Abstract
The initial engagement of host cells by pathogens is often mediated by glycan structures presented on the cell surface. Various components of the glycocalyx can be targeted by pathogens for adhesion to facilitate infection. Glycans also play integral roles in the modulation of the host immune response to infection. Therefore, understanding the parameters that define glycan interactions with both pathogens and the various components of the host immune system can aid in the development of strategies to prevent, interrupt, or manage infection. Glycomaterials provide a unique and powerful tool with which to interrogate the compositional and functional complexity of the glycocalyx. The objective of this review is to highlight some key contributions from this area of research in deciphering the mechanisms of pathogenesis and the associated host response.
Keywords: Glycomaterials, glycobiology, glycocalyx, pathogenesis, immunomodulation, immunology/microbiology/virology
Introduction
Carbohydrates, or glycans, are ubiquitous constituents of the cell surface, serving in a variety of roles that range from establishing protective physical barriers against the outside environment, mediating cell–cell and cell–matrix interactions, or regulating intracellular signaling through organization of membrane receptors.1 Throughout evolution, opportunistic pathogens have developed both the ability to target glycan structures on host cells to facilitate infection, as well as to adopt the host glycosylation machinery to acquire stealth, enabling them to evade immune surveillance.2 As such, glycans present attractive drug targets for infectious disease prevention and treatment.
Despite their promise, glycan-based intervention strategies have proved difficult to attain. This is due to a combination of challenges posed by the glycome. Among those are the complexity and diversity of glycan structures and their non-template-driven biosynthesis. Another crucial factor is the typically weak binding affinity of protein receptors toward individual carbohydrates, which, in a biological setting, is augmented through multivalent presentation of glycans on cell surface proteins or through the dynamic assembly of glycolipid patches in the plasma membrane. This gives rise to a carbohydrate-rich, hierarchical macromolecular system, called the glycocalyx (Figure 1(a)), which glycan-recognizing pathogens must navigate in a highly coordinated manner, to engage receptors on host cells, leading to the initiation of infection.
The nanoscale dimensions and organization of the glycocalyx have confounded determination of the activity of the relevant glycan structures and necessitated the development of material-based approaches to elucidate their biological functions. Advances in synthetic macromolecular chemistry have greatly advanced this field.3 A few themes have emerged as important considerations in the design of materials for probing glycan-mediated interactions such as glycan structure, valency, and macromolecular scaffold architecture (Figure 1(b)). This review highlights some recent contributions from the area of materials science in deciphering the identity and mechanisms of action of host glycans targeted by pathogens to initiate infection and in elucidating the roles of host glycans in regulating the ensuing immune response.
Defining glycan-mediated host–pathogen interactions
A large assortment of viral,4–7 bacterial,8–10 and protozoan11,12 pathogens employ the recognition of carbohydrates displayed on surfaces of epithelial cells to engage, colonize, and infect their target organisms. Therefore, considerable efforts have been directed toward developing technologies for cataloging the complexity of carbohydrate recognition by pathogen receptors. Over the last 15 years, the glycan array has emerged as one of the most general and versatile tools for determining the ligand specificities of glycan binding proteins. Although vital to many divisions of glycobiology, this technology has greatly contributed to the identification of glycan receptors targeted by viruses and microbes alike. In their traditional format, glycan arrays are constructed through immobilization of individual carbohydrate structures, either synthetic or derived from natural sources, onto solid supports to generate two-dimensional multivalent glycan assemblies at sufficient densities to elicit high-avidity interactions with target proteins. This straightforward technological platform has helped unearth a great deal of information regarding the specific roles of glycans within the life cycle of many pathogens. Considering the immense number of glycan binding pathogens, a select few are highlighted below to demonstrate the utility of these arrays alongside current material-based improvements geared toward enhancing our understanding of host–pathogen interactions beyond their recognition of individual glycan structures and in the context of their three-dimensional presentation in the milieu of the cellular glycocalyx.
