Development of a Rapid Immuno-Based Screening Assay for the Detection of Adenovirus in Eye Infections.

Despite progress in fighting infectious diseases, human pathogenesis and death caused by infectious diseases remain relatively high worldwide exceeding that of cancer and cardiovascular diseases. Human adenovirus (HAdV) infects cells of the upper respiratory tract causing flu-like symptoms that are accompanied by pain and inflammation. Diagnosis of HAdV is commonly achieved by conventional methods such as viral cultures, immunoassays, and polymerase chain reaction (PCR) techniques. However, there are a variety of problems with conventional methods including slow isolation and propagation, inhibition by neutralizing antibodies, low sensitivity of immunoassays, and the diversity of HAdV strains for the PCR technique. Herein, we report the development and evaluation of a novel, simple, and reliable nanobased immunosensing technique for the rapid detection of human adenoviruses (HAdVs) that cause eye infections. This rapid and low-cost assay can be used for screening and quantitative tests with a detection limit of 102 pfu/mL in less than 2 min. The sensing platform is based on a sandwich assay that can detect HAdVs visually by a color change. Sensor specificity was demonstrated using other common viral antigens, including Flu A, Flu B, coronavirus (COV), and Middle East respiratory syndrome coronavirus (MERS COV). This cotton-based testing device potentially exhibits many of the desired characteristics of a suitable point-of-care and portable test, which can be carried out by nurses or clinicians especially for low-resource settings.

A Chemoenzymatic Synthon Strategy for Synthesizing N-Acetyl Analogues of O-Acetylated N. meningitidis W Capsular Polysaccharide Oligosaccharides.

O-Acetylated sialic acid has been found in the Neisseria meningitidis serogroup W (NmW) capsular polysaccharide (CPS) and is a required structural component of clinically used NmW CPS-based polysaccharide and polysaccharide-conjugate vaccines. The role of sialic acid O-acetylation in NmW CPS, however, is not clearly understood. This is partially due to the lack of a precise control of the percentage and the location of O-acetylation which is labile and susceptible to migration. We explore chemoenzymatic synthetic strategies for preparing N-acetylated analogues of O-acetylated NmW CPS oligosaccharides which can serve as structurally stable probe mimics. Substrate specificity studies of NmW CPS polymerase (NmSiaDW) identified 4-azido-4-deoxy-N-acetylmannosamine (ManNAc4N3) and 6-azido-6-deoxy-N-acetylmannosamine (ManNAc6N3) as suitable chemoenzymatic synthons for synthesizing N-acetyl analogues of NmW CPS oligosaccharides containing 7-O-acetyl-N-acetylneuraminic acid (Neu5,7Ac2) and/or 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). The synthesis was achieved by NmSiaDW-dependent sequential one-pot multienzyme (OPME) strategy with in situ generation of the corresponding sugar nucleotides from simple monosaccharides or derivatives to form N3-oligosaccharides which were converted to the desired NAc-oligosaccharides by an efficient one-step chemical transformation.

Size-Controlled Chemoenzymatic Synthesis of Homogeneous Oligosaccharides of Neisseria meningitidis W Capsular Polysaccharide.

Neisseria meningitidis (Nm) serogroup W (NmW) is one of the six meningococcal serogroups that cause majority of invasive meningococcal diseases (IMD). Its capsular polysaccharide (CPS) is a virulence factor and is a key component in NmW CPS-protein conjugate vaccines. The current clinically used NmW CPS-protein conjugate vaccines are effective but the costs are high and the products are heterogeneous at both the CPS and the conjugate levels. Towards the development of potentially better NmW CPS vaccines, herein we report the synthesis of homogeneous oligosaccharides of NmW CPS in a size-controlled manner using polysaccharide synthase NmSiaDW in a sequential one-pot multienzyme (OPME) platform. Taking advantage of the obtained structurally defined synthetic oligosaccharides tagged with a hydrophobic chromophore, detailed biochemical characterization of NmSiaDW has been achieved. While the catalytic efficiency of the galactosyltransferase activity of NmSiaDW increases dramatically with the increase of the sialoside acceptor substrate size, the size difference of the galactoside acceptor substrate does not influence NmSiaDW sialyltransferase activity significantly. The ratio of donor and acceptor substrate concentrations, but not the size of the acceptor substrates, has been found to be the major determining factor for the sizes of the oligosaccharides produced. NmW CPS oligosaccharides with a degree of polymerization (DP) higher than 65 have been observed. The study provides a better understanding of NmSiaDW capsular polysaccharide synthase and showcases an efficient chemoenzymatic synthetic platform for obtaining structurally defined NmW CPS oligosaccharides in a size-controlled manner.

