Sialic acids in infection and their potential use in detection and protection against pathogens

In structural terms, the sialic acids are a large family of nine carbon sugars based around an alpha-keto acid core. They are widely spread in nature, where they are often found to be involved in molecular recognition processes, including in development, immunology, health and disease. The prominence of sialic acids in infection is a result of their exposure at the non-reducing terminus of glycans in diverse glycolipids and glycoproteins. Herein, we survey representative aspects of sialic acid structure, recognition and exploitation in relation to infectious diseases, their diagnosis and prevention or treatment. Examples covered span influenza virus and Covid-19, Leishmania and Trypanosoma, algal viruses, Campylobacter, Streptococci and Helicobacter, and commensal Ruminococci.


Introduction
It is increasingly evident that carbohydrates contribute much to biology [1][2][3][4][5] beyond serving as an energy source or structural material.[8][9][10] They are found in a number of structural forms and physiological contexts.Routinely occurring as non-reducing terminal sugar units in diverse glycan structures, sialic acids and the recognition thereof are associated with a range of health and disease scenarios, where they are intimately associated with self-and non-self-recognition.They are commonly associated with infection events -processes mediated by the interaction of microbial or viral surface protein and specific sialic acids [11][12][13][14][15][16][17][18] found on host cell surfaces.

Sialic acid structure and occurrence
Among other factors, host-pathogen specificity can be determined by the large number of naturally occurring sialic acids, of which there are at least 60 different forms. 19Such diversity is achieved by a range of post-glycosylation modifications that involve attachment of functional groups at different sites on the main C9 skeleton.In humans, the most common modification is N-acetylation at position 5 (Neu5Ac, Fig. 1), while in vertebrates that have retained a functional hydroxylase-encoding gene, which has been lost in the human lineage, 20 N-glycolylation predominates (Neu5Gc, Fig. 1).O-Acetylation is found widely across species 21,22 and can occur at positions 4, 7, 8 and 9 of the sialic acid skeleton. 23Other reported modifications include O-methylation, O-sulfation and O-phosphorylation, 19 further expanding the structural and physicochemical diversity of the sialic acids.
The deaminated form of neuraminic acid, 2-keto-3-deoxy-Dglycero-D-galacto-nononic acid or keto-deoxy-nonulosonic acid (KDN, Fig. 1), first identified in rainbow trout eggs, 24 is reported to occur widely among vertebrates and bacteria, 25 while recent studies have also noted its likely widespread occurrence in microalgae. 26In prokaryotes, the nonulosonic acids are commonly implicated in the interaction with pathogens, being involved in the infection process and the disease development including reduced host interaction (exploiting the negative charge), altering the host immune response and molecular mimicry -thought to be a means of avoiding host immune responses.Although much better studied in vertebrates, recent years have seen variations of the sialic acid structure found in bacteria, such as fusaminic acid (Fus5Acthe chirality of which has only been tentatively assigned), 27 the KDN stereoisomeric legionaminic acid (Leg) and its two a Department of Chemistry and Manchester Institute of Biotechnology, University of isomers 4-epilegionaminic acid (4eLeg) and 8-epilegionaminic acid (8eLeg), pseudaminic acid (Pse), acinetaminic acid (Aci) and its isomer 8-epiacinetaminic acid (8eAci), all reported in Fig. 1.Furthermore, the presence of Pse/Leg and KDN in samples from environmental biofilms may indicate additional overlooked roles for the sialic acids, 28 which has consequently prompted renewed interest in the chemical diversity of these acidic sugars, including recent large scale metabolite discovery activities based on mass spectrometry methods. 29

Sialic acids and infection
Sialic acids are present in abundance on host organism cell surfaces as the non-reducing terminal sugar of simple glycolipids and complex glycans.As such, they are often key receptors for pathogens to adhere to host cells -a prelude to infection. 30fluenza viruses, their surface proteins and sialic acid specificities In particular, and by far the most heavily studied, influenza viruses interact with Neu5Ac on the host through its haemagglutinin (HA), a trimeric protein containing the Neu5Ac receptor binding site (RBS), and neuraminidase (NA), a tetrameric protein which is responsible for cleavage of Neu5Ac.These proteins constitute the spikes through which influenza viruses can make contact and then infect their host cells. 30Influenza viruses have been defined as molecular walkers 31 because of their ability to move through the thick sialic acid-containing glycan layer that covers the cells, thanks to the concerted activity 32 of HA and NA:HA binds to the Neu5Ac receptors while NA cleaves it avoiding virus aggregation and allowing the virus to move deeper into the glycan layer until reaching the cell membrane (Fig. 2). 31A generally constitutes ca.80% of surface glycoproteins on influenza virus, the remaining being NA. 33For good viral growth, Fig. 1 Representative sialic acid structural variants.A Symbol Nomenclature for Glycans (SNFG) has been introduced to standardise and simplify glycans drawing.The nonulosonic acids are represented by either a filled diamond shape (NeuAc, KDN etc.) or by a flat diamond shape (Leg, Aci etc.), reported below each of the corresponding chemical structure; a red filled diamond shape is used to indicate a generic sialic acid.