Glycan microarrays have been extensively used to scrutinize the glycan specificity of the Influenza A virus (IAV, Figure 2). The glycan specificity of IAV has been of particular interest, due to its rapidly evolving nature, and the role of recognition toward determining host specificity.13 IAV employs two sialic acid (5-N-acetylneuraminic acid, Neu5Ac)-specific surface proteins, the receptor-binding hemagglutinin (HA) and the receptor-destroying neuraminidase (NA) to, respectively, initiate and complete infection (Figure 2(a)). Avian IAVs generally recognize sialic acid with a α(2,3) glycosidic linkage to an adjacent galactose residue, whereas human viruses bind sialic acid with a α(2,6) glycosidic linkage.7,14 A switch in HA receptor specificity from α(2,3)- to α(2,6)-linked sialic acid is considered a common prerequisite for human transmission,14–16 although not in every case.17–19 Array-based analysis of the glycan-binding phenotype is routinely used as part of IAV surveillance (Figure 2(b))14,20,21; however, discrepancies between the predictions of transmission risks based on glycan array analysis and those determined in infection assays7,22 help motivate continued development of array platforms, with the aim of constructing a more biologically representative array.
Whereas numerous approaches have been explored to optimize the array construction with respect to glycan structure, linker length, grafting chemistry, and surface functionality (these studies have been the subject of several extensive reviews23–26), definite advantageous platforms for accurately probing glycan-binding specificity of pathogen-associated proteins have yet to distinguish themselves. Multiple groups have recently begun to investigate the effects of array design and immobilization strategy by comparing microarray binding results across multiple platforms utilized in various laboratories.27,28 These results demonstrated the variability of carbohydrate recognition across array platforms and the necessity to understand how array construction affects glycan presentation, and, more to the point, how the presentation of these molecules influences the specificity of interactions with protein receptors. Currently, efforts utilizing the principles of macromolecular design are underway to recapitulate the nanoscale three-dimensional presentation of glycans at the cell surface in arrays in order to carefully curate and preserve the contributions of parameters, such as glycan valency and density, on the specificity of glycan recognition (Figure 2(c)).24,29
Bovin and colleagues provided an early demonstration of this approach in their study of glycan-binding specificities of model lectins using a 3D hydrogel array.30,31 Recently, Liu and co-workers adapted this technology to capture distinct bacterial species in carbohydrate-modified hydrogel microarrays.32 The binding specificity of Pseudomonas aeruginosa lectins (e.g. LecA) has also been explored using multivalent glycoconjugate array formats to investigate how both the scaffold itself and the glycan presentation along the scaffold affect lectin binding. The Pieters lab explored galactosyl glycodendrimer arrays to obtain binding profiles of LecA,33 demonstrating a 16-fold increase in potency with a tetravalent β-galactoside dendrimer over a monovalent scaffold, a result similarly echoed while investigating the binding of cholera toxin, another bacterial protein with glycan-binding lectin domains.34 Gildersleeve and his co-workers used synthetically glycosylated bovine serum albumin (BSA) ligands,35 with well-defined glycan valencies, arranged on the array at increasing surface densities to identify high-affinity inhibitors of LecA. In a differing approach, Novoa et al. constructed a library of 625 monovalent and divalent galactose-containing glycans with varying linker components presented on peptide nucleic acid (PNA) scaffolds to screen for LecA recognition. This targeted array aimed to optimize binding of the pathogenic protein by enhancing the ability of LecA to interact with adjacent glycan binding sites separated by 30 Å.36 This array addressed the molecular geometry37 and conformational constrains of glycan recognition, while simultaneously identifying soluble inhibitors capable of inhibiting P. aeruginosa invasion of human lung cells.
