

Figure 10 illustrates the structures of 17 defined glycans used for controls and the predicted structures of 20 selected human milk glycans. The control glycans (left panel) are listed by common names and the HMGs (panels A, B, and C) are listed by fraction names that are the same as the glycan ID on the microarray. Glycans H01 -HI 7 and H-56 were ligands of influenza viruses, glycans H-28-H-36 were ligands of MVMs, and glycans H-99- H-127 were ligands of anti-TRA-1 antibodies. 18 control glycans were printed; however, glycans 11 and 13 were both LNFPI.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression.

As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.

Shotgun glycomic and Metadata- Assisted Glycan Sequencing (MAGS)

Antibodies, lectins, and toxins, also referred to collectively as glycan-binding proteins (GBPs), are examples of GBPs that have been used in indirect approaches to explore features of cell surface glycans. Antibodies to blood group antigens (ABO and Lewis antigens) have been used to identify expression of these determinants on glycoproteins and glycolipids in cells and tissues. Lectins and anti-glycan antibodies have been useful in identifying glycan structures associated with specific glycosylation pathways, as well as to generate mutant cell lines with altered glycosylation identified by their resistance to toxic lectins. Specific GBP binding can provide significant information on the presence of unique monosaccharides (sialic acid and fucose), on the identification of epimers of hexoses (Gal, Man, and Glc), and in many cases anomericity, but they cannot provide complete glycan sequence information.

More direct biochemical approaches to defining glycans involve disassembling the glycocalyx to investigate the structures of specific glycoconjugate classes. Free glycans generated by chemical or enzymatic means or directly available in body fluids, such as milk or urine, may be directly analyzed by MS methods. They are separated by chromatographic techniques, permethylated, and then characterized by MS methods. The amphipathic glycosphingo lipids (GSL), which are small relative to glycoproteins and proteoglycans, are isolated from lipid extracts of cells and tissues, separated by nonaqueous chromatographic techniques, permethylated, and characterized directly by MS methods. Glycans can be released enzymatically from GSL using recombinant endoglycoceramidases and prior to permethylation and MS sequencing. For GSL that have a double bond in the sphingosine moiety, ozonolysis followed by degradation at neutral pH eliminates the glycan and provides a method for obtaining free GSL-derived glycans that can be reduced, reductively aminated with a variety of tags, permethylated, and analyzed by MS methods.

Glycoproteins and proteoglycans may be prepared from biological fluids and aqueous or aqueous-detergent extracts of cells or tissues and then separated by a variety of chromatographic techniques. In cases where a glycomic profile was considered informative, the component glycans may be released by enzymatic or chemical means, such as by treatment with proteases followed with endoglycosidases, e.g. peptide -N-glycosidase-F and O-glycanase, to release N-glycans and the Ser/Thr-linked core 1 O-glycan, respectively, or treated with base, e.g. NaOH or hydrazine, to release Ser/Thr-linked O-glycans by β- elimination using reductive or nonreductive methods. The resulting free glycans, like those derived from GSL, are typically either directly derivatized by fluorescent tags, e.g. 2- aminobenzamide or 2-amino-N-(2-aminoethyl) benzamide (AEAB) for separation by HPLC, or directly reduced, permethylated, and analyzed by MS analysis to obtain structural information. Thus, methods to obtain the glycans comprising the glycoconjugates of a glycome are generally available, but they all have specific limitations.

Glycan sequencing approaches can be combined to aid in defining the glycome. Current ultrasensitive MS methods are routinely used for predicting glycan composition and sequence, and if sufficient standards and sophisticated tandem MS technologies are available to reproducibly identify unique fragment ions, detailed glycan structural analyses are possible. Such complex structural studies can ultimately help to provide the list of glycan structures comprising a glycome.

A variety of separation techniques have been applied to glycan analyses with great success, and if carefully monitored and validated, these methods provide significant structural information based on comparison of chromatographic properties with known standards, as well as direct analyses by MS. Examples of significant success in these areas include ultra- performance liquid chromatography, HPLC-MALDI-TOF MS, high performance anion- exchange chromatography, two-dimensional HPLC, high performance capillary

electrophoresis, and fluorophore-assisted carbohydrate electrophoresis.

Methods employing multiple chromatographic strategies, including hydrophilic interaction liquid chromatography, can be useful in comparative analysis of glycomes, as elegantly applied to serum glycoprotein glycosylation.

Serial lectin affinity chromatography was an early strategy for analyzing glycans based on knowledge of specific structural features in glycans required for their interactions with lectins. In this approach, glycans either metabolically radiolabeled, end radiolabeled, or fluorescently labeled were analyzed by their chromatographic properties and affinity to specific lectins and antibodies. In these studies, glycan structures were determined by their specific binding to defined lectins and antibodies, co-chromatography compared with standards, and the use of highly purified, specific endo- and exo-glycosidase digestions to monitor changes in chromatographic behavior. Anomeric configurations and linkage positions of monosaccharides in a glycan were assessed based on susceptibility or resistance to specific enzyme degradation. This approach using HPLC-based methods is useful for analyzing extremely small amounts of material. In some cases it has been possible to define branching of radiolabeled N-glycans using acetolysis and linkage analyses on extremely small quantities of glycans by subjecting metabolically radiolabeled glycans to

permethylation, hydrolysis, acetylation, and identification of methylated monosaccharides by gas chromatography with detection of radioisotope in the eluted gas. These approaches can generate information on the detailed structure of glycans.

Defined glycan microarrays provide clues to understanding GBP function. The publicly available defined glycan microarray developed by the National Institutes of Health- funded Consortium for Functional Glycomics (CFG) has made a major impact on the advancement of functional glycomic analysis. After the analysis of hundreds of GBPs on this array, investigators in many areas are beginning to appreciate access to glycans in a microarray format that can generate information on GBP function. Because it is not possible to amplify a glycome by a method like PCR, immobilized glycans on an array are analogous to amplified products of the genome, i.e. oligonucleotides, genes, gene fragments, and recombinant proteins that make them available for functional studies.

One approach to defining a glycome, one can derivatize free glycans derived from cells and tissues with a bifunctional tag that is fluorescent and also carries a free amino group (2,6-diaminopyridine, AEAB, or 2-aminobenzamide). Fluorescence provides a method for detecting glycans during their purification, and the amino function provides a reactive center to immobilize glycans for functional analyses on glycan microarrays or other solid phases.

Shotgun glycan microarrays define biologically relevant glycans. Shotgun glycomics is a method to identify physiologically or biologically relevant glycans that are screened as potential glycan ligands for GBPs of interest. Nanoscale methods were used to isolate glycans from natural sources and prepare glycan libraries for direct studies of both their structure and function in terms of GBP recognition. This approach focuses sequencing efforts on functionally relevant glycans recognized by a GBP and results in libraries of naturally occurring glycans that can be archived and retrieved for future studies. In one example of a shotgun glycan microarray (SGM), ozonolysis of the sphingosine portion of a mixture of GSLs generated free aldehydes that readily reacted by reductive amination to create fluorescent GSL derivatives with a primary amino group. The mixture of GSL derivatives composed of bovine brain gangliosides (BBG) was resolved by two-dimensional HPLC into 40 individual derivatives that make up a BBG-tagged glycan library (TGL). The derived glycolipids were quantified based on their fluorescence, characterized by MALDI- TOF/TOF analysis, and printed at equimolar concentrations on N-hydroxysuccinimide- derivatized slides.

In this strategy for human glycomic analysis, the term "shotgun" refers to the fact that glycans are prepared from specific cells or tissues and differs from shotgun genomics in that it does not propose to directly sequence all of the component member glycans in the TGL, but to prioritize structural efforts and identify glycans to be synthesized by chemists for expanding the defined array. As structural definition progresses, the number of defined structures on the SGM will increase. The shotgun glycomics approach may be applied to any organism.

Metadata-assisted Glycan Sequencing (MAGS) is a glycomics approach based on MS analysis of TGLs and defined GBP binding to SGMs. The TGL generated from a tissue or organism represents a significant component of a glycome, and each glycan fraction has an associated mass based on MALDI-TOF analysis carried out prior to printing the SGM. Defined GBPs, e.g. plant and animal lectins and anti-glycan antibodies, provide a rich source of reagents for detecting unique glycan determinants among the glycans printed on an SGM. To validate the printing process, the SGM are typically interrogate with defined GBPs to be sure glycans were printed. Thus, in the process of validating the printing of an SGM with these reagents significant structural information was generated. Fig. 1 provides a description of the unique glycan determinants of a small selection of commercially available lectins and antibodies used to introduce this approach. Concanavalin A (Canavalia ensiformis agglutinin) is capable of detecting N-glycans due to its specificity for a-linked Man in branched Manal-3(Man l-6)Manal-R, as well as its weaker interaction with the internal trimannosyl core of bi-antennary N-glycans, but not with tri- or tetra-antennary or bisected N- glycans. Sambucus nigra agglutinin (SNA) is generally considered specific for Neu5Aca2- 6Gaipi-4GlcNAc, but it binds better to Neu5Aca2-6Gaipi-4GlcNAcpi-3Manal-3Man sequences on N-glycans than to the terminal sequence on the six branch of bi-antennary N- glycans. Maackia amurensis lectin-1 (MAL-1) detects Neu5Aca2-3Gaipi-4GlcNAc, and Erythrina cristagalli lectin (ECL) is specific for Gaipi-4GlcNAc. Among the fucose-binding lectins, the examples provided are Aleuria aurantia lectin (AAL), which has a rather broad specificity for a-linked Fuc, whereas Ulex europaeus agglutinin-I (UEA-I) is specific for H- antigen (Fucal-2Gal-R). A variety of anti-glycan monoclonal antibodies are now

commercially available. Five examples of different antibody specificities among the dozens defined to date are shown in Fig. 1.