Review
][34][35][36] Influenza HA binds not only to terminal Neu5Ac but also to part of the underlying glycan to which Neu5Ac is attached.Indeed, influenza viruses discriminate between prospective hosts through binding with specific sialylated oligosaccharides structures. 38This specificity reflects the predominant glycan composition of the host species.For instance, human influenza viruses bind preferentially to a-2,6-Neu5Ac-Gal receptors, which are prevalent in the human upper respiratory tract (Fig. 3). 38On the other hand, avian influenza viruses bind preferentially to a-2,3-Neu5Ac-Gal receptors, with avian species expressing mainly a-2,3-Neu5Ac-Gal receptors in the respiratory tract (Fig. 3). 38g. 2 The mechanism for influenza molecular walker was firstly described by Sakai et al. 37 The HAs on the influenza virus surface bind to the sialic acid on the host cell receptors with the typical carbohydrate-lectin multimeric interaction.The NA hydrolyse the sialic acid, liberating the virus from binding and triggering the ''rolling'' of the virus on the cell surface.The alternation of HA and NA interaction correspond to an association-disassociation events that generates the crawling and gliding motion of the virus.

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Pigs, on the other hand, express both a-2,6 and a-2,3-Neu5Ac-Gal receptors in their respiratory tract, can be infected with both human and avian influenza viruses and have been consequently defined as ''mixing vessels'' for virus reassortment among avian, swine and human. 38Horses and pigs predominantly express the glycolyl form of sialic acid, as a-2,3-Neu5Gc-Gal, in their trachea.
Given that influenza A viruses can be strictly selective toward Neu5Ac or Neu5Gc, this presents a species jump barrier, given the inability of humans to biosynthesise Neu5Gc. 39or an animal virus to cross the species barrier and infect humans, the virus must be able to bind to both the animal and human sialic acid receptors (Fig. 4).This has been demonstrated for a variety of avian viruses, such as H1N1, H3N2, H5N1 and H7Nx, which have HA mutations that switch its preference from a-2,3-Neu5Ac-Gal to a-2,6-Neu5Ac-Gal. 40However, while receptor specificity is a requirement to cross the species barrier, not all animal viruses can spread between humans by airborne transmission and become pandemic.While it is generally accepted that only viruses with a-2,6-Neu5Ac-Gal affinity transmit efficiently between humans, other factors involved in the airborne transmission are not yet completely understood.The stability of HA mutants, the HA/NA balance, and the efficiency of polymerase-mediated replication are all factors that may contribute to virus adaptation to their new host species. 38nfluenza C, in contrast to influenza A and B, possesses only one surface protein, designated Hemagglutinin-Esterase-Fusion (HEF) protein, that has HA and NA activity, as well as an esterase function. 31HEF recognizes 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac 2 ) and acts as a receptor-destroying enzyme by selectively removing the 9-O-acetyl group (Fig. 5).Similar to influenza C HEF, some coronaviruses (see below) have evolved to specifically recognize 9-O-acetyl-Nacetylneuraminic acid receptors utilizing a spike protein 31 and to facilitate release of viral progeny via the sialic acid O-acetyl esterase activity of their Haemagglutinin-Esterase (HE).

Coronaviruses and sialic acid recognition
Coronaviruses 13,41 cause a range of diseases and symptoms that differ between vertebrate host species.Divided in different subfamilies, a-, b-, g-, and d-coronavirus, they have different affinities for Neu5Ac and its derivatives. 41For example, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus 41 (PRCoV) are both a-coronaviruses; the former (TGEV) 42 shows binding to Neu5Ac with preference for a-2,3linkages, the latter (PRCoV) 41 does not have a sialic acid binding receptor.Human coronavirus41 HCoV-299E and HCoV-NL63, 44 both belong to the a-coronavirus family, and they appear to lack a specific sialic acid receptor, although NL63 uses heparan sulfate as an attachment factor to host cells, highlighting once more the pivotal role of virus-host carbohydrate binding in viral infections.The remaining known human 45 coronaviruses all belong to the b-coronavirus group, showing different specificity towards sialic acid.HCoV-OC43 Fig. 4 Human flu virus binds mainly a-2,6-Neu5Ac-Gal, it can infect humans and can be transmitted (top).Avian virus binds mainly a-2,3-Neu5Ac-Gal, it can infect humans if reaches the lower respiratory tract, where the a-2,3-Neu5Ac-Gal is present, however transmission to other individuals is difficult (middle).Avian virus that infects pigs can switch to a-2,6-Neu5Ac-Gal binding, infect humans and potentially cause a pandemic (bottom).

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RSC Chemical Biology and HKU1 [46][47][48] show preferential binding to Neu5,9Ac 2 and possess an O-acetyl esterase activity on their surface.The mechanism is similar to the receptor-destroying binding of influenza C described in Fig. 5 with the virus removing the 9-OAc group to facilitate release of daughter virions.The Middle East respiratory syndrome coronavirus (MERS-CoV), 49 which emerged in 2012, belongs to the b-coronavirus family and showed binding to sialic acid, with a preference for a-2,3over a-2,6-linked glycans. 49,50Interestingly, the SARS-CoV1 that emerged in 2002 belongs to the b-coronavirus subfamily, but does not have a sialic acid binding receptor.1][52][53] A summary of the sialic acid receptors of coronavirus is reported in the Table 1. 41e structural basis for sialic acid recognition by human coronaviruses 43 via surface glycoproteins has established the basis for 9-O-acetyl-sialoglycan engagement.The spike protein architecture is similar to that of the ligand-binding pockets of coronavirus hemagglutinin esterases and influenza virus C or D hemagglutininesterase fusion glycoproteins.It appears that coronavirus hemagglutinin-esterase and spike proteins have co-evolved to balance and optimise virion avidity. 54Above and beyond the abundant mucin glycan-based respiratory receptors for SARS-CoV2, recent studies have established that sialic acid-containing glycolipids also have the potential to mediate cell binding and viral entry. 55