While IAV specificity has been routinely examined in the traditional glycan array platform, several groups have begun to consider the effects of valency, density, and spatial organization of cell-surface glycans on IAV recognition by utilizing polymeric glycomaterials grafted to solid surfaces to survey viral specificity. Outside the context of microarrays, surface plasmon resonance (SPR) analysis of the binding of HA proteins to surface-immobilized sialylated glycopolymers confirmed the specificity of avian (A/Anhui/1/2005(H5N1)) and human (A/Brisbane/10/2007(H3N2)) IAV forms for the characteristic α(2,3) and α(2,6) sialic acid linkages, respectively.38 Our own group has also explored the use of linear glycopolymers mimicking the architecture of epithelial mucin glycoproteins to explore the effects of sialoglycan presentation on whole IAV binding (Figure 2(c)).39
Glycan mobility within the fluid environment of the cell membrane is also believed to be an important factor in mediating multivalent interactions.40 Current glycan arrays, in which glycans are immobilized to the surface in fixed positions, fail to recreate the dynamic nature of glycan membrane organization. Building on an early report by Disney and Seeberger demonstrating the capture of whole ORN178 Escherichia coli (E. coli) on oligomannose arrays41 through the mannose-specific FimH adhesion protein42 presented at the tips of the bacteria’s type 1 fimbriae, Barth et al. created a fluidic microarray to address concerns of glycan mobility as well as the challenge of tightly controlling the glycan density (Figure 2(c)).43 Utilizing a supported lipid bilayer (SLB) containing mannosylated lipids,44 the researchers observed that a critical mannose density was required for a switch in the avidity of FimH in the ORN178 strain from a monovalent to a trivalent interaction. This result led the authors to hypothesize that each fimbriae binds to a cluster of three mannose residues, in agreement with the known affinity of FimH for a covalently linked trimannose ligand,45 and the enhanced avidity of the resulting multivalent interaction triggers the anchoring of additional fimbriae. Similarly in later work, Shen et al. made use of SPR imaging of SLB arrays46 to investigate the kinetic effects of the membrane surface on the recognition of sialylated glycolipid receptors by recombinant HA proteins from an H5N1 IAV strain.47 In this work, the authors measured the formation of HA–glycan complexes while maintaining the concentration of the preferred receptor, α(2,3) sialyl-N-acetyllactosamine glycolipid and varying a secondary lipid/glycolipid component within the membrane. Receptor binding could be significantly enhanced with increasing amounts of accessible hydrophobic elements; an effect quantitatively attributed to the kinetic formation of a weak HA-membrane interaction prior to glycan recognition. Taken together, these results provide further evidence for the importance of membrane and dynamics in carbohydrate recognition by pathogens.
Inhibiting glycan-mediated host–pathogen interactions
Naturally, inquiries into glycan receptor specificity are ultimately motivated by the desire to neutralize the disease causing agents, and as such, the development of glycomaterials as inhibitors of pathogen binding has had an enduring presence. The Whitesides group was among the earliest to consider the fitness of multivalent glycan ligands as inhibitors of pathogens. Although not alone in this pursuit,48–52 much of their work focused on the creation of polyvalent IAV inhibitors presenting sialic acid on both liposomal53 and polyacrylamide54–58 scaffolds. The principles that govern multivalent glycan interactions, as formulated by Whitesides in the course of these studies, still remain a vital component of pathogen inhibitor design. In particular, good inhibitors make use of additive enthalpic gains stemming from each successful glycan–protein interaction balanced against any entropic penalty caused by restricting the conformational freedom of the polymer scaffold. Progress in this field59,60 has been accelerated by the development of modern synthetic techniques yielding novel scaffolds with ever increasing complexity and control over macromolecular architecture. For instance, reaching beyond the linear architectures of glycopolymers, Papp et al. synthesized sialic acid presenting polyglycerol nanoparticles of varying size and showed that particles roughly the same size as IAV (50–100 nm) were the most effective scaffold for inhibiting IAV infection of Madin–Darby canine kidney cells.61 The diversity in this field is ever growing and is further elaborated by techniques that can create newer diversity in multivalent structure,62 including but not limited to chemoenzymatic,63 gold nanoparticle,64 fullerenes,65 and synthetic peptide carrier66 strategies. Some consideration has also been paid to the use of naturally isolated mucin glycoproteins, which are heavily decorated with pathogen receptor decoys that viruses such as IAV must negotiate en route to their cellular target,67 as inhibitors of infection. One such example demonstrated that a solution of purified porcine gastric mucins could inhibit infection of epithelial cells by human papilloma virus type 16, Merkel cell polyoma-virus, and the A/WSN/1933 (H1N1) IAV strain.68
Soluble glycomaterials are of particular interest as inhibitors of pathogenic bacteria in part because they offer an alternative to current antibiotic treatment, circumventing the pressing challenges associated with the development of antibacterial drug resistance. Designing treatments for bacterial infections with antiadhesive agents offers an attractive alternative to antibiotics by reducing the selective pressure on the pathogen to evolve resistance.69 Being that antiadhesion multivalent materials have been reviewed extensively,70–73 this review will only briefly highlight a few recent examples.
