Structure-Based Design and Synthesis of Lipid A Derivatives to Modulate Cytokine Responses.

Agonists of Toll like receptors (TLRs) have attracted interest as adjuvants and immune modulators. A crystal structure of TLR4/MD2 with E. coli LPS indicates that the fatty acid at C-2 of the lipid A component of LPS induces dimerization of two TLR4-MD2 complexes, which in turn initiates cell signaling leading to the production of (pro)inflammatory cytokines. To probe the importance of the (R)-3-hydroxymyristate at C-2 of lipid A, a range of bis- and mono-phosphoryl lipid A derivatives with different modifications at C-2 were prepared by a strategy in which 2-methylnaphthyl ethers were employed as permanent protecting group that could be readily removed by catalytic hydrogenation. The C-2 amine was protected as 9-fluorenylmethyloxycarbamate, which at a later stage could be removed to give a free amine that was modified by different fatty acids. LPS and the synthetic lipid As induced the same cytokines, however, large differences in activity were observed. A compound having a hexanoyl moiety at C-2 still showed agonistic properties, but further shortening to a butanoyl abolished activity. The modifications had a larger influence on monophosphoryl lipid As. The lipid As having a butanoyl moiety at C-2 could selectively antagonize TRIF associated cytokines induced by LPS or lipid A.

Chemoenzymatic Synthesis of Tri-antennary N-Glycans Terminating in Sialyl-Lewisx Reveals the Importance of Glycan Complexity for Influenza A Virus Receptor Binding.

Sialyl-Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty-two isomeric asymmetrical tri-antennary N-glycans presenting SLex-Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high-affinity ligands for E-selectin. The N-glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5'-diphospho-N-trifluoracetylglucosamine (UDP-GlcNHTFA) that can be transferred by branching N-acetylglucosaminyltransferases to give, after base treatment, GlcNH2-containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E-selectin bound equally well to linear glycans and tri-antennary N-glycans presenting SLex-Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri-antennary N-glycans. Furthermore, several H5 HAs preferentially bound to N-glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N-glycans presenting SLex.

Antibacterial and anti-inflammatory properties of host defense peptides against Staphylococcus aureus.

Cationic host defense peptides (HDPs) are a promising alternative to antibiotics in the fight against Staphylococcus aureus infections. In this study, we investigated the antibacterial and immunomodulatory properties of three HDPs namely IDR-1018, CATH-2, and LL-37. Although all three HDPs significantly inhibited LPS-induced activation of human macrophages, only CATH-2 prevented S. aureus growth. When applied to different infection models focused on intracellularly surviving bacteria, only IDR-1018 showed a consistent reduction in macrophage bacterial uptake. However, this observation did not correlate with an increase in killing the efficiency of intracellular S. aureus. Here, we conclude that despite the promising antibacterial and anti-inflammatory properties of the selected HDPs, macrophages' intrinsic antibacterial functions were not improved. Future studies should either focus on combining different HDPs or using them synergistically with other antibacterial agents to improve immune cells' efficacy against S. aureus pathogenesis.

Heparan Sulfate Proteoglycans as Attachment Factor for SARS-CoV-2.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments, using an extensive heparan sulfate (HS) oligosaccharide library, showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length- and sequence-dependent manner. A hexasaccharide composed of IdoA2S-GlcNS6S repeating units was identified as the minimal binding epitope. Surface plasmon resonance showed the SARS-CoV-2 spike protein binds with a much higher affinity to heparin (K D = 55 nM) compared to the RBD (K D = 1 µM) alone. It was also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. However, exogenous administered heparin or a highly sulfated HS oligosaccharide inhibited RBD binding to cells. Furthermore, an enzymatic removal of HS proteoglycan from physiological relevant tissue resulted in a loss of RBD binding. The data support a model in which HS functions as the point of initial attachment allowing the virus to travel through the glycocalyx by low-affinity high-avidity interactions to reach the cell membrane, where it can engage with ACE2 for cell entry. Microarray binding experiments showed that ACE2 and HS can simultaneously engage with the RBD, and it is likely no dissociation between HS and RBD is required for binding to ACE2. The results highlight the potential of using HS oligosaccharides as a starting material for therapeutic agent development.