Algae-virus interactions and KDN
Looking beyond medicine, sialic acid recognition is also evident in the wider environment.For instance, KDN has emerged

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as a potential key player in viral infection of KDN-producing eukaryotic microalgae [56][57][58][59] and it is thought to play a role in harmful algal bloom dynamics.A potential KDN-containing glycosphingolipid (the stereochemistry of the sugar was not determined; a tentative structure is represented in Fig. 6) has been reported in lipid rafts from the bloom-forming microalga Emiliania huxleyi, 58 which have since been shown to determine the level of susceptibility to lytic viral infection by strains of the giant E. huxleyi Virus (EhV). 59hese studies showed that for all eleven E. huxleyi strains tested, there was a direct relationship between levels of the KDN-like glycoconjugate and susceptibility to viral infection, suggesting that KDN plays an important role in host-pathogen interactions, as seen for other sialic acids in vertebrate infection.Furthermore, recent work has reported the presence of KDN and a dedicated biosynthetic pathway for cytidine-5 0monophospho-KDN (CMP-KDN) biosynthesis in Prymnesium parvum, 57 a haptophyte relative of E. huxleyi.Phylogenetic analyses suggest that all algae of the Haptophyceae and Alveolata phyla have these biosynthetic capabilities. 57Having previously discovered a giant virus that infects this alga, P. parvum DNA Virus (PpDNAV-BW1), 56 and a boom in the discovery of similar giant viruses that infect microalgae, it is tempting to speculate on a broader role for KDN in algae-virus infectionsone with potentially wide impact for brackish inland waterways as well as coastal regions. 60

Parasitic protozoan Trypanosoma cruzi and Neu5Ac
Pathogen-host interactions are often based on well-defined carbohydrate binding events.When Neu5Ac was found in the kinetoplastid parasite Trypanosoma cruzi, 61 the etiologic agent of Chagas' disease, it was accompanied by the discovery of a unique trans-sialidase enzyme (TcTS) associated with the parasite cell surface. 62,63TcTS, a CAZy family GH33 glycoside hydrolase, is attached to the parasite by a glycosylphosphatidylinositol (GPI) anchor.This multifunctional enzyme, considered the major T. cruzi virulence factor, has a central role in both the parasite infection process and modulation of the host immune response towards the parasite. 64Though T. cruzi is unable to synthesise Neu5Ac, this key monosaccharide is incorporated into the parasite surface due to the ability of TcTS to transfer terminal Neu5Ac from host glycoconjugates onto its GPI-anchored mucins, generating a-2,3-linked sialylated b-galactopyranose units (Fig. 7).
It is known that the sialylated mucins contribute directly to the parasite adhesion and invasion of host cells, but the underlying molecular mechanism has not been elucidated. 66ble 1 Summary of coronavirus subfamilies and identified sialic acid and its derivatives receptors 41  Abbreviations: APN -aminopeptidase N; ACE2 -angiotensin converting enzyme 2; CEACAM -murine carcinoembryonic antigen-related adhesion molecule; DPP4 -dipeptidyl peptidase.

Group
Fig. 6 Tentative chemical structure of the novel sialic acid glycosphingolipid. 59