A number of labs have paid particular attention to inhibition of the P. aeruginosa lectins by exploring the use of diverse ligand architectures. In particular, calix-[4]-arenes74 have been ranked among the most effective architectures against LecA (Figure 1(b)).75,76 In a recent report, Boukerb et al. synthesized tetravalent galactosyl and fucosyl calixarenes for the purpose of inhibiting both major P. aeruginosa lectins, LecA and LecB.77 These glycoconjugates were capable of inducing bacterial aggregation, inhibiting adhesion, and protecting against lung injury in a mouse lung model. Importantly, the prevention of biofilm formation was dependent on the multivalency of the glycosylated calixarenes. In addition, other synthetic architectures have been utilized, including carbohydrate-functionalized goldnanoclusters,78 pillararenes,79 dendrimers,80–83 and fullerenes (Figure 1(b)).84 Furthermore, like with viruses,68 the Ribbeck group was also able to inhibit adhesion and biofilm formation of the bacteria by once more utilizing native gastric porcine mucins.85
Although the neutralization of P. aeruginosa with soluble glycopolymers is arguably the most explored, similar approaches have been applied to other bacteria, including E. coli. Multivalent N-heptyl-α-d-mannosylated glycopolymers86 were capable of disrupting binding of invasive E. coli to intestinal epithelial cells by 102–106 fold, ultimately protecting ex vivo mouse colonic loops from adhesion of these cells. In another example, Ryu et al. demonstrated that the dynamic assembly of multivalent nanofibers from amphiphilic glycosylated building blocks could induce aggregation of ORN178 E. coli, by engaging FimH, in a length-dependent fashion. The self-assembly of these nanofibers could be tuned to define lengths87 as well as dynamically shift morphology from linear fibers to spherical micelles.88
Inhibitor strategies can be expanded for effective use in many distinct forms of pathogenic organisms, including the bacterial AB5 toxins,89 as demonstrated in a study by Polizzotti and Kiick on the inhibition of cholera toxin with galactosylated recombinant peptides.90 Similarly, Gram-positive bacteria, such as Streptococcus suis, could be effectively inhibited with tetra- and octavalent galabiose dendrimers91,92 and even the protozoan Entamoeba histolytica could be targeted with linear polyvalent N-acetylgalactosaminides.93,94 These studies nicely illustrate the applicability of soluble glycomaterials as therapeutic and diagnostic tools for pathogen research.
Glycan-mediated regulation of the host immune response
The immune system, including innate and adaptive components, serves as the host defense to combat infection and invasion by foreign pathogens.95 Host glycans play integral roles in mounting host defense to pathogenic threats and, in some cases, their functions can be subverted by pathogens (and even cancer cells) to escape immune surveillance. The burgeoning field of synthetic immunology,96,97 which aims to employ synthetic systems to modulate immune responses, has profoundly impacted this field of study. Among the tools that have been instrumental to its cause are glycomaterials, reflecting the centrality of glycans in orchestrating host immunological responses. The following sections describe several applications of glycomaterials to reveal the mechanisms through which glycans influence the activity of the various components of the immune response and to harness these functions to combat pathogen infections.
Probing glycan functions in innate immunity with glycomaterials
The complement system
A key component of innate immunity is the complement system, which consists of ∼ 30 soluble and membrane-bound proteins. Activation of the complement cascade is the hallmark innate immune effector mechanism present in plasma, cerebral spinal fluid, and mucosa. The complement system can be activated by three initiation pathways: the lectin, classical, and alternative pathways. Glycopolymers with pendant glucose or galactose monosaccharides grafted onto polystyrene nanoparticles have previously been used to assess glycan-mediated complement activation.98 In this study, the nanoparticles were tested for their ability to activate complement by its consumption in normal human serum. In another example, Geng et al. generated well-defined neoglycopolymer-protein biohybrid materials to modulate complement activation through the lectin pathway.99 Complement lectins can bind to mannose-containing structures on the surface of bacteria, fungal pathogens, and viruses. Taking advantage of a single free cysteine residue in BSA, maleimide-terminated synthetic neoglycopolymers decorated with pendant mannose monosaccharide ligands (valency = 74), were conjugated to a single site in the BSA protein. The resulting glycomaterials were used to investigate binding to the complement lectin, MBL (mannose-binding lectin) in an SPR assay. The authors demonstrate that the hybrid mannoglycopolymer–BSA conjugates bound MBL with a much higher affinity (KD = 7.9 + 10-9) and activated complement to a higher extent compared to BSA alone in both SPR and ELISA assays.