MALDI-TOF data provide the molecular masses and the composition of the glycans printed on the SGM. The binding patterns of the different lectins can provide extensive structural information and can be extremely useful for differentiating glycans that have the same mass but a different arrangement of monosaccharides. In Fig. 2, the data are summarize that would be generated from a glycan microarray of 10 isobaric, bi-antennary N-glycans, whose structures are shown with the pattern of binding of the 11 GBP specificities described in Fig. 1. Interestingly, no two patterns were identical despite the fact that all of the glycans were biantennary N-glycans with the same composition. For example, glycans 6 and 7 (Fig. 2) differ only by the linkages of Gal and Fuc in the 3 -branch of the bi-antennary structure, but they are readily distinguished by their binding with anti-Lewis a (Le^a) and anti-Lewis x (Le^x) reagents. The positive MAL binding together with antibody binding confirmed the branched structure because these reagents are specific for terminal structures. The data, however, cannot determine on which branch each determinant resides. After neuraminidase digestion, ECL binding was positive indicating that the a3 -linked sialic acid is located on Gaipi- 4GlcNAc. Space does not permit the interpretation of each data point, but a large amount of data can be associated with each glycan on this example array. These metadata can be compiled in a database and used for predicting monosaccharide sequence and detailed structures of each individual glycan. This approach has been termed metadata-assisted glycan sequencing (MAGS).

MAGS is based on the analyses of many replicate arrays of undefined glycans (SGMs) that are interrogated by many different GBPs (Fig. 3). As the SGM is interrogated with defined GBPs as well as GBPs whose specificity and function are unknown, a database continues to be populated with information on each glycan. When a glycan is determined to be biologically relevant based on a binding event, additional information may be obtained by retrieving the glycan from the TGL for further analysis, although structural information on the entire glycome on the SGM can be addressed by evaluating the binding profile of defined GBPs before and after specific in situ exoglycosidase digestion on the arrays. Analysis of Human Milk SGM by MAGS

A variety of techniques have been applied to solve the structures of the complex mixture of isomeric glycans found in human milk. The neutral, monosialyl, and disialyl glycans of a milk sample were tagged with AEAB, separated by two-dimensional HPLC into 127 nearly homogeneous but not fully characterized glycans that made up the human milk TGL. During the production of the TGL, each fraction was analyzed by MALDI-TOF analysis and data accumulated for each glycan. The TGL was printed as a microarray of 127 glycans (n = 4) on an N-hydroxysuccinimide-derivatized microscope slide to produce the "human milk shotgun glycan microarray" or HM-SGM. To demonstrate the utility of the human milk glycan (HMG) array, it was interrogated with a variety of GBPs, including lectins and specific anti-glycan antibodies. No significant binding was observed with concanavalin A, Vicia villosa lectin, Griffonia simplicifolia lectin II (GSL-II), and M.

amurensis lectin I, consistent with the absence of mannose, terminal GalNAc, terminal GlcNAc, and terminal Neu5Aca2-3Gaipi-4GlcNAc, respectively, in human milk. The other six lectins, A. aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), Lotus

tetragonolobus lectin (LTL), U. europaeus agglutinin-I (UEA-I), Ricinus communis agglutinin I (RCA-I), and Erythrina cristagalli lectin (ECL), exhibited binding to many HMGs on the array.

In interrogations to identify the function of HMGs, a number of interesting features of these glycans were discovered. Some glycans contain epitopes for the monoclonal antibodies TRA-1-60 and TRA-1-81, which are specific for biomarkers of human embryonic pluripotent stem cells. Other specifically sialylated glycans are bound by fluorescently labeled influenza A virus and minute virus of mice (MVM), suggesting that HMGs may function as receptor decoys in an innate defense mechanism against potential pathogens. Overall, influenza A bound to eight glycans; MVM bound to six glycans, and the TRA-1 antibodies bound to six different glycans on the HM-SGM (total of 20 different glycans). Whereas molecular mass data provided compositions of the natural glycan ligands bound by these potential pathogens, more detailed MS data were unable to definitively solve the structures. To obtain more decisive structural characterizations and demonstrate the utility of the MAGS, 22 functionally identified glycans were retrieved from the TGL of the HM-SGM and 17 defined milk glycan standards were printed with them. Individual arrays were interrogated with eight lectins and five anti-glycan monoclonal antibodies that had been analyzed on the CFG glycan microarray to confirm their specificity and binding activity. After obtaining the initial patterns of binding, the subarrays were subjected to digestion with specific exoglycosidases either independently or in combination and subsequently

interrogated again with the appropriate lectins or anti-glycan antibodies with positive and negative binding indicating the presence or absence of the corresponding determinants. The structural data of 22 HMGs on the array were obtained simultaneously.

Several assumptions can be made regarding HMGs. They all have lactose as a reducing disaccharide and are composed of a single glucose residue with Gal and GlcNAc present in linear or branched sequences of Gaipi-3/4GlcNAc (LacNAc). The GlcNAc is linked β 1-3 to Gal in linear glycans with branches occurring when GlcNAc is attached β1-6 to Gal. These core glycans are then substituted with a-linked Fuc and a-linked Neu5 Ac to make up an extremely complex mixture of isomeric and isobaric glycans. The results of interrogation of the disialylated glycan and 10 standard milk glycans with a selection of defined lectins and antibodies are shown in Fig. 4A, and the results of interrogation after exoglycosidase and sequential exoglycosidase digestions are shown in Fig. 4B. The unknown glycan (predicted structure shown in Fig. 4, A and B) is an octasaccharide (mass = 1818.174) composed of three residues of Gal, one Glc, two residues of GlcNAc, and two residues of Neu5Ac based on the fact that it originated from human milk. ECL and the anti-SLe^a/LSTa antibody bind the unknown glycan, indicating that it is a branched structure containing a terminal type 2 glycan (ECL-positive) and a terminal LSTa determinant (absence of fucose excludes the possibility of SLe^a). ECL binding was weak and was presumably due to the steric effect of the sialylated branch, because the ECL binding signal increased by 3 -fold after neuraminidase treatment, and one branch must be a type 1 structure, which is not bound by ECL. Because digestion with a2-3 -neuraminidase leads to no change in ECL binding, it was assumed that the other sialic acid must be a2-6-linked and continues to block the ECL binding even after removal of the a2-3 -sialic acid (Fig. 4B). In addition, anti-type 1 antibody binding is observed only after removal of all the sialic acid by nonspecific neuraminidase. Thus, the disialylated glycan is predicted to contain one terminal type 2 and one terminal disialyl LNT (DSL). In addition, no GSL-II binding is observed after β1-4 galactosidase treatment (Fig. 4B), because GSL-II does not bind GlcNAcpi-6Gal, which would be exposed by sequential a2-3 -neuraminidase and β 1-3 -galactosidase digestion (Fig. 4B). However, sequential nonspecific neuraminidase and β 1-3 -galactosidase treatment did lead to strong binding to GSL-II, consistent with the predicted structure. The low SNA binding is consistent with the lack of SNA binding to the DSL standard (Fig. 4A). Taken together, the MAGS analysis permits a relatively conclusive prediction that the isolated glycan ligand bound by MVM is as shown in the unknown structure in Fig. 4A.

Nutritional and pharmaceutical compositions

In certain embodiments, the disclosure relates to nutritional supplements, such as infant formula, comprising one or more of the human milk glycans selected from those disclosed herein. In certain embodiments, the nutritional supplement comprises or consists of one, two, three, four, five, ten, fifteen, twenty, twenty five, thirty, forty, fifty, sixty, seventy, eighty, ninety, or more of the human milk glycans disclosed herein.

In certain embodiments, the nutritional supplement comprises or consists of H-71, H- 99, H-108, H-l 11, H-125, H-126, or H-127, and combinations thereof. In certain

embodiments, the nutritional supplement comprises or consists of H-28, H-30, H-31, H-36, H-43, or H-60, and combinations thereof. In certain embodiments, the nutritional supplement comprises or consists of H-l, H-5, H-6, H-12, H-14, H-15, H-16, H-17, or H-56, and combinations thereof.

In certain embodiments, the nutritional supplement compositions comprises one or more glycans disclosed herein and a component selected from raw milk, e.g., from a cow or goat, evaporated milk, or hydro lyzed milk.

In certain embodiments, the nutritional supplement compositions comprises one or more glycans disclosed herein and components selected from vitamins, minerals, fiber, fatty acids, or amino acids, and proteins. In certain embodiments, the nutritional supplement compositions comprises one or more glycans disclosed herein and components selected from biotin, choline, inositol, fat, linoleic acid, vitamins: A, C, D, E, K, thiamin (Bl), riboflavin (B2), B6, B12, niacin, folic acid, pantothenic acid, calcium, magnesium, iron, zinc, manganese, copper, phosphorus, iodine, carbohydrates such as sucrose, glucose, dextrins, lactose, and starches.

In certain embodiments, the nutritional supplement compositions comprises one or more glycans disclosed herein and components selected from emulsifiers such as

monoglycerides, diglycerides, and gums.

In certain embodiments, the composition comprises soy protein, egg albumin, glutamine, casomorphin, monomeric amino acids, branched-chain amino acids (BCAA) such as leucine, isoleucine, and valine, glutamine, essential fatty acids, cysteine, maltodextrin, oat fiber, brown rice, wheat flour, glutathione, creatine, creatine monohydrate, creatine ethyl ester, chrysin, and 4-androstene-3,6,17-trione.

In certain embodiments, the disclosure relates to pharmaceutical composition comprising a pharmaceutically acceptable excipient and a glycan with a terminal Neu5Aca2- 6Gaipi-4GlcNAc. In certain embodiments, the glycan is H-28, H-30, H-31 , H-36, H-43, or H-60, and combinations thereof. In certain embodiments, the glycan is H-1, H-5, H-6, H-12, H-14, H-15, H-16, H-17, or H-56, and combinations thereof. In certain embodiments, the pharmaceutical composition of further comprises a second anti-viral or anti-bacterial agent.