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On the other hand, the negatively charged mucin coat of the parasite serves as a shield to protect the infective form of T. cruzi against lysis induced by host anti-a-galactosyl antibodies (Fig. 7). 65In mice, the sialylated mucins also interact with Neu5Ac-binding lectin-E also, Siglec-E (sialic acid-binding ImmunoGlobulin-like LECtins -Fig.8) on host dendritic cells and triggers the suppression of cytokine interleukin 12 (IL-12), the key cytokine in the activation of the immune response. 67 similar mechanism may be associated with Siglec-9 and the production of IL-10 in infected humans. 68The parasites battle to survive and establish a persistent infection is also accompanied by the shedding of TcTS from the parasite surface into the host bloodstream, where it remodels host cell surface sialylation patterns (Fig. 7). 69This can induce dramatic changes in signalling and responses of targeted cells, thus enhancing host vulnerability to infection and disease.
The sialic acid-binding lectins of the immune system The sialic acids represent Self-Associated Molecular Patterns (SAMPs), which are recognized by inhibitory receptors with the objective to diminish unwanted immune reactions. 70These immune modulations can be mediated through interactions of Siglecs with sialylated glycoconjugates.The Siglec family, which includes 14 active members in humans, are type I transmembrane proteins containing an extracellular N-terminal V-set immunoglobulin (Ig) domain that is responsible for sialic acid recognition followed by a variable number (1 to 16) of so-called C2-type Ig-like domains that act as spacers, leading the ligand binding site away from the surface (Fig. 8). 71,72The number of C2-type domains determines the mode of interaction with sialic acid-containing glycans.
In most cases, sialic acid interacts with a Siglec on the same cell surface in cis-mode, whereas Siglec-1, for example, binds sialoglycans in trans, i.e. on adjacent cells. 73As a result, in cis interactions dominate over interactions with trans ligands, without precluding binding of ligands in trans.As such, the Siglecs are integral to maintaining immune homeostasis.However, they also serve to sense pathogen-associated sialic acids, but equally can represent potential vulnerability for the host where pathogens sialyated glycans are concerned.The interplay between Siglecs and sialylated pathogens 74 -bacterial, viral and protozoan -represents an emergent field.It is expected to gain substantial momentum as we better understand how inhibitory Siglec-sialic acid interactions balance immunological activation and tolerance during viral infections, 75 the role of Siglecs in host defense and dissemination of enveloped viruses, 76 and infectious diseases more broadly, 75 including bacteria-induced sepsis 77 and infection associated with parasitic protozoa, such as Leishmania. 781][82] Progress in this field has been reviewed recently by Wennekes et al. 83 The pathogen exploits such host similarity as a camouflage to evade innate and adaptive immune system surveillance, but this molecular mimicry can cause abnormal autoimmune responses in the Fig. 7 The surface of T. cruzi is covered with mucin containing O-linked glycans.The TcTS transfers sialic acid from the host cells surface glycans and serum glycoproteins to the terminal glycan residues of mucin, shielding the parasite from anti a-Gal antibodies.The newly sialylated mucin interacting with Siglec-9 on dendritic cells surface can result in suppressing the release of IL-10.TcTS is released in the blood stream where it alters the glycosylation pattern of surface proteins making the host more susceptible to infections and diseases. 64,65C Chemical Biology Review host, resulting in the generation of auto-antibodies and T cells that attack host tissues. 84he Gram-negative bacterium Campylobacter jejuni is the major cause of bacterial gastro-enteritis worldwide. 85Infection with C. jejuni can lead to neurological complications, including Guillain-Barre ´syndrome (GBS) -an immune-mediated disease affecting the peripheral nerves of the host.The relationship between C. jejuni and GBS has been extensively investigated.The lipo-oligosaccharide (LOS) on the outer surface of the bacteria mimic host cells Neu5Ac-containing ganglioside glycolipid structures (Fig. 9,) which are abundantly expressed on the nervous systems tissues. 86he molecular mimicry between C. jejuni LOS and host gangliosides leads to the formation of cross-reactive antibodies directed against the peripheral nerves of the host.GBSassociated C. jejuni strains bind to Siglec-7, 86 demonstrating that a sialic acid receptor is associated with inflammatory and autoimmune disease (Fig. 10). 86Mass spectrometry analysis demonstrated that the binding was sialic acid-linkage specific, with a preference for a-2,3-linked sialic acid attached to the terminal galactose of the LOS chain, as observed in several gangliosides (e.g.GD1a, GM1b, and GM3). 87Reports also indicate the interaction of Siglec-7 with C. jejuni LOS, especially with strains expressing a di-sialylated ganglioside mimic with a-2,3 or a-2,3/a-2,8 linkages. 88Serological studies using anti-ganglioside antibodies from GBS patients show that they recognise the LOS of C. jejuni, suggesting that they may have been induced by the C. jejuni infection. 89

Potential applications of sialic acids in pathogen detection
The pivotal role of sialic acid and its derivatives in infection 90 (viruses, bacteria, protozoa) discussed in the previous section brings attention to how these glycans could be exploited as a tool to develop new methods for detection.Landa et al. 91 developed a colorimetric assay for the detection of specific strains of Staphylococcus aureus and Pseudomonas aeruginosa, however the majority of the examples reported in literature focus on the detection of viruses, which will be the main focus of the following section.

Neu5Ac binding, virus detection and strain discrimination
The diagnosis of influenza infection is commonly based on nucleic acid-based technologies, such as RT-PCR, or antibodybased technologies applied for instance on lateral flow devices. 92However, both techniques have disadvantages, including cost, the need for specialist equipment, or the need to generate new antibodies to detect emerging strains.The specificity of HA-sialic acid binding can be exploited for alternative technologies in the diagnostic field, including simple agglutination assays and more comprehensive glycan arrays, which may be used to predict the infectiousness and species specificity of a given virus dependent on its glycan binding specificity.

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The use of high information content glycoarrays to assess HA and intact influenza virus glycan specificity is well documented and provides invaluable underpinning information for the discrimination between viral strains. 93,94A more focussed glycan Fig. 9 (A) Schematic representation of human ganglioside structure containing sialic acid residues bound to a ceramide inner core and (B) schematic representation of C. jejuni LOS structures containing sialic acid derivatives that act as structural mimic of the human ganglioside (A), in this case the glycan derivatives are bound to an inner core and lipid A transmembrane tail. 86g. 10 The interaction of Siglec-7 with C. jejuni strains expressing disialylated LOS 86 may be related to an anti-GQ1b cross-antibody activation, leading to oculomotor weakness in patients with Guilliam-Barre ´syndrome or the related Miller Fisher syndrome. 88GQ1b disialylated structures are contained in ganglioside of the human peripheral nervous system.