Phagocytic immune components (neutrophils, monocytes, macrophages)
Neutrophils, white blood cells, which can engulf, or phagocytose, foreign pathogens present in the bloodstream, were once thought to be non-discriminatory components of the immune system. Recent studies have shown that neutrophils can distinguish between structurally similar glucan components of the fungal cell wall. In a landmark study, Rubin-Bejearno et al. coated polystyrene beads with either β(1–3) or β(1–6) glucans and evaluated the activation of human neutrophils in response to their exposure.100 Although both structural isomers are present in the fungal cell wall, β(1–6) glucans comprise only 9–20% of the total glucan composition. Notably, the 6 µm polystyrene beads used in this study, match the size of Candida albicans yeast form cells (5 µm). The authors demonstrate that the β(1–6) beads led to significantly enhanced production of heat shock proteins, and that the presentation on particles (versus soluble polysaccharides) was necessary to elicit this effect. These beads also caused greater ingestion and reactive oxygen species (ROS) production by neutrophils. The authors go on to demonstrate that the β(1–6) beads recruited the complement factor C3d (proteolytic fragements of C3b) to a larger extent compared to the β(1–3) beads, and that this recruitment led to the binding of the neutrophil cell surface complement receptor CR3. Finally, the authors show that selectively glycan-digested whole C. albicans cells also exhibited this behavior, wherein cells exposed to an endo β(1–6) glucanase exhibited a 50% reduction in phagocytosis ROS production and HSP expression. Similar polystyrene beads have also been used to decode the mechanism through which human monocyte-derived macrophages phagocytose virulent strains of Mycobacterium tuberculosis (Erdman and H37Rv).101 The authors demonstrate that additional terminal (single, di-, or tri-) mannosyl units on lipoarabinomannan glycolipids, which are somewhat unusual for mycobacteria, but are present on virulent strains, are directly responsible for binding to the macrophage mannose receptor.
Dendritic cells (DCs)
DCs play critical roles in the rapid response toward the presence of foreign pathogens and also bridge the innate and adaptive immune systems. The molecular signature of pathogens, often referred to as pathogen-associated molecular patterns (PAMPs), is recognized by immune pattern recognition receptors (PRRs) on DCs. Sugar-complexed PAMPs102 are the largest constituents of PAMPs and include lipopolysaccharide (LPS), N-acetylglucosamine (GlcNAc), peptidoglycan, and glucan-containing cell walls. Activation of PRRs results in the expression of antigen-presenting molecules (MHCII), co-stimulatory factors (CD80/86, CD40), and pro-inflammatory cytokines. Various glycoconjugates have been used to modulate DC responses. Hotaling et al. recently conducted a study on BSA conjugates of various glycans to clarify the molecular determinants (glycan composition, density, carrier cationicity) that influence DC responses.103
The LPS capsule of Gram-negative bacterial pathogens, which includes a glucosamine disaccharide moiety, is a potent activator of Toll-like receptor-4 s (TLR4s), a type of PRR present on DCs. The interaction of LPS and TLR4 can be beneficial, as it signals the presence of a foreign pathogen, but it can also be harmful as it can lead to potentially lethal septic shock. Thus, LPS or TLR4 antagonists have long been proposed as therapeutic agents to treat septic shock. Shaunak et al. have developed partially glycosylated dendrimers as TLR4 antagonists that can inhibit the inflammation response.104 Anionic carboxylic acid-terminated polyamidoamine dendrimers modified with ∼ 8 surface glucosamine residues inhibited LPS-induced TLR4 responses with µM inhibitory activities (e.g. production of inflammatory cytokines and chemokines) in immature human DCs and macrophages. In a follow-up study, the authors provide computation-based guidelines for the design of other types of macromolecular structures for the inhibition of TLR4.105