Pharmaceutical compositions disclosed herein may be in the form of pharmaceutically acceptable salts, as generally described below. Some preferred, but non-limiting examples of suitable pharmaceutically acceptable organic and/or inorganic acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citric acid, as well as other pharmaceutically acceptable acids known per se (for which reference is made to the references referred to below).

When the compounds of the disclosure contain an acidic group as well as a basic group, the compounds of the disclosure may also form internal salts, and such compounds are within the scope of the disclosure. When a compound contains a hydrogen-donating heteroatom (e.g. NH), salts are contemplated to covers isomers formed by transfer of said hydrogen atom to a basic group or atom within the molecule.

Pharmaceutically acceptable salts of the compounds include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non -toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate,

bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen

phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporated herein by reference.

The compounds described herein may be administered in the form of prodrugs. A prodrug can include a covalently bonded carrier which releases the active parent drug when administered to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Prodrugs include, for example, compounds wherein a hydroxyl group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl group. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol functional groups in the compounds. Methods of structuring a compound as prodrugs can be found in the book of Testa and Mayer, Hydrolysis in Drug and Prodrug Metabolism, Wiley (2006). Typical prodrugs form the active metabolite by transformation of the prodrug by hydrolytic enzymes, the hydrolysis of amide, lactams, peptides, carboxylic acid esters, epoxides or the cleavage of esters of inorganic acids.

Pharmaceutical compositions for use in the present disclosure typically comprise an effective amount of a compound and a suitable pharmaceutical acceptable carrier. The preparations may be prepared in a manner known per se, which usually involves mixing the at least one compound according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is again made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences. Generally, for pharmaceutical use, the compounds may be formulated as a

pharmaceutical preparation comprising at least one compound and at least one

pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active compounds.

The pharmaceutical preparations of the disclosure are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Generally, such unit dosages will contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the disclosure, e.g. about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

The compounds can be administered by a variety of routes including the oral, ocular, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes, depending mainly on the specific preparation used. The compound will generally be administered in an "effective amount", by which is meant any amount of a compound that, upon suitable administration, is sufficient to achieve the desired therapeutic or prophylactic effect in the subject to which it is administered. Usually, depending on the condition to be prevented or treated and the route of administration, such an effective amount will usually be between 0.01 to 1000 mg per kilogram body weight of the patient per day, more often between 0.1 and 500 mg, such as between 1 and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg, per kilogram body weight of the patient per day, which may be administered as a single daily dose, divided over one or more daily doses. The amount(s) to be administered, the route of administration and the further treatment regimen may be determined by the treating clinician, depending on factors such as the age, gender and general condition of the patient and the nature and severity of the disease/symptoms to be treated. Reference is again made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences.

Depending upon the manner of introduction, the compounds described herein may be formulated in a variety of ways. Formulations containing one or more inhibitors can be prepared in various pharmaceutical forms, such as granules, tablets, capsules, suppositories, powders, controlled release formulations, suspensions, emulsions, creams, gels, ointments, salves, lotions, or aerosols and the like. Preferably, these formulations are employed in solid dosage forms suitable for simple, and preferably oral, administration of precise dosages. Solid dosage forms for oral administration include, but are not limited to, tablets, soft or hard gelatin or non-gelatin capsules, and caplets. However, liquid dosage forms, such as solutions, syrups, suspension, shakes, etc. can also be utilized. In another embodiment, the formulation is administered topically. Suitable topical formulations include, but are not limited to, lotions, ointments, creams, and gels. In a preferred embodiment, the topical formulation is a gel. In another embodiment, the formulation is administered intranasally.

Formulations containing one or more of the compounds described herein may be prepared using a pharmaceutically acceptable carrier composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. As generally used herein "carrier" includes, but is not limited to, diluents, binders, lubricants, disintegrators, fillers, pH modifying agents, preservatives, antioxidants, solubility enhancers, and coating compositions.

Carrier also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. Delayed release, extended release, and/or pulsatile release dosage formulations may be prepared as described in standard references such as "Pharmaceutical dosage form tablets", eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al, (Media, PA: Williams and Wilkins, 1995). These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl

methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDPvAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients present in the drug-containing tablets, beads, granules or particles include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also referred to as "fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatimzed starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.

Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatimzed starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatimzed starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross- linked PVP (Polyplasdone XL from GAF Chemical Corp).

Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium

dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenyl ether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-beta-alanine, sodium N-lauryl-beta- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

If desired, the tablets, beads, granules, or particles may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, or preservatives.

In certain embodiments, the pharmaceutical composition comprises one or more glycans disclosed herein and an the anti-viral agent such as one selected from abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, complera, darunavir, delavirdine, didanosine, docosanol, dolutegravir, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfmavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin , raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, stavudine, stribild, tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate (TAF), tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, or zidovudine, and combinations thereof.

In certain embodiments, the pharmaceutical composition comprises one or more glycans disclosed herein and an anti-bacterial agent such as sulphadiazine, sulfones - [Dapsone (DDS) and Paraaminosalicyclic (PAS)], sulfanilamide, sulfamethizole,

sulfamethoxazole, sulfapyridine, trimethoprim, pyrimethamine, nalidixic acids, norfloxacin, ciproflaxin, cinoxacin, enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, ofloxacin, pefloxacin, sparfloxacin, trovafloxacin, penicillins (Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin ,Flucloxacillin, Hetacillin, Oxacillin, Mezlocillin, Penicillin G, Penicillin V, Piperacillin), cephalosporins (Cefacetrile, Cefadroxil, Cefalexin, Cefaloglycin, Cefalonium, Cefaloridin, Cefalotin, Cefapirin, Cefatrizine, Cefazaflur, Cefazedone, Cefazolin, Cefradine, Cefroxadine, Ceftezole, Cefaclor, Cefonicid, Ceforanide, Cefprozil, Cefuroxime, Cefuzonam, Cefinetazole, Cefoteta, Cefoxitin, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefixime,

Cefinenoxime, Cefodizime, Cefoperazone, Cefotaxime, Cefotiam, Cefpimizole, Cefpiramide, Cefpodoxime, Cefteram, Ceftibuten, Ceftiofur, Ceftiolen, Ceftizoxime, Ceftriaxone,

Cefoperazone, Ceftazidime, Cefepime), moxolactam, carbapenems ( Imipenem, Ertapenem, Meropenem) monobactams (Aztreonam ), oxytetracycline, chlortetracycline, clomocycline, demeclocycline, tetracycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, chloramphenicol, amikacin, gentamicin, framycetin, kanamycin, neomicin, neomycin, netilmicin, streptomycin, tobramycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin, polymyxin-B, colistin, bacitracin, tyrothricin, notrifurantoin, rurazolidone, metronidazole, imidazole, isoniazid, pyrazinamide, ethionamide, nystatin, amphotericin-B, hamycin, miconazole, clotrimazole, ketoconazole, fluconazole, rifampacin, lincomycin, clindamycin,

spectinomycin, chloramphenicol, clindamycin, colistin, rosfomycin, loracarbef,

metronidazole, nitrofurantoin, polymyxin B, polymyxin B sulfate, procain, spectinomycin, imidazole, trimethoprim, ramoplanin, teicoplanin, vancomycin, trimethoprim,

sulfamethoxazole, or nitrofurantoin and combinations thereof. Methods of use

In certain embodiments, the disclosure relates to methods comprising feeding a subject younger than one or two years old a nutritional supplement of disclosed herein. In certain embodiments, the nutritional supplement is feed with a composition comprising one or more glycans disclosed herein optionally in combination with an infant formula.

In certain embodiments, the disclosure relates to methods of treating or preventing a pathogenic infection comprising administering a pharmaceutical composition disclosed herein to a subject at risk of, exhibiting symptoms of, or diagnosed with a pathogenic infection. In certain embodiments, the composition is administered in combination with an antiviral or antibiotic agent.

EXAMPLES

Preparation of the Human Milk Glycome (HMG) Shotgun Glycan Microarray (SGM)

The generation of a SGM of HMGs from a single individual milk sample is illustrated in Fig. 5. To extract the human milk free glycome, the lipids, proteins, and most of the lactose were removed by centrifugation and ethanol precipitation. The glycans were obtained after size exclusion chromatography and then separated into three distinct groups (neutral, monosialyl, and disialyl) by ion-exchange chromatography. Adopting the shotgun glycomics approach, the glycan mixtures in each group were conjugated with AEAB and separated by two-dimensional HPLC into a total of 156 individual fractions. Selected fractions were subjected to an additional round of reverse-phase HPLC to further improve the purity and obtain 127 glycans in the TGL. The array contains 73 (57%) sialylated glycans and 54 (43%) neutral glycans. It should be noted that although the number of sialylated glycans exceeds the neutral ones, the total abundance of neutral glycans is much higher than that of sialylated ones. Analysis of the observed masses of all samples revealed glycans ranging in size from 2 residues (2 hexoses (Hex)), i.e. lactose, up to 12 residues (6 Hex + 4 HexNAc + 2

Fuc/Neu5Ac) and in mass from 506.337 [M+H]+ (2 Hex) to 2550 [M+H]+ (6 Hex + 4 HexNAc + 2 Neu5Ac). Of the 54 neutral glycans, >80%> carry 1-3 fucose residues, whereas less than half of the sialylated glycans are fucosylated. Of the 73 sialylated glycans, 17 were purified from the monosialyl group, and 56 were from the disialyl group. However, mass results indicated that some of the samples purified from the disialyl group only carry one sialic acid. After quantification using AEAB fluorescence, the 127 glycans were adjusted to the same concentration (regardless of their abundance) and printed (replicates of n = 5) on N- hydroxysuccinimide-activated glass slides along with 11 structurally defined glycans that serve as controls for binding experiments. Subsequently, the HMG microarray was interrogated with lectins and antibodies to evaluate whether the glycans were effectively printed.