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array with potential diagnostic applications was developed by Iyer and co-workers, 95 based on a range of C-, S-, and triazolelinked, monomeric sialosides, designed and demonstrated to resist influenza NA action (Fig. 11(A) and (B)).The authors showed that these sialosides were stable to NA and could bind intact viruses at room temperature without the need for adding NA inhibitor.Furthermore, good sensitivity and distinct fingerprint binding patterns were observed (Fig. 11(C)-(F)). 95alylglycan-magnetic nanoparticle pull-downs for PCR analysis The HA-sialic acid binding interaction can also be exploited in sample preparation/enrichment (Fig. 12), 96 with a microfluidic chip system in tandem with glycan-coated magnetic beads to isolate influenza A viruses from complex biological samples, which were then analysed and quantified by RT-PCR.

Colorimetric assays with sialic acid-containing glyconanoparticles and nanorods
Nanobiosensors have been developed exploiting HA-sialic acid binding specificity.For instance, gold nanoparticles coated with trimeric a-2,6-thio linked Neu5Ac-Gal ligands bind selectively to human influenza virus H3N2-X31. 97A change in extinction of the colloidal suspension of gold nanoparticles upon recognition and binding to the virus occurred within 30 minutes after addition of the virus.Importantly, gold nanoparticles functionalised with a-2,6-thio-linked Neu5Ac-Gal ligand were able to discriminate between human and avian influenza viruses, allowing for species-specific virus detection (Fig. 13-I).These reagents were subsequently adapted for use in lateral flow tests for influenza viruses (Iceni Glycoscience, unpublished results).The sialic acid binding properties of SARS-CoV2 have been exploited with gold nanorod plasmonic particles, 98 which were functionalised with a PHEA polymer decorated with a-2,3 0 -Neu5Ac-lactose.These nanorods have dual absorption bands (520 nm and 785 nm), compared to a single band (520 nm) for gold nanoparticles, which offers advantage as the 520 nm band is impacted by sample matrix effects (Fig. 13-II).The glyco-nanorods successfully detected positives in clinical samples in a dose dependent manner, showing proof of concept application of the system.

Electrochemical sensors presenting sialic acid
Another example of direct, label-free detection of influenza virus lies in the development of self-assembled monolayerpresented a-2,6 0 -Neu5Ac-lactose immobilised on gold electrodes. 99 significant signal is observed only upon binding of human influenza virus, showing the ability of the system to detect and discriminate between influenza virus strains.Importantly, in terms of hemagglutination titre (HAU), the sensitivity of this system (2 À4 HAU) is much higher than that of immunochromatographic assay (2 2 -2 4 ) or PCR (2 0 ).Horiguhi et al. 99 made a comparison of sensitivity, detection time and average cost with other detection methodology, summarised in Table 2.    (II) Gold nanorods functionalised with a-2,3 0 -Neu5Aclactose exploited for the rapid detection of SARS-CoV2. 98ith a limit of detection of 0.013 HAU (for astandard immunochromatographic assay the LOD is 1.13 HAU).

Sialic acid and SARS CoV2 detection on nitrocellulose strips
A rapid test has been reported for SARS-CoV2 detection that is based on sialic acid recognition in the form of a paper-based assay. 53Colloidal gold nanoparticles were functionalised with a poly N-hydroxyethyl acrylamide (PHEA) polymer equipped with either a-2,6 0 -Neu5Ac-lactose, a-2,3 0 -Neu5Ac-lactose or simply a Neu5Ac residue.The sample is deposited on a nitrocellulose strip and sialic-acid functionalised gold nanoparticles are eluted along the strip, generating a red spot in case of positive detection.Follow-through work 101 tested the concept using clinical samples based on nasal swabs originating from COVID-19 positive patients showing the simple Neu5Ac decorated particles to have the best performance.In this instance, an additional silver staining step enhanced the limit of detection, with the unoptimized test achieving 85% sensitivity and 93% specificity, with cycle threshold (Ct) values as high as 25.

Potential applications of sialic acids in protection against infection
The interplay between glycan structure, pathogen receptors and enzymes, and the host immune system lectin repertoire is key to both health and infection.With sialic acid as a dominant non-reducing terminal unit in many animal glycans, direct inhibition of its recognition or blocking or effecting the removal of this class of sugar has therapeutic potential in several ways.In addition, sialic acid-containing glycans produced naturally in the host can also have a protective role in preventing infection.

