Understanding the Chemistry and Biology of Glycosylation with Glycan Synthesis.

Glycoscience research has been significantly impeded by the complex compositions of the glycans present in biological molecules and the lack of convenient tools suitable for studying the glycosylation process and its function. Polysaccharides and glycoconjugates are not encoded directly by genes; instead, their biosynthesis relies on the differential expression of carbohydrate enzymes, resulting in heterogeneous mixtures of glycoforms, each with a distinct physiological activity. Access to well-defined structures is required for functional study, and this has been provided by chemical and enzymatic synthesis and by the engineering of glycosylation pathways. This review covers general methods for preparing glycans commonly found in mammalian systems and applying them to the synthesis of therapeutically significant glycoconjugates (glycosaminoglycans, glycoproteins, glycolipids, glycosylphosphatidylinositol-anchored proteins) and the development of carbohydrate-based vaccines.

Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B, and G.

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer.

A Neutralizing Antibody Recognizing Primarily N-Linked Glycan Targets the Silent Face of the HIV Envelope.

Virtually the entire surface of the HIV-1-envelope trimer is recognized by neutralizing antibodies, except for a highly glycosylated region at the center of the "silent face" on the gp120 subunit. From an HIV-1-infected donor, #74, we identified antibody VRC-PG05, which neutralized 27% of HIV-1 strains. The crystal structure of the antigen-binding fragment of VRC-PG05 in complex with gp120 revealed an epitope comprised primarily of N-linked glycans from N262, N295, and N448 at the silent face center. Somatic hypermutation occurred preferentially at antibody residues that interacted with these glycans, suggesting somatic development of glycan recognition. Resistance to VRC-PG05 in donor #74 involved shifting of glycan-N448 to N446 or mutation of glycan-proximal residue E293. HIV-1 neutralization can thus be achieved at the silent face center by glycan-recognizing antibody; along with other known epitopes, the VRC-PG05 epitope completes coverage by neutralizing antibody of all major exposed regions of the prefusion closed trimer.

Increased expression of SSEA-4 on TKI-resistant non-small cell lung cancer with EGFR-T790M mutation.

Non-small cell lung cancer (NSCLC), a major life-threatening disease accounting for 85% of all lung cancer cases, has been treated with tyrosine kinase inhibitors (TKIs), but often resulted in drug resistance, and approximately 60% of TKI-resistant cases are due to acquired secondary (epithelial growth factor receptor) EGFR-T790M mutation. To identify alternative targets for TKI-resistant NSCLC with EGFR-T790M mutation, we found that the three globo-series glycosphingolipids are increasingly expressed on this type of NSCLC cell lines, and among them, the increase of stage-specific embryonic antigen-4 (SSEA-4) expression is the most significant. Compared to TKI-sensitive cell lines, SSEA-4 and the key enzyme ß3GalT5 responsible for the synthesis of SSEA3 are more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and the expression levels strongly correlate with poor survival in patients with EGFR mutation. In addition, we demonstrated that a SSEA-4 targeted monoclonal antibody, especially the homogeneous glycoform with well-defined Fc glycan designed to improve effective functions, is highly effective against this subpopulation of NSCLC in cell-based and animal studies. These findings provide a direction for the prediction of tumor recurrence and treatment of TKI-resistant NSCLC with EGFR-T790M mutation.

Glycans Function as Anchors for Antibodies and Help Drive HIV Broadly Neutralizing Antibody Development.

Apex broadly neutralizing HIV antibodies (bnAbs) recognize glycans and protein surface close to the 3-fold axis of the envelope (Env) trimer and are among the most potent and broad Abs described. The evolution of apex bnAbs from one donor (CAP256) has been studied in detail and many Abs at different stages of maturation have been described. Using diverse engineering tools, we investigated the involvement of glycan recognition in the development of the CAP256.VRC26 Ab lineage. We found that sialic acid-bearing glycans were recognized by germline-encoded and somatically mutated residues on the Ab heavy chain. This recognition provided an "anchor" for the Abs as the core protein epitope varies, prevented complete neutralization escape, and eventually led to broadening of the response. These findings illustrate how glycan-specific maturation enables a human Ab to cope with pathogen escape mechanisms and will aid in optimization of immunization strategies to induce V2 apex bnAb responses.