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation.

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

Heparan sulfate proteoglycans as attachment factor for SARS-CoV-2.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments using an extensive heparan sulfate (HS) oligosaccharide library showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length- and sequence-dependent manner. Hexa- and octa-saccharides composed of IdoA2S-GlcNS6S repeating units were identified as optimal ligands. Surface plasma resonance (SPR) showed the SARS-CoV-2 spike protein binds with much higher affinity to heparin (KD = 55 nM) compared to the RBD (KD = 1 uM) alone. We also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. Our data supports a model in which HS functions as the point of initial attachment for SARS-CoV-2 infection. Tissue staining studies using biologically relevant tissues indicate that heparan sulfate proteoglycan (HSPG) is a critical attachment factor for the virus. Collectively, our results highlight the potential of using HS oligosaccharides as a therapeutic agent by inhibiting SARS-CoV-2 binding to target cells.

Chemoenzymatic Synthesis of Campylobacter jejuni Lipo-oligosaccharide Core Domains to Examine Guillain-Barré Syndrome Serum Antibody Specificities.

Guillain-Barré syndrome is often caused by Campylobacter jejuni infection that has induced antibodies to the lipo-oligosaccharide (LOS) that cross-react with gangliosides at peripheral nerves causing polyneuropathy. To examine fine specificities of anti-ganglioside antibodies and develop a more robust platform for diagnosis and disease monitoring, we developed a chemoenzymatic approach that provided an unprecedented panel of oligosaccharides composed of the inner-core of the LOS of C. jejuni extended by various ganglioside mimics. The compounds and corresponding ganglio-oligosaccharides were printed as a microarray to examine binding specificities of lectins, anti-ganglioside antibodies, and serum antibodies of GBS patients. Although lectins and anti-ganglioside antibodies did not differentiate the ganglio-oligosaccharides and mimics, the patient serum samples bound much more strongly to the ganglioside mimics. The data indicate that antibodies have been elicited to a foreign epitope that includes a heptosyl residue unique of bacterial LOS and that these antibodies subsequently cross-react with lower affinity to gangliosides. The microarray detected anti-GM1a antibodies with high sensitivity and will be attractive for diagnosis, disease monitoring, and immunological research.

Mono- and Di-Fucosylated Glycans of the Parasitic Worm S. mansoni are Recognized Differently by the Innate Immune Receptor DC-SIGN.

The parasitic worm, Schistosoma mansoni, expresses unusual fucosylated glycans in a stage-dependent manner that can be recognized by the human innate immune receptor DC-SIGN, thereby shaping host immune responses. We have developed a synthetic approach for mono- and bis-fucosylated LacdiNAc (LDN-F and LDN-DF, respectively), which are epitopes expressed on glycolipids and glycoproteins of S. mansoni. It is based on the use of monosaccharide building blocks having carefully selected amino-protecting groups, facilitating high yielding and stereoselective glycosylations. The molecular interaction between the synthetic glycans and DC-SIGN was studied by NMR and molecular modeling, which demonstrated that the α1,3-fucoside of LDN-F can coordinate with the Ca2+ -ion of the canonical binding site of DC-SIGN allowing for additional interactions with the underlying LDN backbone. The 1,2-fucoside of LDN-DF can be complexed in a similar manner, however, in this binding mode GlcNAc and GalNAc of the LDN backbone are placed away from the protein surface resulting in a substantially lower binding affinity. Glycan microarray binding studies showed that the avidity and selectivity of binding is greatly enhanced when the glycans are presented multivalently, and in this format Lex and LDN-F gave strong responsiveness, whereas no binding was detected for LDN-DF. The data indicates that S. mansoni has developed a strategy to avoid detection by DC-SIGN in a stage-dependent manner by the addition of a fucoside to a number of its ligands.