Sialic acid-containing milk oligosaccharides
Human milk oligosaccharides (HMOs) are a biologically active component of breast milk that exert prebiotic effects (i.e.promote the growth and replication of commensal microorganisms), as well as other health promoting benefits to new-born infants. 102Over 200 different structures of HMOs have been identified in human breast milk, 103 significantly more than are present in the milk of livestock and most primates. 104Roughly 50-70% of known HMOs are fucosylated, with ca 10-20% sialylated.The reverse is true for bovine and porcine milk oligosaccharides, where many milk oligosaccharides contain sialic acid. 105umerous health benefits are thought to be associated with sialylated HMOs, with relevance to bacterial and viral infection, utilisation by gut commensals, direct modulation of the immune system and enhanced cognition and brain development.Animal milk oligosaccharides comprise the sialic acids Neu5Ac and/or Neu5Gc, 106 including HMOs 107 where the Neu5Gc is dietary derived due to the expression of an inactive CMP-Neu5Ac hydroxylase in man. 108The Neu5Gc from dietary sources, such as diary and red meat, is also found in human tissue, as indicated by circulating anti-Neu5Gc-antibodies and its incorporation into cancerous tumours. 109,110MOs are principally thought of as prebiotics -promoting the growth of beneficial microorganisms in the gut, 102 particularly the commensals Bifidobacteria spp, B. longum and B. bifidum. 111Sialylated HMOs, specifically a-2,3 0 -Neu5Aclactose and a-2,6 0 -Neu5Ac-lactose which induce sialidase activity in B. spp and various B. longum strains, enable them to metabolise and grow on Neu5Ac and produce acidic fermentation products, lactate and short-chain fatty acids (SCFAs). 1125][116] Additionally, previous work has shown that the hexasaccharide disialyllacto-N-tetraose (DSLNT, Fig. 14) contributes to the prevention of necrotising enterocolitis in a neonatal rat model. 117,118Acidic HMOs, particularly DSLNT, LS-tetrasaccharide a (LST-a,) and LS-tetrasaccharide c (LST-c) (Fig. 14), also have a pronounced effect on the modulation of intestinal epithelial cell maturation. 119tudies have indicated that sialylated HMOs can inhibit hemagglutination mediated by uropathogenic E. coli (UPEC) and enterotoxigenic E. coli (ETEC), thereby blocking bacterial adhesion. 120A similar process can be seen with Helicobacter pylori, where a-2,3 0 -Neu5Ac-lactose inhibits binding to the gastrointestinal epithelium 121 and, in rotavirus, decrease replication. 122Acidic HMOs are also thought to possess antiviral properties.In in vitro hemagglutination inhibition assays of avian influenza viruses, a-2,3 0 -Neu5Ac-lactose exhibited antiviral properties.In addition, in vivo studies of pathogen-free chicken models treated with a-2,3 0 -Neu5Ac-lactose showed a reduction in symptoms when infected with H9N2 influenza virus, with the virus being completely eradicated within 24 hours. 123mmensal intramolecular trans-sialidase While most trans-sialidases have been studied from bloodborne trypanosomes, 64 a novel intramolecular trans-sialidase has been identified in the commensal gut bacterium  Fig. 15 The IT-sialidase of R. gnavus cleaves sialic acid from host cell surface in the gut and rearrange it into 2,7 anhydro sialic acid, providing an advantage over other bacterial species in the gut able to metabolise the standard sialic acid. 126

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Ruminococcus gnavus. 124,125Studies revealed a new mode of action for this enzyme, which cleaves terminal a-2,3-linked sialic acid from human gut mucins and releases 2,7-anhydro-Neu5Ac instead of Neu5Ac.R. gnavus possesses a specific uptake mechanism for 2,7-anhydro-Neu5Ac that is not prevalent in nature, thus providing specific advantage for R. gnavus in scavenging sialic acid from sialylated glycans in the gut (Fig. 15). 126This in turn helps to maintain species balance and hence gut homeostasis, although a higher level of this intramolecular trans-sialidase enzyme has been found in patients with inflammatory bowel diseases. 127u5Ac and small(er) molecule anti-influenza therapeutics Synthetic glycans, 128 glycopeptides and glycopolymers 129,130 have been informative with regards of the impact of glycan density and presentation (Fig. 16) on influenza virus infection.These studies demonstrate that glycan structure, valency and density (crowding) has a profound impact on NA binding and activity, and influenza virus adhesion and infectivity, respectively.Taken together, these studies suggest that multivalent inhibitors present obvious opportunities for therapeutic intervention. 131While this prospect has yet to be realised in the clinic, several studies have demonstrated approaches to high affinity ligands for the influenza surface-presented NA and, to a lesser extent, HA.The coordinated action of NA and HA 132,133 is responsible for binding of influenza virus to the sialylated cellular receptors, facilitating viral internalisation into host epithelial cells (HA) 38 as well as daughter virion release (NA).Two currently approved anti-NA drugs, Zanamivir (Relenza) and Oseltamivir phosphate (Tamiflu) (Fig. 17), act as transition state analogue inhibitors targeting the active site of NA.
However, mutations in and around the active site of the NA can lead to development of drug resistant strains resulting in the drugs being less effective in treatment and prevention of influenza virus infection.Weight et al. 134 demonstrated that a multivalent polymer-bound Zanamivir binds 2000 times more strongly than its monomeric equivalent to the Zanamivirresistant turkey/MN (Fig. 18).