Another PRR that has been a popular target is DC-SIGN (specific intercellular adhesion molecule-3-grabbing non-integrin), a C-type (or calcium-dependent) lectin,106 which binds to terminal fucose or mannose saccharides. DC-SIGN is often exploited by pathogens to facilitate infection and transmission, as in the cases of HIV, Ebola, or Dengue viruses. Thus, targeting or disrupting the pathogen–DC-SIGN interaction has been proposed as a route to vaccines and therapies (Figure 3(a)). Varga and co-workers demonstrated that glycodendrons incorporating multiple copies of mannose saccharides inhibited DC-SIGN binding to mannose-conjugated BSA in an SPR assay, as well as inhibited HIV and Dengue infection of DC-SIGN transfected cells with activities in the low micromolar range.107 Lewis X oligosaccharides conjugated to ovalbumin have also been evaluated for their activities to inhibit DC-SIGN and stimulate immune responses.108 Recently, glycofullerenes have generated considerable interest as inhibitors of Ebola virus infection.109 Using hexakis adducts of [60]fullerene as building blocks, Munoz et al. generated water-soluble tridecafullerenes decorated with 120 peripheral mannose subunits with subnanomolar inhibitory activity against Ebola virus.
Sialic acid-binding immunoglobin-like lectins (Siglecs) and B cell activation
Siglecs are a family of cell surface transmembrane receptors present on immune cells (e.g. NK, DCs).110 Various members of the Siglec family have served as targets for the development of immunotherapies, because of their restricted expression patterns on immune cell types, high expression, and the ability to modulate receptor signaling. Due to their ability to bind sialic acids, numerous scaffolds bearing various sialoside glycans have been developed as Siglec ligands. Historically, polyacrylamide conjugates of α(2–6) sialyllactose glycans have been used to reveal the sialic-acid binding lectin activity of Siglec-2 (CD22) on B lymphocytes.111 As competitive trans ligands of Siglec-2, these multivalent probes possess sufficient avidity to overcome “masking” of the Siglec binding site by endogenous cis ligands. Much work has been devoted to targeting Siglecs as anticancer therapies (e.g. B-cell lymphomas). Toxins conjugated to the sialoglycan probes are efficiently taken up by B-cells, resulting in toxin-mediated killing.112 Rillahan and co-workers have developed glycan microarrays as a high-throughput screen for synthetic structural analogs of sialoglycans against Siglecs-7, 9, and 10.113,114 They then conjugated the high affinity ligand to PEG lipids to generate liposomal nanoparticles and evaluated targeting of Siglec-expressing cells in human blood. The liposomal nanoparticle strategy has also been used to deliver antigens to macrophages expressing Siglec-1.115
Interestingly, Siglec-2 is also an inhibitory co-receptor of B-cell receptors (BCRs), which recognize dinitrophenyl (DNP) antigens. Upon engagement of the BCR, a signaling cascade ensues to endocytose the BCR–hapten complex and facilitate antigen uptake for processing and display on the cell surface. Siglec-2 functions to prevent erroneous B cell activation by down-regulating signaling and inhibits the BCR signaling cascade. Courtney and co-workers have previously generated homopolymers of DNP (stimulatory antigen) and its co-polymers with pendant α(2–6) sialyllactose groups (inhibitory antigen) to modulate BCR signaling and establish the relationship between signaling and endocytosis116 (Figure 3(a)). Hudak et al. have shown that sialic acid glycopolymers end-functionalized with phospholipids can passively incorporate into cell membranes of tumor cells to engage, in a trans interaction, Siglec-7 presented on natural killer (NK) cells, resulting in the remodeled cell’s altered susceptibility to NK killing.117 This study provided an insight into the functional role of the hypersialylated phenotype acquired by many adenocarcinoma cells to evade immune surveillance by NK cells.