Factors Affecting Anti-Glycan IgG and IgM Repertoires in Human Serum.

Serum anti-glycan antibodies play important roles in many immune processes and are of particular interest as biomarkers for many diseases. Changes in anti-glycan antibodies can occur with the onset of disease or in response to stimuli such as pathogens and vaccination. Understanding relationships between anti-glycan antibody repertoires and genetic and environment factors is critical for basic research and clinical applications, but little information is available. In this study we evaluated the effects of age, race, gender, and blood type on anti-glycan antibody profiles in the serum of 135 healthy subjects. As expected, IgG and IgM antibody signals to blood group antigens correlated strongly with blood type. Interestingly, antibodies to other non-ABH glycans, such as the alpha-Gal antigen, also correlated with blood type. A statistically significant decline in IgM signals with age was observed for many antibody subpopulations, but not for IgG. Moreover, statistically significant correlations between race and IgG levels to certain LacNAc-containing glycans were observed. The results have important implications for designing studies and interpreting results in the area of biomarker discovery and for the development of vaccines. The study also highlights the importance of collecting and reporting patient information that could affect serum anti-glycan antibody levels.

ABO blood type correlates with survival on prostate cancer vaccine therapy.

Immunotherapies for cancer are transforming patient care, but clinical responses vary considerably from patient to patient. Simple, inexpensive strategies to target treatment to likely responders could substantially improve efficacy while simultaneously reducing health care costs, but identification of reliable biomarkers has proven challenging. Previously, we found that pre-treatment serum IgM to blood group A (BG-A) correlated with survival for patients treated with PROSTVAC-VF, a therapeutic cancer vaccine in phase III clinical trials for the treatment of prostate cancer. These results suggested that ABO blood type might influence efficacy. Unfortunately, blood types were not available in the clinical records for all but 8 patients and insufficient amounts of sera were left for standard blood typing methods. To test the hypothesis, therefore, we developed a new glycan microarray-based method for determining ABO blood type. The method requires only 4 µL of serum, provides 97% accuracy, and allows simultaneous profiling of many other serum anti-glycan antibodies. After validation with 220 healthy subjects of known blood type, the method was then applied to 74 PROSTVAC-VF patients and 37 control patients from a phase II trial. In this retrospective study, we found that type B and O PROSTVAC-VF patients demonstrated markedly improved clinical outcomes relative to A and AB patients, including longer median survival, longer median survival relative to Halabi predicted survival, and improved overall survival via Kaplan-Meier survival analysis (p = 0.006). Consequently, blood type may provide an inexpensive screen to pre-select patients likely to benefit from PROSTVAC-VF therapy.

Competition between serum IgG, IgM, and IgA anti-glycan antibodies.

Anti-glycan antibodies are an abundant subpopulation of serum antibodies with critical functions in many immune processes. Changes in the levels of these antibodies can occur with the onset of disease, exposure to pathogens, or vaccination. As a result, there has been significant interest in exploiting anti-glycan antibodies as biomarkers for many diseases. Serum contains a mixture of anti-glycan antibodies that can recognize the same antigen, and competition for binding can potentially influence the detection of antibody subpopulations that are more relevant to disease processes. The most abundant antibody isotypes in serum are IgG, IgM, and IgA, but little is known regarding how these different isotypes compete for the same glycan antigen. In this study, we developed a multiplexed glycan microarray assay and applied it to evaluate how different isotypes of anti-glycan antibodies (IgA, IgG, and IgM) compete for printed glycan antigens. While IgG and IgA antibodies typically outcompete IgM for peptide or protein antigens, we found that IgM outcompete IgG and IgA for many glycan antigens. To illustrate the importance of this effect, we provide evidence that IgM competition can account for the unexpected observation that IgG of certain antigen specificities appear to be preferentially transported from mothers to fetuses. We demonstrate that IgM in maternal sera compete with IgG resulting in lower than expected IgG signals. Since cord blood contains very low levels of IgM, competition only affects maternal IgG signals, making it appear as though certain IgG antibodies are higher in cord blood than matched maternal blood. Taken together, the results highlight the importance of competition for studies involving anti-glycan antibodies.