Preliminary Characterization of the HMGs on the SGM by Lectins and Antibodies Defined glycan microarrays are used to explore the specificity of GBPs, including lectins and anti-glycan antibodies. In turn, GBPs with well-defined binding specificity can assist in the elucidation of glycan structures. To interrogate the SGM and evaluate the structural diversity of isolated HMGs, 10 biotinylated lectins, each at several concentrations in the range of 0.001-10 μg/ml, were applied to the array. No significant binding was observed with concanavalin A (Con A), Vicia villosa lectin (VVL), Griffonia simplicifolia lectin II (GSL-II), and Maackia amurensis lectin- 1 (MAL-1). These lectins recognize glycans containing mannose, terminal GalNAc, terminal GlcNAc, and terminal Neu5Aca2-3Gaipi- 4GlcNAc, respectively; the absence of their binding is consistent with the lack of such structures in human milk free glycans. Independently the binding of these lectins to a defined glycan microarray from the CFG (v5.0) was evaluated, and the data are available online as glycan array data on the CFG website. The other six lectins, Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), Lotus tetragonolobus lectin (LTL), Ulex europaeus agglutinin-I (UEA-I), Ricinus communis agglutinin-I (RCA-I), and Erythrina cristagalli Lectin (ECL), exhibited binding to many of the HMGs on the SGM as discussed below, and the binding histograms showing average relative fluorescence units (RFU) for selected concentrations of each lectin are shown in Fig. 6.

Three fucose binding lectins, AAL, LTL, and UEA-I, were used to reveal the fucosylated glycans on the SGM. Approximately 61 glycans (48%) showed strong binding with AAL (RFU > 10,000), which binds to terminal a-linked 1-fucose in 1-2, 1-3, and 1-4 linkages, and an additional 24 glycans (19%) had weaker binding (RFU 1,000-8,000).

Together, the fucosylated glycans recognized by AAL made up 67% of the total isolated HMGs, which is close to the percentage found by using HPLC-ChlP/MS method. In addition, most of the neutral glycans and about 40% of the sialylated glycans are fucosylated, in agreement with the results from glycan composition analysis based on mass. UEA-I, which is specific for αΙ-2-linked fucose on a type 2 chain, bound very weakly (<4,000 RFU) to several multifucosylated, neutral glycans (Fig. 6B), suggesting the presence of a 1-2 fucose on the HMG array. However, this weak binding was presumably due to the cross-reactivity of UEA-I with other fucose-containing glycans and actually not from Fuc l-2. This conclusion was supported by the observation that glycans were resistant to a 1-2 fucosidase digestion in solution. This result indicated the absence of H type 2 (Fucal-2Gaipi-4GlcNAc) and Ley (Fucal-2Gaipi-4(Fucal-3)GlcNAc) structures in the SGM. LTL recognizes al-3-linked fucose within type 2 glycans, like Le^x and Le^y determinants. It showed strong binding to three neutral glycans (H-75, H-83, and H-87) and weak binding to another five glycans (Fig. 6C). The three high affinity binders likely contain terminal Lex determinants because the absence of UEA-I binding (Fig. 6B) excludes the possibility of αΙ-2-linked fucose.

SNA, which binds to the Neu5Aca2-6Gaipi-4GlcNAc determinant, bound well to 14 (of 73) sialylated glycans on the SGM along with three control glycans (2-6-DS-NA2, LSTc, and fetuin), indicating the existence of a2-6-sialylated type 2 structure in 19% of the glycans (Fig. 6D). Both RCA-I and ECL recognize terminal Gaipi-4GlcNAc, but the former has much higher affinity and also binds to Neu5Aca2-6Gaipi-4GlcNAc with slightly lower affinity, whereas the latter can also bind Fucal-2Gaipi-4Glc. Consistent with these features, the binding pattern of RCA-I on this SGM was similar to the combined pattern of SNA and ECL (Fig. 6, D-F) together, suggesting that ~46% of glycans have terminal type 2 or sialylated type 2 structures. In summary, the results of lectin binding both validated the preparation of the SGM and provided significant structural information on individual HMGs.

The structures of HMGs reflect the Lewis blood type and secretor status of the mothers. Certain fucosylated glycans that were reported to possess biological functions that occur only in the milk of Lewis positive and/or secretor-positive mothers due to the expression of FUT-3 and FUT-2, respectively. Because there are no lectins specific for Lewis blood group antigens, the SGM were analyzed with several blood group-related monoclonal antibodies. As shown in Fig. 7A, the mAb to Lea bound to 34 glycans, including 9 sialylated ones, with fluorescence signals in the range of 500-15,000 RFU. To reveal additional Lea-containing glycans, the array was interrogated with an anti-sialyl Lea (SLe^a) antibody. According to the data from the structurally defined CFG glycan microarray, this antibody also binds to LSTa (Neu5Aca2-3Gaipi-3GlcNAcpi-3Gaipi-4Glc) moiety with lower signal. At the concentration of 10 μg/ml, sialylated glycans showed binding signals higher than 30,000 RFU with the antibody, and another 19 glycans had signals >5,000 RFU (Fig. 7B). Although one could not simply assign SLe^a and LSTa structures by signal intensity, it is certain that many of these sialyl glycans are fucosylated based on their AAL binding (Fig. 7A) and may contain the SLea moiety. Nevertheless, the abundance of Lea- containing glycans showed that the SGM was from a Lewis positive donor. To determine the secretor status of the donor, the SGM was interrogated with anti-Le^b and anti-blood group H type 1 antibodies. There was little to no binding observed with the anti-Le^b antibody at any concentration, which indicates that the milk sample is from a secretor negative donor. This finding was confirmed by assaying with anti-Hl antibody, which bound only the control LNFP I (Fucal-2Gaipi-3GlcNAcpi-6Gaip-4Glc) (Fig. 7C), the precursor of Le^b antigen.

The array was also interrogated with anti-CD 15 antibody, known to recognize Le^x antigen. Strong binding was observed to glycans H-98 and H-103 with weaker binding to several other neutral glycans (Fig. 7D). When comparing CD 15 antibody with lectin LTL, the two Le^x-recognizing proteins showed distinct specificity toward different HMGs, although with some overlap. This could indicate that the recognition does not solely depend on the Le^x determinant for complex glycans, and the nearby residues or branches could affect the binding. Detection of HMGs That Are Epitopes of Anti-TRA-1 Antibodies, Specific for Human Pluripotent Stem Cells

The predicted epitopes were reported that for mAbs anti-TRA-1-60 and anti-TRA-1- 81 based on binding data from Version 4.2 of the CFG glycan microarray. These mAbs, which are specific for human pluripotent stem cells, bound only to two glycans, both containing the type 1 lactosamine epitope, Gaipi-3GlcNAcpi-3Gaipi-4GlcNAc, on that version of the CFG glycan microarray. The two mAbs were further examined on Version 5.0 of the CFG array, which contains many multiantennary glycans with poly-N- acetyllactosamine, and strong binding by three additional glycans were observed.

Importantly, these three glycans (#522, #572, and #573) are multiantennary glycans with two to three type 1 lactosamine repeats at their non-reducing ends. In addition, weak but significant binding was observed to 387, a glycan with a fucosylated type 1 lactosamine chain. Considering that HMG is a rich source of type 1 and type 2 lactosamine structures, the SGM was interrogated with the two anti-TRA-1 antibodies at several concentrations (1-100 μg/ml), and the results at 50 μg/ml are shown in Fig. 7, E and F. Consistent with the CFG array data, TRA-1-60 and TRA-1-81 share similar receptor specificity as both bind glycans H-71, H-99, H-108, H-l 11, H-125, and H-127. However, unlike the CFG results, which showed no significant difference in the signal intensity and binding patterns for the two mAbs at 50 μg/ml, it was observed that with the SGM the signal intensity for TRA-1-60 binding was always several-fold higher than that of TRA-1-81 at the same concentration and that there are three more low affinity binders for TRA-1-60, H-109, H-l 12, and H-126 (Fig. 7E). To define the glycan epitope in HMG for TRA-1 antibodies, glycans H-71, H-99, H-108, fil l 1, H-125, H-126, and H-127 were retrieved from the TGL for further characterization. With the exception of sialylated glycan H-71, all of the TRA-1 -bound glycans are neutral fucosylated structures consisting of 2-4 lactosamine repeats. These glycans are predicted to possess type 1 lactosamine. The data also indicate that the two anti-TRA-1 mAbs recognize complex glycans, as described below.

Investigation of Virus Binding to the SGM

The HMG-derived SGM provides a library of 127 naturally occurring glycans that permits us to investigate the binding properties of biologically relevant proteins and pathogens and to provide interesting insights into the potential function of HMGs. To explore the general application of this HMG-derived SGM for exploring pathogen interactions, MVM and influenza virus were examined, both of which attach to the sialic acid on the surface of their target cells at the initial stage of infection.

Two prototype strains, empty capsid (MVMp-WT) and virus-like particle (MVMp- VLP), and two immunosuppressive strain mutants, MVMi-agD and MVMi-ggA, were tested at 200 μg/ml concentration and detected by a rabbit anti-MVM capsid antibody. All of the MVM viruses recognized glycans H-30, H-31, H-34, H-43, H-60, and H-73 with each non- WT strain binding several additional glycans (Fig. 8). Interestingly, the initial MALDI data showed that the 6 common binders are disialylated glycans, and the lectin and antibody binding data revealed that all of the binders were also recognized by the anti-SLe^a/LSTa antibody, suggesting that the terminal sialyl a2-3 -linked type 1 motif might be part of the binding determinant. This is a new finding compared with the previous report with CFG Glycan Array Version 3.0 (— 180 glycans), which concluded that MVMs specifically recognized a2-3-linked type 2 motif and MVMi also bound to a2-8-linked multi-sialylated glycans. These data are complementary to the data from the CFG array as there is very little overlap between the structures found on the CFG array and HMG-derived SGM. In fact, these observations indicate that the repertoire of glycan receptors of MVM is broader than originally reported, and these viruses prefer highly charged or possibly multi-sialylated glycans. Glycans were retrieved that were bound by all MVM strains from the TGL for more detailed structural analysis discussed below.