Low-sugar universal mRNA vaccine against coronavirus variants with deletion of glycosites in the S2 or stem of SARS-CoV-2 spike messenger RNA (mRNA).

Since the outbreak of Severe Acute Respiratory Syndrome Virus-2 (SARS-CoV-2) in 2019, more than 15 million spike protein sequences have been identified, raising a new challenge for the development of a broadly protective vaccine against the various emerging variants. We found that the virus, like most other human viruses, depends on host-made glycans to shield the conserved epitopes on spike protein from immune response and demonstrated that deletion of the glycan shields exposed highly conserved epitopes and elicited broadly protective immune responses. In this study, we identified 17 conserved epitopes from 14 million spike protein sequences and 11 of the conserved epitopes are in the S2 domain, including the six most conserved epitopes in the stem region. We also demonstrated that deletion of the glycosites in the spike messenger RNA (mRNA) S2 domain or the stem region exposed the highly conserved epitopes and elicited broadly protective immune responses, particularly CD-8+ T cell response against various SARS-CoV-2 variants, and other human coronaviruses including MERS, SARS viruses, and those causing common cold.

A Prominent Site of Antibody Vulnerability on HIV Envelope Incorporates a Motif Associated with CCR5 Binding and Its Camouflaging Glycans.

The dense patch of high-mannose-type glycans surrounding the N332 glycan on the HIV envelope glycoprotein (Env) is targeted by multiple broadly neutralizing antibodies (bnAbs). This region is relatively conserved, implying functional importance, the origins of which are not well understood. Here we describe the isolation of new bnAbs targeting this region. Examination of these and previously described antibodies to Env revealed that four different bnAb families targeted the (324)GDIR(327) peptide stretch at the base of the gp120 V3 loop and its nearby glycans. We found that this peptide stretch constitutes part of the CCR5 co-receptor binding site, with the high-mannose patch glycans serving to camouflage it from most antibodies. GDIR-glycan bnAbs, in contrast, bound both (324)GDIR(327) peptide residues and high-mannose patch glycans, which enabled broad reactivity against diverse HIV isolates. Thus, as for the CD4 binding site, bnAb effectiveness relies on circumventing the defenses of a critical functional region on Env.

Early Antibody Lineage Diversification and Independent Limb Maturation Lead to Broad HIV-1 Neutralization Targeting the Env High-Mannose Patch.

The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways.

Glycosite-deleted mRNA of SARS-CoV-2 spike protein as a broad-spectrum vaccine.

Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein vaccine, and there is no need to manufacture the protein in vitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response. Here, we showed that the mRNA vaccine of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with deletion of glycosites in the receptor-binding domain (RBD) or especially the subunit 2 (S2) domain to expose more conserved epitopes elicited stronger antibody and CD8+ T cell responses with broader protection against the alpha, beta, gamma, delta, and omicron variants, compared to the unmodified mRNA. Immunization of such mRNA resulted in accumulation of misfolded spike protein in the endoplasmic reticulum, causing the up-regulation of BiP/GRP78, XBP1, and p-eIF2α to induce cell apoptosis and strong CD8+ T cell response. In addition, dendritic cells (DCs) incubated with S2-glysosite deleted mRNA vaccine increased class I major histocompatibility complex (MHC I) expression. This study provides a direction for the development of broad-spectrum mRNA vaccines which may not be achieved with the use of expressed proteins as antigens.

Identification of Common Features in Prototype Broadly Neutralizing Antibodies to HIV Envelope V2 Apex to Facilitate Vaccine Design.

Broadly neutralizing antibodies (bnAbs) directed to the V2 apex of the HIV envelope (Env) trimer isolated from individual HIV-infected donors potently neutralize diverse HIV strains, but strategies for designing immunogens to elicit bnAbs have not been identified. Here, we compared four prototypes (PG9, CH01, PGT145, and CAP256.VRC26.09) of V2 apex bnAbs and showed that all recognized a core epitope of basic V2 residues and the glycan-N160. Two prototype bnAbs were derived from VH-germlines that were 99% identical and used a common germline D-gene encoded YYD-motif to interact with the V2-epitope. We identified isolates that were neutralized by inferred germline (iGL) versions of three of the prototype bnAbs. Soluble Env derived from one of these isolates was shown to form a well-ordered Env trimer that could serve as an immunogen to initiate a V2-apex bnAb response. These studies illustrate a strategy to transition from panels of bnAbs to vaccine candidates.