Mutation of the second sialic acid-binding site of influenza A virus neuraminidase drives compensatory mutations in hemagglutinin.

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential.

Chemoenzymatic synthesis of the oligosaccharide moiety of the tumor-associated antigen disialosyl globopentaosylceramide.

Disialosyl globopentaosylceramide (DSGb5) is often expressed by renal cell carcinomas. To investigate properties of DSGb5, we have prepared its oligosaccharide moiety by chemically synthesizing Gb5 which was enzymatically sialylated using the mammalian sialyltransferases ST3Gal1 and ST6GalNAc5. Glycan microarray binding studies indicate that Siglec-7 does not recognize DSGb5, and preferentially binds Neu5Acα(2,8)Neu5Ac containing glycans.

N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses.

A species barrier for the influenza A virus is the differential expression of sialic acid, which can either be α2,3-linked for avians or α2,6-linked for human viruses. The influenza A virus hosts also express other species-specific sialic acid derivatives. One major modification at C-5 is N-glycolyl (NeuGc), instead of N-acetyl (NeuAc). N-glycolyl is mammalian specific and expressed in pigs and horses, but not in humans, ferrets, seals, or dogs. Hemagglutinin (HA) adaptation to either N-acetyl or N-glycolyl is analyzed on a sialoside microarray containing both α2,3- and α2,6-linkage modifications on biologically relevant N-glycans. Binding studies reveal that avian, human, and equine HAs bind either N-glycolyl or N-acetyl. Structural data on N-glycolyl binding HA proteins of both H5 and H7 origin describe this specificity. Neuraminidases can cleave N-glycolyl efficiently, and tissue-binding studies reveal strict species specificity. The exclusive manner in which influenza A viruses differentiate between N-glycolyl and N-acetyl is indicative of selection.

Protecting-Group-Controlled Enzymatic Glycosylation of Oligo-N-Acetyllactosamine Derivatives.

We describe a chemoenzymatic strategy that can give a library of differentially fucosylated and sialylated oligosaccharides starting from a single chemically synthesized tri-N-acetyllactosamine derivative. The common precursor could easily be converted into 6 different hexasaccharides in which the glucosamine moieties are either acetylated (GlcNAc) or modified as a free amine (GlcNH2 ) or Boc (GlcNHBoc). Fucosylation of the resulting compounds by a recombinant fucosyl transferase resulted in only modification of the natural GlcNAc moieties, providing access to 6 selectively mono- and bis-fucosylated oligosaccharides. Conversion of the GlcNH2 or GlcNHBoc moieties into the natural GlcNAc, followed by sialylation by sialyl transferases gave 12 differently fucosylated and sialylated compounds. The oligosaccharides were printed as a microarray that was probed by several glycan-binding proteins, demonstrating that complex patterns of fucosylation can modulate glycan recognition.

Synthesis and Immunological Evaluation of a Multicomponent Cancer Vaccine Candidate Containing a Long MUC1 Glycopeptide.

A fully synthetic MUC1-based cancer vaccine was designed and chemically synthesized containing an endogenous helper T-epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC). It also activated cytotoxic T-lymphocytes. Collectively, the immunological data demonstrate engagement of helper T-cells in immune activation. A synthetic methodology was developed for a penta-glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1-based cancer vaccines that elicited potent immune responses employed exogenous helper T-epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T-epitope will be more attractive, because it will activate cognate CD4+ T-cells that will provide critical tumor-specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory.

Heparan Sulfate Microarray Reveals That Heparan Sulfate-Protein Binding Exhibits Different Ligand Requirements.