It has been reported that the highest binding of H1N1 isolates was toward sialylated poly-N-acetyllactosamine structures, which are abundant among HMGs. To determine if HMGs contained natural glycans capable of binding influenza A virus, the SGM were interrogated with three seasonal human H1N1 strains (A/Brisbane/59/2007,

A/Pennsylvania/08/2008, and A/Oklahoma/447/08), one human pandemic H1N1 strain (A/California/04/2009), and one H3N2 virus (A/Oklahoma/483/08) for comparison.

Consistent with previous reports, A/Brisbane isolate showed the broadest binding specificity and preferred glycans with terminal a2-6-linked sialic acid (Fig. 9A). Interestingly, the glycans recognized by A/Brisbane/59/2007 (H1N1) were the same glycans that bound SNA, which is specific for the determinant, Neu5Aca2-6Gaipi-4GlcNAc. The binding profile of A/Oklahoma/447/08 (H1N1) virus is similar to the A/Brisbane virus but displayed a much higher signal to noise ratio (Fig. 9B). When compared with binding data from lectins and antibodies, A/Oklahoma/447/08 (H1N1) displayed a clear preference for glycans with a2-6 sialic acid, binding strongly to 2-6-DS-NA2, LSTc, and all of the glycans bound by SNA. Additionally, like A/Brisbane/59/2007, several glycans were recognized by the anti- SLe LSTa antibody, indicating certain specificity toward a2-3 sialic acid-containing glycans. A/Pennsylvania/08/2008, which was shown to preferentially bind glycans having terminal a2-6 sialic acid when assayed on the CFG defined glycan array, differed from the other H1N1 strains, A/Oklahoma/447/08 and A/Brisbane/59/2007, in that it did not bind some of the HMGs (such as glycan H-12, H-15, H-16, H-53, and H-55) that possess the Neu5Aca2-6Gaipi-4 motif (Fig. 9C). This result indicates that the virus binding does not solely rely on the sialic acid linkage. In the case of A/California/04/2009, a human pandemic H1N1 isolate, the binding pattern overlaps with SNA (Fig. 9D), which confirms the results from CFG microarray that A/California/04/2009 has a restricted binding preference for a2-6 sialic acid-linked type 2 glycans. Furthermore, as a comparison to the H1N1

A/Oklahoma/447/08 virus, the A/Oklahoma/483/08 H3N2 virus isolate were tested (Fig. 9E) and a more restricted binding pattern was obtained where all the bound glycans contain Neu5Aca2-6Gaipi-4GlcNAc structure and were recognized by the H1N1

A/Oklahoma/447/08.

Finally, the binding properties of three human parainfluenza viruses were evaluated on the human milk SGM. It was reported that these viruses require the presence of

Neu5Aca2-3Gaipi-4GlcNAc motif. However, the M. amurensis lectin I binding data showed that this structure is not a component of the HMG. Thus, no binding was observed with the type 1 and 3 (hPIVl and hPIV3) viruses. By contrast, unlike type 1 and 3, the type 2 parainfluenza virus (hPIV2) displayed a very strict preference for a2-6-sialic acid- containing glycans (Fig. 9F), with receptor specificity similar to the H1N1 strains. Overall, the virus binding experiments demonstrated that many of the HMGs might function as decoys for cell- bound receptors and that the elements in the HMGs might be found on cell-bound receptors. Glycans that bound viruses were selected for more detailed characterization as described below.

Structural Characterization of Selected HMGs

Interrogation of the partially characterized HMG-derived SGM with antibodies against biological markers and viruses demonstrated the potential to identify the receptors of GBPs, including lectins, anti-glycan antibodies, and GBPs in pathogens. Although the existing lectin/antibody binding data already provided some common features of the receptors, detailed structural analysis is necessary to relate the specific structures with biological functions. Relevant glycans were selected from the human milk TGL and the use of tandem mass spectrometry and/or serial enzymatic digestion was attempted to decipher these structures. It was found that although MALDI analysis of the glycan derivatives generated excellent data and in some cases good secondary fragmentation data, more sophisticated analyses of permethylated glycan- AEAB derivatives were difficult to interpret due to the complexity of the spectra generated from partial methylation of the primary and secondary amines introduced by the AEAB. These complexities limited the detailed structural analysis of selected glycans from the TGL by MS/MS.

It was realized that the lectin-based analysis of the human milk SGM provided significant structural information in a rather high throughput format as all 127 glycans could be analyzed in a single assay. It was reasoned that digestion of the glycans on the microarray would be an approach to do in situ structural analysis by combining specific exoglycosidase digestion with defined lectin binding. To demonstrate this, a total of 22 functionally identified glycans were selected, and their HPLC profiles and MALDI-TOF spectra were taken. These structures include seven glycans bound by anti-TRA-1 antibodies (H-71, H-99, H-108, H-111, H-125, H-126, and H-127), six glycans bound by MVM (H-28, H-30, H-31, H-36, H-43, and H-60) and nine glycans bound by influenza viruses (H-1, H-5, H-6, H-12, H- 14, H-15, H-16, H-17, and H-56). These glycans were printed as a separate array designated "HMG subarray" on N-hydroxysuccinimide-derivatized slides along with 18 structurally defined glycan standards. The 18 control glycans represent some typical structural motifs found in human milk, such as type 1 and type 2 glycans and Lewis blood group glycans (structures shown in Fig. 10). The results from these glycans were used to monitor the behaviors of reagents and to direct structure predictions.

To obtain on-array sequence information, the non-reducing terminal structures of the selected glycans were first determined by screening the HMG subarray with a variety of defined lectins and antibodies whose specificities were defined by analysis on v5.0 of the CFG defined glycan array. It was reasoned that collection of these data along with the predicted compositional data from mass spectrometry could be combined as a collection of metadata and would provide information about the specific structures of glycans that mass spectrometry alone might not easily resolve. This approach has been referred to as MAGS. To this end, positive/negative binding by lectins or antibodies to each glycan indicates the presence/absence of the corresponding moiety that each lectin or antibody recognizes, e.g. ECL for Gaipi-4GlcNAc, AAL for fucose, and anti-Le^a antibody for Le^a epitope. The binding data from multiple GBPs were analyzed in detail to assign the structures. For example, if SNA, RCA-I, and anti-type 1 chain antibody, but not ECL, showed binding toward a glycan, it would suggest that this glycan might possess a terminal Neu5 Aca2- 6Gaipi-4GlcNAc determinant (SNA and RCA-I positive; ECL negative) together with a terminal Gaipi-3GlcNAc determinant (ECL negative and anti-type 1 antibody-positive). These data do not distinguish between an asymmetric biantennary glycan and the presence of two glycans; however, other metadata can be associated with the individual printed glycans such as a MALDI analysis to determine the number of molecular ions and composition and the shape of the individual peak(s) during HPLC to evaluate glycan homogeneity. The next series of experiments involved the use of a group of specific exoglycosidases to treat the glycans directly on the microarray followed by interrogating with defined lectins or antibodies. The gain and loss of binding can provide composition and/or linkage information for the terminal and penultimate sugar residues. To accomplish this the reaction condition of five exoglycosidases were optimized for on-array digestion, including the nonspecific neuraminidase from A. ureafaciens, the recombinant a2-3 -neuraminidase from Salmonella typhimurium, the jack bean βΐ— 4/6 galactosidase, the recombinant βΙ-3-galactosidase from Xanthomonas manihotis, and the recombinant βΙ-4-galactosidase from Bacteroides fragilis. It was found that longer incubation times and certain enzyme concentrations were needed to achieve effective digestion when compared with the reactions in solution. The binding results after enzymatic treatment can be divided into two categories, loss and gain. The loss of signal after digestion confirms the prediction from the positive signals before digestion, as in the case of neuraminidase treatment, where the loss of SNA or anti-SLe^a antibody binding confirms the presence of a specific sialic acid linkage. Similarly, β-galactosidase digestion confirms a type 1 or type 2 chain structure. For the type 1 chain, the binding of anti-type 1 chain antibody is specifically diminished by β 1-3 -galactosidase treatment, whereas β1-4- galactosidase has no effect. On the contrary, the binding of ECL to a type 2 chain is lost only after βΙ-4-galactosidase digestion. Furthermore, the type 1 and type 2 structures can also be distinguished by the gain of G. simplicifolia lectin-II binding after βΙ-3/4-galactosidase digestion removes a terminal Gal revealing a terminal GlcNAc.

Beyond the single enzymatic treatment, sequential digestion involving desialylation was also conducted first with neuraminidase or a2-3 -specific neuraminidase followed by specific β-galactosidase treatment. This set of experiments is particularly useful for sialylated glycans and HMGs that are comprised of many isomers of type 1 and type 2 linear and branched lactosamines and poly-N-acetyllactosamines. Finally, all of the collected metadata including molecular ions, fragmentation MS or MS/MS data, and the behavior on ion exchange chromatography and HPLC are combined to provide predictions of structures that were not possible with MALDI-TOF and MALDI-TOF/TOF analyses alone. Using this MAGS approach, the structural moieties were predicted and most of the linkages for 20 of the 22 HMGs as shown in Fig. 10. The structures of H-06 and H-71 were not proposed because these glycans appeared to be mixtures. Six pairs of glycans were found to be the same structure (H1/H5, H14/H16, H15/H17, and H28/H30) or have the same general structures (H43/H60 and HI 11/H126) based on HPLC profiles, MALDI analysis, and binding data. This is thought to be due to the overlap of glycans in the fractions obtained during the multi-dimensional chromatography. In addition, several samples were

contaminated with minor impurities. Nevertheless, the correlation of proposed structures with the function defined by antibody and virus binding revealed interesting findings. The five influenza virus receptors, including monosialylated H-01/05, H12, H14/16, H15/17, and disialylated H-56 all contain the Neu5Aca2-6Gaipi-4Glc/GlcNAc moiety as indicated by SNA, RCA-1, ECL, neuraminidase, and β1-4 galactosidase data. Except for the sialyl lactose (HO 1/05), the other four structures are biantennary glycans with one type 2 chain branch. It appears that the other branch can be diverse structures, as the presence of Le^a, Le^x, type 2 chain, and sialylated type 1 chain were observed. The on-array structural analysis also revealed common features for the MVM receptors. Mostly relying on antibodies (anti- SLe LSTa and anti-type 1) and exoglycosidases (specific and unspecific neuraminidase) data, it is proposed that the four disialyl structures all carry an a2-3 -sialylated type 1 chain with an additional sialic acid attached to the GlcNAc in a2-6 linkage (Neu5Aca2-3Gaipi- 3(Neu5Aca2-6)GlcNAc). It is possible that the disialyl LNT motif is one of the recognition determinants for MVM. Similar to influenza virus, modifications such as fucosylation and branching on this motif did not block the virus recognition. Furthermore, these results together with the CFG data indicated that the recognition of MVMs is beyond the sialic acid as the viruses did not bind to all the multisialylated glycans.