Glycoconjugates: Synthesis, Functional Studies, and Therapeutic Developments.

Glycoconjugates are major constituents of mammalian cells that are formed via covalent conjugation of carbohydrates to other biomolecules like proteins and lipids and often expressed on the cell surfaces. Among the three major classes of glycoconjugates, proteoglycans and glycoproteins contain glycans linked to the protein backbone via amino acid residues such as Asn for N-linked glycans and Ser/Thr for O-linked glycans. In glycolipids, glycans are linked to a lipid component such as glycerol, polyisoprenyl pyrophosphate, fatty acid ester, or sphingolipid. Recently, glycoconjugates have become better structurally defined and biosynthetically understood, especially those associated with human diseases, and are accessible to new drug, diagnostic, and therapeutic developments. This review describes the status and new advances in the biological study and therapeutic applications of natural and synthetic glycoconjugates, including proteoglycans, glycoproteins, and glycolipids. The scope, limitations, and novel methodologies in the synthesis and clinical development of glycoconjugates including vaccines, glyco-remodeled antibodies, glycan-based adjuvants, glycan-specific receptor-mediated drug delivery platforms, etc., and their future prospectus are discussed.

Homogeneous antibody and CAR-T cells with improved effector functions targeting SSEA-4 glycan on pancreatic cancer.

Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.

Identification of existing pharmaceuticals and herbal medicines as inhibitors of SARS-CoV-2 infection.

The outbreak of COVID-19 caused by SARS-CoV-2 has resulted in more than 50 million confirmed cases and over 1 million deaths worldwide as of November 2020. Currently, there are no effective antivirals approved by the Food and Drug Administration to contain this pandemic except the antiviral agent remdesivir. In addition, the trimeric spike protein on the viral surface is highly glycosylated and almost 200,000 variants with mutations at more than 1,000 positions in its 1,273 amino acid sequence were reported, posing a major challenge in the development of antibodies and vaccines. It is therefore urgently needed to have alternative and timely treatments for the disease. In this study, we used a cell-based infection assay to screen more than 3,000 agents used in humans and animals, including 2,855 small molecules and 190 traditional herbal medicines, and identified 15 active small molecules in concentrations ranging from 0.1 nM to 50 µM. Two enzymatic assays, along with molecular modeling, were then developed to confirm those targeting the virus 3CL protease and the RNA-dependent RNA polymerase. Several water extracts of herbal medicines were active in the cell-based assay and could be further developed as plant-derived anti-SARS-CoV-2 agents. Some of the active compounds identified in the screen were further tested in vivo, and it was found that mefloquine, nelfinavir, and extracts of Ganoderma lucidum (RF3), Perilla frutescens, and Mentha haplocalyx were effective in a challenge study using hamsters as disease model.

Mechanism of Antigen Presentation and Specificity of Antibody Cross-Reactivity Elicited by an Oligosaccharide-Conjugate Cancer Vaccine.

Polysaccharides have been successfully used as immunogens for the development of vaccines against bacterial infection; however, there are no oligosaccharide-based vaccines available to date and no previous studies of their processing and presentation. We reported here the intracellular enzymatic processing and antigen presentation of an oligosaccharide-conjugate cancer vaccine prepared from the glycan of Globo-H (GH), a globo-series glycosphingolipid (GSL). This oligosaccharide-conjugate vaccine was shown to elicit antibodies against the glycan moieties of all three globo-series GSLs that are exclusively expressed on many types of cancer and their stem cells. To understand the specificity and origin of cross-reactivity of the antibodies elicited by the vaccine, we found that the vaccine is first processed by fucosidase 1 in the early endosome of dendritic cells to generate a common glycan antigen of the GSLs along with GH for MHC class II presentation. This work represents the first study of oligosaccharide processing and presentation and is expected to facilitate the design and development of glycoconjugate vaccines based on oligosaccharide antigens.

A non-neutralizing antibody broadly protects against influenza virus infection by engaging effector cells.