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To avoid drug resistance, a new strategy has been developed, where Zanamivir was covalently conjugated to a biocompatible water-soluble polymer and exhibited up to a 20 000-fold improvement in anti-influenza potency compared with the Zanamivir parent against human and avian viral strains, including both wild-type and drug-resistant mutants. 135ultivalent sialic acid-based HA lectin inhibitors 136,137 can also provide anti-influenza activity by virtue of their ability to block virus-cell interactions.With HA ligands, this is more challenging that for NA ligands, as the monomeric ligand affinities for the latter are very much higher to start with.Nonetheless, polymer-stabilized sialylated nanoparticles can bind potently to and discriminate between influenza haemagglutinins. 138Inhibition of influenza A virus adhesion has been demonstrated for di-and tri-valent haemagglutinin inhibitors, 139 (Fig. 19).By linking sialylated LacNAc units to di-and trivalent scaffolds, inhibitors were obtained that demonstrated 4400-fold enhanced inhibition.Clearly, ligand presentation is central to achieving optimised affinity, as it is evident with natural glycan binding by influenza viruses.For instance, H3N2 viruses have specificity for a-2,6-sialylated branched N-glycans with at least three N-acetyllactosamine units (tri-LacNAc); the length of the glycan chain can be used to target enhance discrimination between virus strains. 140rucidal sialic acid materials Precise presentation of sialic acid is an important factor in achieving inhibition of influenza virus infection.Decorating a b-cyclodextrin scaffold with three copies of sialic acid derivatives (Fig. 20), either a-2,6 0 -or a-2,3 0 -Neu5Ac-lactose, achieves effective inhibition of human and avian influenza virus infection, respectively. 141The authors carefully examined the impact of the sialic acid linker used to immobilise the carbohydrate moiety on the cyclodextrin scaffold, with a hydrophobic linker being more effective than a hydrophilic one.Significantly, the cyclodextrin-sialic acid constructs showed excellent virucidal properties -i.e. the compounds disrupted the virus structure, rather than just binding to HA.They also proved to be effective both as prophylactic agent when administered

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Fig. 19 (A) The position of the ligands is elaborated from the 3D structure of the protein; (B) in terms of distance between ligands and orientation; the information is transferred to suitable trimeric structure (C) with scaffold and spacer to achieve the correct orientation and distance to get ligands binding simultaneously. 139g. 20 b-Cyclodextrin was used as scaffold to immobilise a-2,6 0 -Neu5Ac-lactose decorated with different linker.The most efficient configuration in terms of therapeutics and prophylactic activity was obtained with a hydrophobic linker.The construct was efficient in both in vitro, ex vivo and in vivo (mice) experiments against human influenza H1N1 infection. 141view RSC Chemical Biology pre-infection, and as a therapeutic when administered postinfection in mice.

Multivalent sialic acid-binding lectins block recognition of host cells by influenza virus
Reports show that masking host cell sialic acid receptors with engineered multivalent sialic acid-recognising carbohydrate binding modules (CBMs) (Fig. 21) provided protection to mice against the 2009 pandemic H1N1 influenza virus 142 and the influenza A (H7N9) virus. 143The authors suggested that this host-targeted approach could provide a front-line prophylactic that has the potential to protect against any current and future influenza virus and possibly against other respiratory pathogens that use sialic acid as a receptor.Furthermore, the same Fig.21 The binding of CBMs to sialic acid act as a shield, preventing the virus from bind to the same receptors; DAS181, instead, prevents the binding of the virus by cleaving the terminal sialic acid effectively destroying the surface cell receptors. 142,147g. 22 The dual Zanamivir-dinitrophenyl conjugate binds to surface NAs of the virus inhibiting the neuraminidase activity and suppressing virus budding from the host cell.The dinitrophenyl (DNP) hapten is highly immunogenic and recruits endogenous anti-DNP antibodies both on the virus-free and the virus-infected cell resulting in their opsonization and the consequent immune-mediated clearance. 149BM constructs were shown to possess immunoregulatory properties, 144,145 supporting the notion that they could be used not only to protect from on-going disease, but that they could modulate immune responses to prevent future infections and potentially find application as adjuvants 146 for vaccines.

Therapeutic sialidase-mediated removal of sialic acid prevents influenza infection
An alternative strategy 147 employed to block influenza infection targets the host cell sialic acids, rather than the two viral surface proteins HA and NA discussed above, has been reported.DAS181 (Fludase) is composed of a sialidase catalytic domain, cleaving a-2,6-as well as a-2,3-sialic acid, fused with a cell surface-anchoring sequence, which destroys essential sialic acid receptors and consequently blocks viral adhesion (Fig. 21).In vitro assays of laboratory strains and clinical isolates of influenza A and B viruses showed EC 50 values range from 0.04 to 0.9 nM.In another similar study, DAS181 showed strong inhibition against a panel of Oseltamivir resistant H1N1 using plaque number reduction assay on MDCK cells. 135Currently, DAS181 has completed pre-clinical development and has entered clinical Phase I and Phase II trials, with the latest clinical data showing that DAS181 significantly reduces viral load in participants infected with influenza virus, thus justifying future clinical development of this novel host-directed therapy. 148uraminidase inhibitor-mediate immunotherapy for influenza infection Harnessing the host immune response to specifically target influenza virus presents a novel approach to anti-viral therapy.
A synthetic bifunctional small molecule was prepared by conjugating the NA inhibitor Zanamivir with the highly immunogenic dinitrophenyl group, 149 which specifically targets the surface of free virus and viral-infected cells (Fig. 22).This approach has dual function, in that the Zanamivir blocks daughter virion release from host cells, while the primed immune response serves to attack and clear virus from the body.In relation to severe infections, this therapeutic regimen remains effective up to three days post lethal inoculation, suggesting that it may be useful for infections refractory to established therapies.