Selectins
Upon encountering a pathogen, macrophages express pro-inflammatory cytokines that induce cell surface expression of selectins, which are also members of the C-type lectin family. Interactions between selectins expressed on the surfaces of endothelial cells and their sialylated (and often sulfated) ligands presented on circulating leukocytes induce “leukocyte rolling,” a phenomenon, which allows leukocytes to scan a blood vessel and enter an infection site. Competitive inhibitors of selectin in the form of glycoconjugates (polymers of synthetic glycoproteins) have been proposed as anti-inflammatory agents. Sanders and co-workers have designed glycopolymers generated by ring-opening metathesis that efficiently inhibited L-selectin binding.118 This study revealed that inhibition is dependent on the specific sulfation pattern of the glycan moieties, as well as multivalency. Importantly, this study points to the need to consider the effects of shear forces influencing glycan binding under physiologically relevant flow conditions. A subsequent study by Rele and co-workers used poly(ethylene)-oxide dendrimers of sulfated lactose to inhibit L-selectin binding, as well as reduce inflammatory cell recruitment.119
Glycomaterials for the activation of the adaptive immune system
Prophylactic and/or vaccine treatments have been poised as solutions to infectious diseases that have been difficult to diagnose and treat, due to the lack of specific clinical symptoms and rapid emergence of antibiotic resistance. A number of glycan-based vaccines have been developed to immunize against pathogenic microbes, such as bacteria, fungi, and viruses.120 Often, these approaches employ protein glycoconjugates121 to elicit stronger immunogenic responses from otherwise weakly immunogenic glycans isolated from pathogenic cell surfaces (Figure 3(b)). In many cases, the carrier protein, often itself immunogenic, can also serve as a scaffold for the multivalent display of glycans. Such conjugate vaccines, including Prevnar (Wyeth Pharmaceuticals, against Streptococcus pneumoniae) and Pentacel (Sanofi Pasteur, against Haemophilus influenzae), are now commercially available, or in development, and have been proven to produce long-lasting protection against pathogens.
Perhaps the most prominent example of antibodies combating infection via a glycan-mediated mechanism is that of the broadly neutralizing antibody 2G12. 2G12 is a human monoclonal antibody that specifically recognizes the HIV envelope glycoprotein, gp120, via its mannose oligosaccharides.122,123 The discovery of 2G12 and other glycan-targeting antibodies was remarkable, as the dense canopy of glycans on the HIV envelope, often called the “glycan shield” for its defensive ability to obscure peptide epitopes from antibody recognition, was now breachable and could even be used as a targeting moiety.124,125 Over the years, several groups have created various multivalent constructs of this oligosaccharide to generate high-avidity 2G12 binders that could serve as haptens for the development of anti-HIV antibodies. Such constructs include protein or immunogen conjugates of a cyclic glycopeptide mimotope of the oligomannose saccharides,126,127 dendrons,128 viral capsids,129,130 and even nucleic acid-based displays.131 The latter have especially garnered considerable interest, due to the ability for combinatorial screening and the capability to precisely tune interligand distances and valencies. Using PNA conjugates of mannose oligosaccharides to display the glycan epitopes, Gorska et al. demonstrated that carbohydrate spacing matching the binding sites of the dimeric 2G12 antibody is crucial for avidity.132 Because DC-SIGN also has affinity for the same oligomannose saccharides, anti-HIV therapeutics with the inhibition of the DC-SIGN-gp120 interaction in mind have also been proposed. Dendrimers,133,134 glycopolymers,135 and other platforms that do so have previously been developed.