In the case of anti-TRA-1 antibodies, the sialylated binder H-71 was found to have relatively low purity, and thus its structure was not elucidated. The other six binders (H-99, H-108, H-l 11, H-125, H-126, and H-127) are neutral complexed glycans, especially for the latter four, which are multi -branched structures. Although all of the linkage information for these large glycans were not obtain, it was found that all the receptors contain the common motif: type 1 lactosamine epitope. Although H-99 is a relatively simple lactosamine glycan, similar to the structure identified from CFG array, the others contain an additional type 2, Le^a or Le^x branch, and it seems that these extra branches do not prevent the binding of the antibodies. The structures corresponding to glycans are defined in Fig. 10. Glycan 19 (H-01) - The HPLC profile of H-01 shows a single symmetrical peak and the MALDI-TOF analysis shows a molecular ion at 797.5 [M+H]+, consistent with a trisaccharide containing two hexoses and one sialic acid. Because these are human milk oligosaccharides, it is assumed that the two hexoses are lactose and this glycan must be a sialyl lactose (Neu5Aca2-3/6Gai i-4Glc). When looking at the lectin/antibody binding data, H-01 only shows weak binding to SNA and RCA-I, which indicates the sialic acid might be an a2-6 linkage because replacement of the GlcNAc in the recognition motif (Neu5Aca2-6Gaipi-4GlcNAc) with Glc greatly reduces the binding affinity of SNA. When H-01 is incubated with non-specific

neuraminidase, a product that co-migrates with lactose-AEAB is observed by HPLC. In contrast, no new product occurs with a2-3 -specific neuraminidase. Therefore, it is conclude that H-01 is 6'- sialyllactose.

5 p

H-01

Glycan 20 (H-05) - H-05 shows the same molecular ion 819.501 [M+Na+], HPLC retention time and binding profile as H-01 and no new product occurs with a2-3-specific neuraminidase digestion, therefore it is considered to have the same structure as H-01.

Redundancies were anticipate such as this due to the first dimension of HPLC being collected by time instead of individual peaks. Glycan 21 (H-06) - H-06 is a mixture of two components as shown by the second dimension HPLC on PGC. The major peak is around 60% and the second peak (-20%) shares the same retention time with H-01. MALDI-TOF showed a mass of 807.485 which does not match any HMG composition. Like H-01 and H-05, H-06 only weakly binds to SNA and RCA-I. Due to the low purity of H-06, its structure is not predicted, but it is assumed that it contains some of the H-01 (6'-sialyllactose) which contributed to the influenza virus binding.

Glycan 22 (H-12) MALDI-TOF analysis of H-12 shows molecular ions at 1717.938 [M+2Na]+ and 1404.834 [M+Na]+ that is consistent with compositions of

Hex4HexNAc2FuclNeu5Acl and the desialylated form (Hex4HexNAc21Fucl),

respectively. Because neutral and sialyl sugars are easily separated by HPLC and H-12 profile shows a single symmetrical peak, the desialylation presumably occurred during the mass fragmentation and H-12 is composed of 1 lactose, 2 Gai i-3/4GlcNAc units, 1 fucose and 1 Sialic Acid; but, the MS data cannot provide information on the arrangement of these units. However, the lectin and antibody binding data can provide some information on their arrangement. For example, since H-12 is bound strongly by SNA, anti-Le^a antibody and anti- SLe LSTa antibody, it presumably contains three different terminal structures: Neu5Aca2- 6Gaipi-4GlcNAc based on SNA binding; Gaipi-3(Fuca-4)GlcNAc based on anti-Le^a antibody; and Neu5Aca2-3Gaipi-3(Fuca-4)GlcNAc and/or Neu5Aca2-3Gaipi-3GlcNAc based on anti-SLe^a/LSTa antibody. However, a three-branched structure does not agree with the composition. Therefore, it is predict that H-12 is actually a mixture of two isomers that were not separated by two-dimensional HPLC. Since the MALDI analysis indicates only one composition containing one fucose and one Sialic Acid, the two isomers must have one sialylated branch (sialyl type 2 or SLe^a/LSTa) and one neutral branch (type 2 or Le^a). Based on previous structural analyses of HMG, in a branched HMG, the lactosamine branch attached to the 6-position of the Gal in lactose is always a type 2 glycan (Gaipi-4GlcNAcpi- 6) or sialylated/fucosylated type 2 glycan. Thus, one isomer must have a Neu5Aca2-6Gaipi- 4GlcNAcpi-6 branch and a Le^a branch consistent with fucose positive and Le^a positive structure A. For the isomer B, one of the branches must be SLe^a or LSTa, a sialylated type 1 structure, and the other branch should be a type 2 glycan. Further, since there is no Le^x binding observed, the fucose must be on the type 1 glycan and the SLe^a is on the other branch of structure B.

H-

The enzyme digestion results also support the prediction since SNA binding is eliminated by non-specific neuraminidase, but not by the a2-3 -specific neuraminidase, consistent with the A structure. The weak RCA-I, ECL binding to untreated H-12 glycan and the weak ECL binding to the desialylated H-12 are believed to be due to the steric effect of the nearby Le^a branch on H-12 A and SLe^a branch on H-12 B. For the same reason, the combination of neuraminidase and jack bean galactosidase (only β1-4 galactosidase activity) treatment also reveal very little gain of GSL-II binding. In the case of antibody binding, both non-specific neuraminidase and a2-3 -specific neuraminidase digestion increase the binding by anti-Le^a antibody, the result of exposure of the Le^a terminal of H-12 B glycan. At the same time, anti-SLe^a/LSTa antibody binding is mostly lost after neuraminidase and a2-3-specific neuraminidase digestion, also consistent with the proposed structures.

Glycan 23 (H-14) - A mass of 1571.898 [M+Na]+ is observed for H-14 consistent with a composition of Hex4HexNAc2Neu5Acl, which could represent one N-acetyl lactosamine (Gai i-3/4GlcNAc), one sialyl lactosamine (Neu5Aca2-3/6 Gai i-3/4GlcNAc) and one lactose. Although these three units could assemble to either a linear or a branched structure, strong binding of SNA, RCA-I and ECL by the untreated glycan indicates that H- 14 contains both Neu5Aca2-6Gaipi-4GlcNAc and Gaipi-4GlcNAc at terminal positions making this a branched glycan with the structure proposed below. In addition, sequential digestion with non-specific neuraminidase, but not a2-3 -specific neuraminidase, and β1-4 galactosidase, results in strong GSL-II binding which is consistent with exposing a GlcNAc on the 3 branch of Lactose since GSL-II does not bind GlcNAc i-6Gal. Interestingly, GSL-II binding to H-14 after β1-4 galactosidase digestion is not observed, consistent with the free terminal Gal of the proposed H-14 structure being on the 6-branch of the Lactose since GSL- II does not bind GlcNAc i-6Gal. No antibody binding is observed for H-14, which is also consistent with the predicted structure.

Glycan 24 (H-15) - H-15 has a mass of 1718.004 [M+2Na]+ and thus the

composition of Hex4HexNAc2FuclNeu5Acl . H-15 showed strong binding of SNA, RCA-I, AAL and anti-Le^x antibody. Accordingly, a branched structure was assigned with terminal Neu5Aca2-6Gaipi-4GlcNAc recognized by SNA and RCA-I and not by ECL, and Le^x motif (Gaip 1 -4(Fucal -3)GlcNAc) recognized by anti-Le^x. This prediction is supported by the binding results after enzyme digestions. SNA binding is eliminated and ECL binding is generated by the non-specific neuraminidase, but not by the a2-3-specific neuraminidase. Digestion with galactosidase alone has no effect on the lectin binding since the enzyme does not work on sialylated or fucosylated type 1 or type 2. Sequential digestion with non-specific neuraminidase and β1-4 galactosidase treatment exposes the GlcNAc on the Neu5Aca2- 6Gaipi-4GlcNAc and a strong signal is observed indicating that this moiety is on the 3- branch of the Lactose moiety.

Glycan 25 (H-16) - H-16 displays the same molecular ion (1571.915 [M+Na]+), HPLC retention time and lectin binding profile as H-14, therefore it is considered to have the same structure as H-14.

Glycan 26 (H-17) - H-17 displays the same molecular ion (1717.984 [M+Na]+), HPLC retention time and lectin binding profile as H-15, therefore it is considered to have the same structure as H-15.