Summary
Given the ever-increasing demonstration of roles for glycans in immune health and disease, 150 with impact for infection and the prevention thereof, the need for much further investigation of the glycobiology of cell surfaces is called for.In addition, in terms of therapeutic intervention, biopharmaceuticals, which are often sialylated, are very much to the fore at present.The glycosylation, and in particular sialylation 151 thereof, is crucial to the optimisation of efficacious, cost-effective, and safe medicines. 152As far back as 2001, with reference to glycoscience it was projected that ''Cinderella's coach is ready''. 153In the intervening period fundamental glycomics studies continue to advance at pace, 154 with sialic acids central to the investigation of infection studies.As a field, however, glycomics lies some way behind other omics topics (Table 3), highlighting the challenge, the opportunity and the unmet need that glycoscience presents.Translational impact for sialic acids faces a number of challenges going forwards.While the scalable enzymatic synthesis of sialic acid [155][156][157][158] and sialylated glycans [159][160][161] has been achieved, the correct presentation of this key sugar recognition element is critical to achieving physiologically or therapeutically relevant biological recognition.For instance, the valency of NeuAc presentation 97 as well as secondary interactions from the glycan chain to which it is attached 140 can have a profound impact on target engagement.In addition, the efficiency of glycan recognition is context dependent, in that monovalent affinity does not directly correlate with polyvalent avidity. 162So consideration needs to be given not only to glycan structure, but also to the assay format used when considering cell adhesion events, for instance.
A further challenge lies in the myriad of sialic acid modifications found in nature, some of which (e.g.sialylation) are labile 163 or prone to intramolecular O-acetyl migration, 164 but which may have a profound effect on enhancing or masking sialic acid recognition events.Further still, sialic acids are key players in the immune system, where true physiological effect and therapeutic potential can only be achieved through in vivo studies -and all animals are not equal in glycoimmunology. 165,166Nonetheless, as highlighted in this article, substantial advances are being made to open up sialic acid biology and therapeutics.
The central role of sialic acids in infections is clear cut and there has been longstanding success with inhibitors of sialic acid metabolism in the prevention of influenza infection, in particular.The current state of play provides much in the way of foundational tools and initial leads, leading to much optimism about the prospect of a rich future of sialic acid-related diagnostics, prophylactics and therapeutics going forwards.

Fig. 5
Fig.5The Hemagglutinin-Esterase-Fusion surface protein on influenza C virus (left) surface binds to the glycans on host cell surface and the virus is internalised (A), replicated (B) and released (C) outside the cells, facilitated by the esterases action that destroy the binding glycan moiety.The same function is performed by the two different surface proteins HA and NA in influenza A and B viruses (right), D, E and F.

Fig. 8
Fig. 8 Schematic representation of Siglecs. 72Human Siglec receptors contain one N-terminal V-set Ig domain that is responsible for sialic acid binding and several C2-type Ig-like domains acting as spacers and determining the mode of interaction.Siglecs with ITIM (magenta) motifs are inhibitory proteins, whereas Siglecs containing ITAM (purple) motifs are activating receptors, interacting with activation partners DAP10/12.[Figure and caption reproduced from ref. Lenza et al. 72 from open access MDPI, copyright 2020.]

Fig. 11 (
Fig.11 (A) Sialic acid core equipped with uncleavable linker; (B) substituent of the sialic acid core; (C) library is printed in the array; (D) binding to the viruses is inhibited in the presence of NA/HA inhibitor; (E) influenza viruses of different strains are assayed against the glycan array; (F) virus of different strains react differently with each glycan generating a signal intensity fingerprint that can be used to characterise the virus/strain.95

Fig. 12
Fig. 12 Integrated system combining microfluidic, magnetic nanoparticles and RT-PCR.(A) The glyco-nanoparticles are loaded into the microfluidic system; (B) the sample is then loaded in the microfluidic chip; (C) viruses binding to the specific glycan are captured by the magnetic nanoparticles; (D) the unbound material is eluted; (E) the RT-PCR reagents are loaded; (F) the readout provides information of the captured virus(es).96

96
Fig. 12 Integrated system combining microfluidic, magnetic nanoparticles and RT-PCR.(A) The glyco-nanoparticles are loaded into the microfluidic system; (B) the sample is then loaded in the microfluidic chip; (C) viruses binding to the specific glycan are captured by the magnetic nanoparticles; (D) the unbound material is eluted; (E) the RT-PCR reagents are loaded; (F) the readout provides information of the captured virus(es).96

Fig. 14
Fig. 14 Chemical structures of HMO DSLNT and LST-a and LST-c.

Fig. 16
Fig.16 Schematic representation of how sialic acid and its derivatives have been exploited to generate multivalent materials to target receptors Siglecs, selectins and virus proteins to achieve immune modulation, targeted drug delivery and anti-virus treatments [reproduced with permission from Biomaterial Science, Royal Society of Chemistry, copyright 2013].131

Fig. 17
Fig. 17 Chemical structure of the two NA inhibitors, Oseltamivir and Zanamivir, both mimic the oxacarbenium ion of the sialic acid intermediate formed during the NA action.

Fig. 18
Fig. 18 Schematic representation of how Zanamivir bound to a flexible polymer can maximise the interaction with surface NA and increase its binding strength.

Table 2
Detection system comparison between new and existing technologies a Quartz crystal microbalance.

Table 3
Comparison of the volume of 'omics' publications by discipline.Web of Knowledge literature search 08/07/2023.[inspired by presentation from Ole Hindsgaul]