Chemoenzymatic synthesis of sialosides containing C7-modified sialic acids and their application in sialidase substrate specificity studies.

Modifications at the glycerol side chain of sialic acid in sialosides modulate their recognition by sialic acid-binding proteins and sialidases. However, limited work has been focused on the synthesis and functional studies of sialosides with C7-modified sialic acids. Here we report chemical synthesis of C4-modified ManNAc and mannose and their application as sialic acid precursors in a highly efficient one-pot three-enzyme system for chemoenzymatic synthesis of α2-3- and α2-6-linked sialyl para-nitrophenyl galactosides in which the C7-hydroxyl group in sialic acid (N-acetylneuraminic acid, Neu5Ac, or 2-keto-3-deoxynonulosonic acid, Kdn) was systematically substituted by -F, -OMe, -H, and -N3 groups. Substrate specificity study of bacterial and human sialidases using the obtained sialoside library containing C7-modified sialic acids showed that sialosides containing C7-deoxy Neu5Ac were selective substrates for all bacterial sialidases tested but not for human NEU2. The information obtained from sialidase substrate specificity can be used to guide the design of new inhibitors that are selective against bacterial sialidases.

Glycan microarrays: powerful tools for biomarker discovery.

Over the last 10 years, glycan microarray technology has emerged as a powerful high-throughput tool for studying the interactions of carbohydrates with a variety of biomolecules. The array format allows one to screen thousands of binding interactions in a single experiment using minimal amounts of scarce materials. More recently, this technology has been applied to the discovery of biomarkers for diagnosis, prognosis, risk prediction, and monitoring immune responses. Biomarker discovery using glycan arrays has primarily focused on monitoring changes to the anti-glycan antibody repertoires in serum, since the populations of antibodies can change significantly with the onset of disease, exposure to pathogens, or vaccination. Herein, we review efforts to use glycan arrays to identify new biomarkers for cancer, infections, autoimmune diseases, and immune responses.

Divergent behavior of glycosylated threonine and serine derivatives in solid phase peptide synthesis.

Solid phase peptide coupling of glycosylated threonine derivatives was systematically evaluated. In contrast to glycosylated serine derivatives which are highly prone to epimerization, glycosylated threonine derivatives produce only negligible amounts of epimerization. Under forcing conditions, glycosylated threonine analogs undergo ß-elimination, rather than epimerization. Mechanistic studies and molecular modeling were used to understand the origin of the differences in reactivity.

Cross-comparison of protein recognition of sialic acid diversity on two novel sialoglycan microarrays.

DNA and protein arrays are commonly accepted as powerful exploratory tools in research. This has mainly been achieved by the establishment of proper guidelines for quality control, allowing cross-comparison between different array platforms. As a natural extension, glycan microarrays were subsequently developed, and recent advances using such arrays have greatly enhanced our understanding of protein-glycan recognition in nature. However, although it is assumed that biologically significant protein-glycan binding is robustly detected by glycan microarrays, there are wide variations in the methods used to produce, present, couple, and detect glycans, and systematic cross-comparisons are lacking. We address these issues by comparing two arrays that together represent the marked diversity of sialic acid modifications, linkages, and underlying glycans in nature, including some identical motifs. We compare and contrast binding interactions with various known and novel plant, vertebrate, and viral sialic acid-recognizing proteins and present a technical advance for assessing specificity using mild periodate oxidation of the sialic acid chain. These data demonstrate both the diversity of sialic acids and the analytical power of glycan arrays, showing that different presentations in different formats provide useful and complementary interpretations of glycan-binding protein specificity. They also highlight important challenges and questions for the future of glycan array technology and suggest that glycan arrays with similar glycan structures cannot be simply assumed to give similar results.