Glycan 27 (H-28) - The HPLC profile indicates that H-28 is a mixture of two glycans. The minor component (20%) has the same retention time as H-30. MALDI also shows two components (1306.474 [M-H]+ and 1619.515 [M-Na]+) that match a monosialyl composition Hex3HexNAclFuclNeu5Acl and a disialyl composition

Hex3HexNAclFuclNeu5Ac2. The two components only differ by one sialic acid, so it is highly possible that the monosialyl glycan originated from the partial desialylation of the disialyl glycan. The composition indicates that H-28 must be a linear structure with one lactose and one Gah31-3/4GlcNAc unit. One sialic acid should be at the terminal and the other one is presumably attached to the GlcNAc, which is a common linkage in human milk oligosaccharides.

The lectin binding does not provide much information, and only strong AAL binding is observed, consistent with the presence of a single fucose. Specific antibody binding proved to be very useful for this structural analysis. The strong binding of anti-SLe^a/LSTa antibody suggests that a sialic acid is attached to a Gaip 1-3 GlcNAc in an a2-3 linkage. Even if there are two sialic acids present, it is know that anti-SLe^a/LSTa antibody can bind to disialyl glycans, since DSLNT is bound by this antibody. The exoglycosidase digestion data provide additional structural information. Removal of the sialic acids with non-specific

neuraminidase abolishes the binding of anti-SLe^a/LSTa antibody and gives positive binding to the anti-Le^a antibody, which confirms the presence of the Le^a motif. In addition, digestion with a2-3 neuraminidase diminishes the binding to anti-SLe^a/LSTa antibody as well, but this enzyme digestion does not result in anti-Le^a binding, which confirms the a2-3 sialic acid linkage, and also indicates the presence of an a2-6 sialic acid linkage, which interferes with the anti-Le^a binding. Taken together, it is predicted that H-28 contains structure A, a disialyl Le^a and structure B, a monosialyl Le^a. Most likely the B structure came from the loss of the a2-3 sialic acid of the A structure. B does not contribute to any binding until the a2-6 sialic acid is removed by non-specific neuraminidase, not the a2-3 neuraminidase.

Glycan 28 (H-30) - H-30 has a molecular mass of 1600.136 [M+H]+, which matches a composition of Hex3HexNAclFuclNeu5Ac2. Its HPLC profile shows a relatively pure peak. The binding profile of H-30 is the same as H-28 except for the signal intensity. It is bound by anti-SLe^a/LSTa antibody with 3 -fold higher signal at 1 μg/ml (data not shown), presumably due to the higher purity. Since the antibody binding after neuraminidase digestions are also the same as H-28, it is predicted that H-30 is the pure disialyl Le^a or disialyl-LNFII (DSLNFII), also structure A of H-28.

Glycan 29 (H-31) - H-31 has a major molecular ion of 1582.119[M+H]+ that matches with the composition of H-30 (Hex3HexNAclFuclNeu5Ac2 minus H₂0), therefore, it is also a disialyl linear glycan with one fucose, like H-28 and H-30. However, the lectin and antibody binding behavior of H-31 is slightly different. It binds to AAL weakly compared to H-28 and H-30, which suggests the fucose may be attached to the Glc instead of the GlcNAc. Once again, the antibody binding provides the linkage information of the sialic acids. H-31 is bound by anti-SLe^a/LSTa antibody, and this binding is diminished by either non-specific neuraminidase or a2-3 neuraminidase treatment, indicating the a2-3 linkage of the terminal sialic acid. The weak anti-Le^a antibody binding after removal of sialic acids also confirms that the fucose is not linked to the GlcNAc and presumably linked to the glucose at the reducing end, which is a common structure in human milk oligosaccharides. It is proposed that H-31 is a disialyl LNT with a fucose linked a 1-3 to the Glucose at the reducing end.

Glycan 30 (H-36) - H-36 has a molecular mass of 1818.174 [M+H]+ that matches an octasaccharide with the composition of Hex4HexNAc2Neu5Ac2. RCA-I, ECL and anti- SLe LSTa antibody binds H-36, indicating that it is a branched structure containing a terminal type 2 glycan (ECL positive) and a terminal LSTa determinant, since the absence of fucose excludes the possibility of SLe^a motif. The weak ECL binding may be due to the steric effect of the sialylated branch since the ECL binding signal increases by three fold after neuraminidase treatment and one branch has to be a type 1 structure. In addition, since digestion with a2-3 neuraminidase leads to no change in ECL binding, it is assumed that the other sialic acid is in an a2-6 linkage and continues to block the ECL binding even after removal of the a2-3 sialic acid. More evidence is observed from the anti-type 1 antibody binding, which is observed only after removal of all the sialic acids by non-specific neuraminidase. Taken together, H-36 is predicted to contain one terminal type 2 and one terminal disialyl LNT. In addition, No GSL-II binding is observed after β1-4 galactosidase treatment because GSL-II does not bind GlcNAc i-6Gal which would be exposed by β 1-3 galactosidase digestion of H-36. However, the sequential neuraminidase and β1-3

galactosidase treatment lead to strong binding to GSL-II, consistent with the DSL structure. The low SNA binding is probably the result of steric hindrance from the LacNAc on the 6 position of the Lactose moiety.

Glycan 31 (H-43) - H-43 has a molecular mass of 2111.394 [M+H]+, corresponding to the composition of Hex4GlcNAc2Fuc2Neu5Ac2. HPLC profile shows it has a closely- migrating impurity. When compared with H-30, H-43 displays the same binding properties. The native glycan only binds AAL and anti-SLe^a/LSTa antibody. After neuraminidase treatment, strong anti-Le^a antibody binding is revealed and the anti-SLe^a binding is diminished, indicating the presence of SLe^a structure. Because no other lectin or antibody binding is observed, H-43 must have no non-sialylated terminal regardless of whether it is branched or not. The possibility of two sialyl Le^a termini was excluded because no anti-Le^a antibody binding is gained after a2-3 neuraminidase treatment. The possibility of one sialyl Le^a and one sialyl Le^x terminus was also exclude because no anti-CD 15 antibody binding is observed after non-specific neuraminidase digestion. Finally, it is predicted that H-43 is similar to H-30 but with an addition LacNAc unit. The second fucose could be either attached to the GlcNAc or Glc.

Glycan 32 (H-56) - H-56 is a disialyl glycan based on its behavior on DEAE cellulose chromatography, and while positive mode of MALDI is not able to provide clear signal for the disialyl moiety, negative mode of MALDI gives a molecular mass of 1838.687 [M+Na]^" that is consistent with an octasaccharide composition of Hex4HexNAc2Neu5Ac2, like glycan H-36. A monosialyl form is also seen in the MS spectrum and is presumably due to the fragmentation in the mass analysis. H-56 is bound strongly by SNA, RCA-I and anti- SLe^a/LSTa antibody, which again indicates a branched structure with terminal Neu5Aca2- 6Gaipi-4GlcNAc and a terminal LSTa motif (Neu5Aca2-3Gaip 1-3 GlcNAc). However, binding of AAL suggests that this glycan is probably slightly contaminated with a fucose- containing glycan. After neuraminidase treatment of H-56, SNA binding is lost and ECL binding is observed, which is consistent with the Neu5Aca2-6Gaipi-4GlcNAc structure. Although weak ECL binding is observed after a2-3 neuraminidase digestion, this is probably due to non-specific hydrolysis by this enzyme after a relatively long incubation time since some decrease in the SNA signal was also observed. Because of the lack of fucose in the composition of the major component, the second branch is assumed to be an LSTa structure, which is supported by the observation that nonspecific neuraminidase digestion eliminates binding the anti-SLe^a/LSTa antibody while binding by anti-type 1 antibody appears. These data are consistent with the major component of H-56 being:

However, significant GSL-II binding is revealed after combined non-specific neuraminidase and β1-4 galactosidase digestion, which is not consistent with this structure since such treatment would generate a terminal GlcNAc l-6Gal that is not bound by GSL-II. In addition, the a2-3 neuraminidase digestion does not generate binding by anti-type 1 antibody, possibly due to the steric effect from the nearby branch, but also possibly due to it being a low concentration. Thus, H-56 might be better represented as a mixture of two closely related structures H-56 A and B:

H-56

Glycan 33 (H-60) - H-60 shows the same molecular mass, HPLC retention time and lectin binding profile as H-43, therefore it is considered to have the same structure as H-43.

Glycan 34 (H-71) - Both MALDI and HPLC shows that H-71 is a mixture of several glycans. The binding profile also indicates the mix of multiple glycan structures. Due to the low purity, H-71 structure is not predicted.

Glycan 35 (H-99) - H-99 is a neutral glycan. MALDI spectrum shows the existence of two major fractions with molecular masses 1258.788 [M+Na]+ and 1623.991 [M+Na]+, which correspond to Hex4HexNAc2 and Hex5HexNAc3, respectively. The HPLC profile also shows that a minor fraction migrates very closely with the major fraction. Thus, H-99 should contain a lactose reducing terminal and 2-3 additional disaccharides consisting of Gai i-3/4GlcNAc. The additional disaccharides (either type 1 or type 2) could be in a linear or branched configuration, with a number of branching possibilities.

The lectin binding analysis shows that H-99 did not bind SNA, MAL-1, GSL-II, AAL, UEA-I, or LTL, which is consistent with the absence of sialic acid and fucose based on MS. Strong binding by RCA-I, ECL and by the type 1 antibody, indicates that H-99 has two distinct non-reducing termini - a terminal Gai i-4GlcNAc detected with RCA-I and ECL, and a terminal Gai l-3GlcNAc detected by the anti-type 1 antibody. In addition, digestion of H-99 with galactosidase specific for Gai i-3GlcNAc or with galactosidase under conditions specific for Gai i-4GlcNAc results in GSL-II binding, thus supporting the existence of both type 1 and type 2 termini. However, the GSL-II binding resulting from digestion with β 1-3 galactosidase is 15 -fold higher than the GSL-II binding resulting from digestion with β1-4 galactosidase, suggesting a predominance of Gai i-3GlcNAc in this fraction. Based on the results, H-99 is predicted to be an octasaccharide mixed with a small amount of

hexasaccharide.