Heparan sulfates (HS) are linear sulfated polysaccharides that modulate a wide range of physiological and disease-processes. Variations in HS epimerization and sulfation provide enormous structural diversity, which is believed to underpin protein binding and regulatory properties. The ligand requirements of HS-binding proteins have, however, been defined in only a few cases. We describe here a synthetic methodology that can rapidly provide a library of well-defined HS oligosaccharides. It is based on the use of modular disaccharides to assemble several selectively protected tetrasaccharides that were subjected to selective chemical modifications such as regioselective O- and N-sulfation and selective de-sulfation. A number of the resulting compounds were subjected to enzymatic modifications by 3-O-sulfotransferases-1 (3-OST1) to provide 3-O-sulfated derivatives. The various approaches for diversification allowed one tetrasaccharide to be converted into 12 differently sulfated derivatives. By employing tetrasaccharides with different backbone compositions, a library of 47 HS-oligosaccharides was prepared and the resulting compounds were used to construct a HS microarray. The ligand requirements of a number of HS-binding proteins including fibroblast growth factor 2 (FGF-2), and the chemokines CCL2, CCL5, CCL7, CCL13, CXCL8, and CXCL10 were examined using the array. Although all proteins recognized multiple compounds, they exhibited clear differences in structure-binding characteristics. The HS microarray data guided the selection of compounds that could interfere in biological processes such as cell proliferation. Although the library does not cover the entire chemical space of HS-tetrasaccharides, the binding data support a notion that changes in cell surface HS composition can modulate protein function.

Synthesis of asymmetrical multiantennary human milk oligosaccharides.

Despite mammalian glycans typically having highly complex asymmetrical multiantennary architectures, chemical and chemoenzymatic synthesis has almost exclusively focused on the preparation of simpler symmetrical structures. This deficiency hampers investigations into the biology of glycan-binding proteins, which in turn complicates the biomedical use of this class of biomolecules. Herein, we describe an enzymatic strategy, using a limited number of human glycosyltransferases, to access a collection of 60 asymmetric, multiantennary human milk oligosaccharides (HMOs), which were used to develop a glycan microarray. Probing the array with several glycan-binding proteins uncovered that not only terminal glycoepitopes but also complex architectures of glycans can influence binding selectivity in unanticipated manners. N- and O-linked glycans express structural elements of HMOs, and thus, the reported synthetic principles will find broad applicability.

MUC1 Vaccines, Comprised of Glycosylated or Non-Glycosylated Peptides or Tumor-Derived MUC1, Can Circumvent Immunoediting to Control Tumor Growth in MUC1 Transgenic Mice.

It remains challenging to produce decisive vaccines against MUC1, a tumor-associated antigen widely expressed by pancreas, breast and other tumors. Employing clinically relevant mouse models, we ruled out such causes as irreversible T-cell tolerance, inadequate avidity, and failure of T-cells to recognize aberrantly glycosylated tumor MUC1. Instead, every tested MUC1 preparation, even non-glycosylated synthetic 9mer peptides, induced interferon gamma-producing CD4(+) and CD8(+) T-cells that recognized glycosylated variants including tumor-associated MUC1. Vaccination with synthetic peptides conferred protection as long as vaccination was repeated post tumor challenge. Failure to revaccinate post challenge was associated with down-regulated tumor MUC1 and MHC molecules. Surprisingly, direct admixture of MUC1-expressing tumor with MUC1-hyperimmune T-cells could not prevent tumor outgrowth or MUC1 immunoediting, whereas ex vivo activation of the hyperimmune T-cells prior to tumor admixture rendered them curative. Therefore, surrogate T-cell preactivation outside the tumor bed, either in culture or by repetitive vaccination, can overcome tumor escape.

Controlled Multi-functionalization Facilitates Targeted Delivery of Nanoparticles to Cancer Cells.