Hemagglutinin (HA) is the immunodominant protein of the influenza virus. We previously showed that mice injected with a monoglycosylated influenza A HA (HAmg) produced cross-strain-reactive antibodies and were better protected than mice injected with a fully glycosylated HA (HAfg) during lethal dose challenge. We employed a single B-cell screening platform to isolate the cross-protective monoclonal antibody (mAb) 651 from mice immunized with the HAmg of A/Brisbane/59/2007 (H1N1) influenza virus (Bris/07). The mAb 651 recognized the head domain of a broad spectrum of HAs from groups 1 and 2 influenza A viruses and offered prophylactic and therapeutic efficacy against A/California/07/2009 (H1N1) (Cal/09) and Bris/07 infections in mice. The antibody did not possess neutralizing activity; however, antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis mediated by natural killer cells and alveolar macrophages were important in the protective efficacy of mAb 651. Together, this study highlighted the significance of effector functions for non-neutralizing antibodies to exhibit protection against influenza virus infection.

Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria.

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers.

Broadly neutralizing HIV antibodies are much sought after (a) to guide vaccine design, both as templates and as indicators of the authenticity of vaccine candidates, (b) to assist in structural studies, and (c) to serve as potential therapeutics. However, the number of targets on the viral envelope spike for such antibodies has been limited. Here, we describe a set of human monoclonal antibodies that define what is, to the best of our knowledge, a previously undefined target on HIV Env. The antibodies recognize a glycan-dependent epitope on the prefusion conformation of gp41 and unambiguously distinguish cleaved from uncleaved Env trimers, an important property given increasing evidence that cleavage is required for vaccine candidates that seek to mimic the functional HIV envelope spike. The availability of this set of antibodies expands the number of vaccine targets on HIV and provides reagents to characterize the native envelope spike.

Chemical Synthesis of a Keto Sugar Nucleotide.

Keto sugar nucleotides (KSNs) are common and versatile precursors to various deoxy sugar nucleotides, which are substrates for the corresponding glycosyltransferases involved in the biosynthesis of glycoproteins, glycolipids, and natural products. However, there has been no KSN synthesized chemically due to the inherent instability. Herein, the first chemical synthesis of the archetypal KSN TDP-4-keto-6-deoxy-d-glucose (1) is achieved by an efficient and optimized route, providing feasible access to other KSNs and analogues, thereby opening a new avenue for new applications.

Effective Organotin-Mediated Regioselective Functionalization of Unprotected Carbohydrates.

Regioselective functionalization of unprotected carbohydrates at a secondary OH group in the presence of primary OH groups based on the commonly used organotin-mediated reaction has been improved. We found that the preactivation of the dibutylstannylene acetal intermediate with tetrabutylammonium bromide in toluene is a key to the improved condition for the efficient, high-yielding, and regioselective tosylation, benzoylation, or benzylation of unprotected carbohydrates. The counteranion of tetrabutylammonium ion with a weak coordination ability plays a crucial role in the improved regioselective reactions. A convenient access to the intermediates of synthetic value is also demonstrated in the organotin-mediated regioselective tosylation of unprotected carbohydrates, followed by the nucleophilic inversion reaction to give sulfur-containing and azide-modified carbohydrates.

Isolation of Anti-SARS-CoV-2 Natural Products Extracted from Mentha canadensis and the Semi-synthesis of Antiviral Derivatives.

Traditional herbal medicine offers opportunities to discover novel therapeutics against SARS-CoV-2 mutation. The dried aerial part of mint (Mentha canadensis L.) was chosen for bioactivity-guided extraction. Seven constituents were isolated and characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Syringic acid and methyl rosmarinate were evaluated in drug combination treatment. Ten amide derivatives of methyl rosmarinate were synthesized, and the dodecyl (13) and 3-ethylphenyl (19) derivatives demonstrated significant improvement in the anti-SARS-CoV-2 plaque reduction assay, achieving IC50 of 0.77 and 2.70 µM, respectively, against Omicron BA.1 as compared to methyl rosmarinate's IC50 of 57.0 µM. Spike protein binding and 3CLpro inhibition assays were performed to explore the viral inhibition mechanism. Molecular docking of compounds 13 and 19 to 3CLpro was performed to reveal potential interaction. In summary, natural products with anti-Omicron BA.1 activity were isolated from Mentha canadensis and derivatives of methyl rosmarinate were synthesized, showing 21- to 74-fold improvement in antiviral activity against Omicron BA.1.