Glycan 36 (H-108) - MALDI-TOF analysis of H-108 shows a single molecular ion (1269.014) that does not match any known HMG composition. The closest composition for (1258.48[M+Na+]) is Hex4HexNAc2, however, the binding data does not fit such a composition. H-108 strongly binds RCA-I, ECL and AAL, indicating the presence of type 2 glycan and fucose. The moderate binding toward anti-Le^x antibody and the very weak binding toward anti-Le^a antibody also shows that there is fucose in the H-108 fraction, which is possible since the HPLC shows additional peaks beside the major peak. In addition to the type 2 and the fucosylated glycan, H-108 also binds to the anti-type 1 antibody and GSL-II binding is gained after β1-3 galactosidase treatment. The strong signals suggest that the type 1 glycan should be in the major fraction. β1-4 galactosidase treatment reveals weak GSL-II binding, which is observed multiple times from other glycans and is attributed to the linkage preference of GSL-II lectin. Despite the positive binding from the fucose-related lectin/antibodies, the major fraction of H-108 is predicted to be a hexasaccharide with one terminal type 1 glycan and one terminal type 2 glycan.

Glycan 37 (H-lll) - MALDI-TOF analysis indicates that this glycan is comprised of two distinct molecular ions with masses of 1623.917[M+Na]+ and 2135.205 [M+Na]+, which are consistent with compositions Hex5HexNAc3 and Hex6HexNAc4Fucl, respectively. H-l l l is bound by RCA-I, AAL, anti-type 1 antibody and anti-Le^x antibody. The single fucose is assigned to the Le^x motif (Gaipi-4(Fucal-3)GlcNAc), and the data also support the presence of both type 1 and type 2 glycans in the glycan mixture. After β1 -3 galactosidase treatment, very strong GSL-II binding is observed, indicating a type 1 glycan on a 3-branch must be in the major fraction or even present as multiple termini. The enzyme digestion and lectin binding data of the octasaccharide with the composition Hex5HexNAc3 is difficult to interpret since it is in a mixture, and the RCA-I and ECL binding to this fraction are not typical of a terminal Gai i-4GlcNAc. It is possible that terminal Gai i-4GlcNAc is simply a minor determinant in H-l 11. The absence of fucose from the octasaccharide prevents us from identifying more than one termini and this could be a linear structure, but linear structures of this size seem to be rare. It is predict that the octasaccharide component of H-l 11 is shown as H-l 11 A:

H-111

For the glycan with the composition Hex6GlcNAc4Fucl, there are a number of possibilities, but based on previous structural studies (Ref. 1 and 2), it is predicted that the undecasaccharide is most probably the structure shown as H-l 11 B. This prediction is consistent with what is known about HMG structures and explains the weak binding by ECL and the strong binding of GSL-II after β 1-3 galactosidase digestion and no GSL-II binding after i-4galactosidase digestion.

Glycan 38 (H-125) - HPLC profile of H-125 shows a relatively symmetrical peak, but MALDI analysis indicates two major components (2281.374 and 2135.278 [M+Na]+) that differ by a single fucose (mass=146) suggesting that the compositions are

Hex6HexNAc4Fuc2 and Hex6HexNAc4Fucl, respectively. Strong binding of H-125 by the lectins RC A-I, ECL and AAL, as well as by antibodies against type 1 , Le^a and Le^x, strongly suggests glycan H-125 contains four distinct non-reducing termini: the Le^a determinant detected by anti-Le^a antibody, the Le^x determinant detected by the anti-Le^x antibody, a terminal Gai l-4GlcNAc detected with RCA-I and ECL, which do not bind the Le^x determinant, and a terminal Gai i-3GlcNAc detected by the positive binding of the anti-type 1 antibody. Digestion of glycan H-125 with β 1-3 -specific galactosidase or with β 1 -4-specific galactosidase results in some increase in GSL-II binding, supporting the existence of both type 1 and type 2 termini in the preparation. Since the size of the glycan(s) is not large enough to accommodate 4 terminal disaccharides, the presence of 4 distinct terminal determinants indicates glycan H-125 is a mixture of isobaric structures and most probably represented by the structures shown in A, B, and C below, where the glycans can have one or two fucose residues located in 3 possible positions with either type 1 or type 2 disaccharides on the middle branch. Inspecting this structure, one notices that even with the limitation of core structures by the assumption that all extensions of the β 1-6 branch points are type 2 (Gai i-4GlcNAc) and that fucose is substituted only on GlcNAc in a terminal disaccharide, there are still 12 possible isobaric structures with one fucose, and 24 possible isobaric structures with 2 fucoses. Nevertheless, based on data from defined GBP binding the array before and after specific glycosidase digestions, it is possible to make logical predictions and limit the possible isobaric structures to a manageable number to assist in interpretation of MS data from "deeper" analysis. H-125

Glycan 39 (H-126) - H-126 has a molecular mass of 2135.303[M+Na]+ that is consistent with composition of Hex6HexNAc4Fucl, identical to H-111. However, unlike H- 111 , it is a relatively pure fraction and the retention time in HPLC is about the same as the major fraction of H-111. H-126 is bound by RCA-I, anti-type 1 antibody and anti-Le^x antibody, and weakly by RCA-I and poorly by ECL, suggesting that it may have two or three distinct branches. Because the lectin and antibody binding profile of H-126 match with H- 111, it is considered to have the same general structure as H-111 B.

Glycan 40 (H-127) - H-127 appears to be comprised of a major component with at least one other glycan as shown by HPLC. The major component has a molecular mass of 1989.203 [M+Na]+, which is consistent with a composition of Hex6HexNAc4. The minor fraction has an additional fucose (2281.388[M+Na]+) that matches with a composition of Hex6HexNAc4Fucl found in H-111. H127 is bound by RCA-I, ECL, AAL, anti-Le^a antibody and anti-type 1 antibody, suggesting the existence of type 1, type 2 glycan and Le^a motif, Gah31-3(Fucal-4)GlcNAc. The Structure proposed below for H-127 is consistent with the strong RCA-I and ECL binding that is not observed for glycans H-126 and H-111. The AAL and anti-Le^a binding detects the fucosylated component. The strong GSL-II binding found after β1-3 galactosidase treatment and the absence of binding of GSL-II after β1-4 galactosidase digestion is also consistent with the proposed structures.

Claims

CLAIMS What we claim:

1. An array of comprising a plurality of zones wherein each zone contains a conjugate comprising a glycan conjugated to a fluorescent tag, wherein the glycan is derived from milk of a mammal.

2. The array of claim 1 , wherein the mammal is a human.

3. The array of claim 1, wherein the number of zones is greater than 20, 50, 100, 200, or 300.

4. The array of claim 3, wherein the glycan is selected from HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, H18, H19, H20, H21, H22, H23, H24, H25, H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H36, H37, H38, H39, H40, H41, H42, H43, H44, H45, H46, H47, H48, H49, H50, H51, H52, H53, H54, H55, H56, H57, H58, H59, H60, H61, H62, H63, H64, H65, H66, H67, H68, H69, H70, H71, H72, H73, H74, H75, H76, H77, H78, H79, H80, H81, H82, H83, H84, H85, H86, H87, H88, H89, H90, H91, H92, H93, H94, H95, H96, H97, H98, H99, H10, H101, H102, H103, H104, H105, H106, H107, H108, H109, H110, Hi l l, H112, H113, H114, H115, H116, H117, H118, H119, H120, H121, H122, H123, H124, H125, H126, and H127.

5. A computer readable medium comprising data on the sequence of the glycan in each zone of an array of claim 1.

6. A device comprising an array of claim 1, a source of electromagnetic irradiation, and a fluorescence detector.

7. A nutritional supplement comprising one or more of the human milk glycans selected from HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, H18, H19, H20, H21, H22, H23, H24, H25, H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H36, H37, H38, H39, H40, H41, H42, H43, H44, H45, H46, H47, H48, H49, H50, H51, H52, H53, H54, H55, H56, H57, H58, H59, H60, H61, H62, H63, H64, H65, H66, H67, H68, H69, H70, H71, H72, H73, H74, H75, H76, H77, H78, H79, H80, H81, H82, H83, H84, H85, H86, H87, H88, H89, H90, H91, H92, H93, H94, H95, H96, H97, H98, H99, H10, H101, H102, H103, H104, H105, H106, H107, H108, H109, Hl lO, Hi l l, H112, H113, H114, H115, H116, H117, H118, H119, H120, H121, H122, H123, H124, H125, H126, and H127.

8. The nutritional supplement of claim 7 comprising five, twenty five, thirty, forty, fifty, sixty, seventy, eighty, ninety, or more of the human milk glycans.

9. The nutritional supplement of Claim 8 comprising or consisting of H-71, H-99, H- 108, H-l 11, H-125, H-126, or H-127, and combinations thereof.

10. The nutritional supplement of Claim 8 comprising or consisting of H-28, H-30, H-31, H-36, H-43, or H-60, and combinations thereof.

11. The nutritional supplement of Claim 8 comprising or consisting of H-l, H-5, H-6, H-

12. H-14, H-15, H-16, H-17, or H-56, and combinations thereof.

12. A method comprising feeding a subject younger than one or two years old a nutritional supplement of claim 8.

13. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a glycan with a terminal Neu5Aca2-6Gaipi-4GlcNAc.

14. The pharmaceutical composition of Claim 13, wherein the glycan is H-28, H-30, H- 31, H-36, H-43, or H-60, and combinations thereof.

15. The pharmaceutical composition of Claim 13, wherein the glycan is H-l, H-5, H-6, H-12, H-14, H-15, H-16, H-17, or H-56, and combinations thereof.

16. The pharmaceutical composition of Claim 13, further comprising a second anti -viral or anti-bacterial agent.

17. A method of treating or preventing a pathogenic infection comprising administering a pharmaceutical composition of claim 16 to a subject at risk or, exhibiting symptoms of, or diagnosed with a pathogenic infection.