Liposomal adjuvants generated by incorporation of glycolipid components of pathogens into nanoparticles have also been developed. Lipid A, a common component of Gram-negative bacterial cell walls, and trehalose 6,6′-dimycolate (TDM), a cord factor present in mycobacterial cell walls are prominent in adjuvant formulation. Monophosphoryl lipid A, a TLR4 agonist, has been shown to elicit CD8 ( + ) T-cell responses in vivo.136 A synthetic analog of TDM, trehalose 6,6′-dibehenate (TDB) has been inserted into cationic liposomes of the quaternary ammonium lipid N,N′-dimethyl-N,N′-dioctadecylammonium, to generate the adjuvant CAF01.137 Using ovalbumin as a model vaccine antigen, the authors show that CAF01 primed complex immune responses, and these responses were above those obtained with currently used adjuvants (e.g. monophosphoryl lipid A, Alum). They also demonstrate that vaccines based on CAF01 elicited significant immunity against three models of infection, M. tuberculosis, Chlamydia trachomatis, and malaria (Plasmodium yoelii), as well as HIV.138,139 In addition to its immunoprotective effects, TDB has also been shown to play a critical role in promoting the stability of such cationic liposomes.140 Due to its capability to retain water and stabilize membranes, the trehalose disaccharide has been implicated in endowing organisms to survive anhydrous and freezing conditions.141 Indeed, trehalose glycopolymers have been shown to promote stability of protein conjugates.142
Torosantucci et al. have generated laminarin conjugates of the diphtheria toxoid CRM197 as antifungal prophylactic vaccines.143 Laminarin is a β-glucan with β(1,3) and some β(1,6) branching, (isolated from plant algae) that mimics the viability-critical β-glucan polysaccharides present on the cell walls of all human pathogenic fungi. The authors show that mice immunized with the laminarin–CRM conjugates produced significant titers of IgG and IgM antibodies against β(1,3) and β(1,6) glucans. They show that the laminarin–CRM conjugates effectively reduced fungal burden and conferred significant protection against lethal systemic Candida albicans and Aspergillus fumigatus infections. Importantly, they also show that the protection was mediated mostly (if not all) via the elicited anti-β-glucan (not anti-CRM) IgG antibodies. These antiglucan antibodies inhibited fungal growth by directly binding to the fungal hyphae cell surfaces. In another study, Paulovicova et al. demonstrated that mice immunized with heptamannoside BSA conjugates induced Th1, Th2, and Th17 immune responses to induce candidacidal activity.144
Recently, researchers at GlaxoSmithKline, Novartis, and the University of Oxford developed a glycoconjugate vaccine against Salmonella enterica serovar typhimurium, for which none is currently available.145 The authors conjugated the Salmonella LPS O-antigen (OAg) to CRM197 and evaluated the production of anti-OAg antibodies in mice after immunization, as well as the serum bactericidal activity against Salmonella. Glycoconjugates with single or double attachments were found to elicit significant levels of anti-OAg antibodies with serum bactericidal activity. Notably, the researchers explored various site-selective conjugation methods and found that the conjugation site of the antigen to CRM197 greatly affected immunogenicity. These findings are consistent with the model proposed by Avci et al.,146 wherein carbohydrate presentation within peptides can differentially affect immune responses by influencing the communication between B-cells and T-cells.
Summary
The elegant studies described in this review clearly illustrate the central role of materials science in providing key insights into how the organization of glycans at the cell surface influences host–pathogen interactions, and how glycans may be targeted to prevent infection or to modulate the host response to pathogenic threats. There is no doubt that future innovation in the design and synthesis of ever more sophisticated glycomaterials will yield novel macromolecular architectures, providing advanced tools enabling the interrogation of glycan function. The staggering compositional and functional complexity of the glycocalyx remains one of the most exciting, yet largely unexplored, frontiers in glycobiology research. While the contributions of its individual components have increasingly come into focus, the glycocalyx in its entirety, as a dynamic hierarchically organized biomolecular system, still harbors many discoveries to be made. New avenues of glycobiology research are emerging that integrate chemical, materials, and molecular biological approaches aimed at defining how the nanoscale organization of the glycocalyx instructs biological events occurring at the cellular boundary. A particular challenge in this field is the translation of information obtained from various analytical platforms within the complexity of biological systems. Cell surface glycan engineering strategies that integrate concepts of materials design are beginning to emerge and are likely to play a central role in revealing the mechanisms through which the glycocalyx influences pathogenesis and to provide new paradigms for the formulation of therapeutic interventions.
Acknowledgements
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: CJF was supported in part by the UCSD Graduate Training Program in Molecular Biophysics through an institutional training grant from the National Institute of General Medical Sciences, T32 GM08326. This work was supported in part by the National Institutes of Health through grants 5 R00 EB013446-05 (NIBIB) and 1DP2HD087954-01 (NICHD).
Authors’ contributions
MLH, CJF, and KG designed and wrote the manuscript. KG had primary responsibility for its final content.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.s
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