A major objective of nanomedicine is to combine in a controlled manner multiple functional entities into a single nanoscale device to target particles with great spatial precision, thereby increasing the selectivity and potency of therapeutic drugs. A multifunctional nanoparticle is described for controlled conjugation of a cytotoxic drug, a cancer cell targeting ligand, and an imaging moiety. The approach is based on the chemical synthesis of polyethylene glycol that at one end is modified by a thioctic acid for controlled attachment to a gold core. The other end of the PEG polymers is modified by a hydrazine, amine, or dibenzocyclooctynol moiety for conjugation with functional entities having a ketone, activated ester, or azide moiety, respectively. The conjugation approach allowed the controlled attachment of doxorubicin through an acid-labile hydrazone linkage, an Alexa Fluor dye through an amide bond, and a glycan-based ligand for the cell surface receptor CD22 of B-cells using strain promoted azide-alkyne cycloaddition. The incorporation of the ligand for CD22 led to rapid entry of the nanoparticle by receptor-mediated endocytosis. Covalent attachment of doxorubicin via hydrazone linkage caused pH-responsive intracellular release of doxorubicin and significantly enhanced the cytotoxicity of nanoparticles. A remarkable 60-fold enhancement in cytotoxicity of CD22 (+) lymphoma cells was observed compared to non- targeted nanoparticles.

Mucin architecture behind the immune response: design, evaluation and conformational analysis of an antitumor vaccine derived from an unnatural MUC1 fragment.

A tripartite cancer vaccine candidate, containing a quaternary amino acid (α-methylserine) in the most immunogenic domain of MUC1, has been synthesized and examined for antigenic properties in transgenic mice. The vaccine which is glycosylated with GalNAc at the unnatural amino acid, was capable of eliciting potent antibody responses recognizing both glycosylated and unglycosylated tumour-associated MUC1 peptides and native MUC1 antigen present on cancer cells. The peptide backbone of the novel vaccine presents the bioactive conformation in solution and is more resistant to enzymatic degradation than the natural counter part. In spite of these features, the immune response elicited by the unnatural vaccine was not improved compared to a vaccine candidate containing natural threonine. These observations were rationalized by conformational studies, indicating that the presentation and dynamics of the sugar moiety displayed by the MUC1 derivative play a critical role in immune recognition. It is clear that engineered MUC1-based vaccines bearing unnatural amino acids have to be able to emulate the conformational properties of the glycosidic linkage between the GalNAc and the threonine residues. The results described here will be helpful to the rational design of efficacious cancer vaccines.

Linear synthesis and immunological properties of a fully synthetic vaccine candidate containing a sialylated MUC1 glycopeptide.

A strategy for the linear synthesis of a sialylated glycolipopeptide cancer vaccine candidate has been developed using a strategically designed sialyl-Tn building block and microwave-assisted solid-phase peptide synthesis. The glycolipopeptide elicited potent humoral and cellular immune responses. T-cells primed by such a vaccine candidate could be restimulated by tumor-associated MUC1.

Immune and anticancer responses elicited by fully synthetic aberrantly glycosylated MUC1 tripartite vaccines modified by a TLR2 or TLR9 agonist.

The mucin MUC1 is overexpressed and aberrantly glycosylated by many epithelial cancer cells manifested by truncated O-linked saccharides. Although tumor-associated MUC1 has generated considerable attention because of its potential for the development of a therapeutic cancer vaccine, it has been difficult to design constructs that consistently induce cytotoxic T-lymphocytes (CTLs) and ADCC-mediating antibodies specific for the tumor form of MUC1. We have designed, chemically synthesized, and immunologically examined vaccine candidates each composed of a glycopeptide derived from MUC1, a promiscuous Thelper peptide, and a TLR2 (Pam3 CysSK4 ) or TLR9 (CpG-ODN 1826) agonist. It was found that the Pam3 CysSK4 -containing compound elicits more potent antigenic and cellular immune responses, resulting in a therapeutic effect in a mouse model of mammary cancer. It is thus shown, for the first time, that the nature of an inbuilt adjuvant of a tripartite vaccine can significantly impact the quality of immune responses elicited against a tumor-associated glycopeptide. The unique adjuvant properties of Pam3 CysSK4 , which can reduce the suppressive function of regulatory T cells and enhance the cytotoxicity of tumor-specific CTLs, are likely responsible for the superior properties of the vaccine candidate 1.