Enhanced epimerization of glycosylated amino acids during solid-phase peptide synthesis.

Glycopeptides are extremely useful for basic research and clinical applications, but access to structurally defined glycopeptides is limited by the difficulties in synthesizing this class of compounds. In this study, we demonstrate that many common peptide coupling conditions used to prepare O-linked glycopeptides result in substantial amounts of epimerization at the α position. In fact, epimerization resulted in up to 80% of the non-natural epimer, indicating that it can be the major product in some reactions. Through a series of mechanistic studies, we demonstrate that the enhanced epimerization relative to nonglycosylated amino acids is due to a combination of factors, including a faster rate of epimerization, an energetic preference for the unnatural epimer over the natural epimer, and a slower overall rate of peptide coupling. In addition, we demonstrate that use of 2,4,6-trimethylpyridine (TMP) as the base in peptide couplings produces glycopeptides with high efficiency and low epimerization. The information and improved reaction conditions will facilitate the preparation of glycopeptides as therapeutic compounds and vaccine antigens.

Probe sialidase substrate specificity using chemoenzymatically synthesized sialosides containing C9-modified sialic acid.

A library of α2-3- and α2-6-linked sialyl galactosides containing C9-modified sialic acids was synthesized from C6-modified mannose derivatives using an efficient one-pot three-enzyme system. These sialosides were used in a high-throughput sialidase substrate specificity assay to elucidate the importance of C9-OH in sialidase recognition.

Pasteurella multocida CMP-sialic acid synthetase and mutants of Neisseria meningitidis CMP-sialic acid synthetase with improved substrate promiscuity.

Cytidine 5'-monophosphate (CMP)-sialic acid synthetases (CSSs) catalyze the formation of CMP-sialic acid from CTP and sialic acid, a key step for sialyltransferase-catalyzed biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates. More than 50 different sialic acid forms have been identified in nature. To facilitate the enzymatic synthesis of sialosides with diverse naturally occurring sialic acid forms and their non-natural derivatives, CMP-sialic acid synthetases with promiscuous substrate specificity are needed. Herein we report the cloning, characterization, and substrate specificity studies of a new CSS from Pasteurella multocida strain P-1059 (PmCSS) and a CSS from Haemophillus ducreyi (HdCSS). Based on protein sequence alignment and substrate specificity studies of these two CSSs and a Neisseria meningitidis CSS (NmCSS), as well as crystal structure modeling and analysis of NmCSS, NmCSS mutants (NmCSS_S81R and NmCSS_Q163A) with improved substrate promiscuity were generated. The strategy of combining substrate specificity studies of enzymes from different sources and protein crystal structure studies can be a general approach for designing enzyme mutants with improved activity and substrate promiscuity.

Modifications of glycans: biological significance and therapeutic opportunities.

Carbohydrates play a central role in a wide range of biological processes. As with nucleic acids and proteins, modifications of specific sites within the glycan chain can modulate a carbohydrate's overall biological function. For example, acylation, methylation, sulfation, epimerization, and phosphorylation can occur at various positions within a carbohydrate to modulate bioactivity. Therefore, there is significant interest in identifying discrete carbohydrate modifications and understanding their biological effects. Additionally, enzymes that catalyze those modifications and proteins that bind modified glycans provide numerous targets for therapeutic intervention. This review will focus on modifications of glycans that occur after the oligomer/polymer has been assembled, generally referred to as post-glycosylational modifications.

Efficient chemoenzymatic synthesis of sialyl Tn-antigens and derivatives.

An N-terminal and C-terminal truncated recombinant α2-6-sialyltransferase cloned from Photobacterium sp. JH-ISH-224, Psp2,6ST(15-501)-His(6), was shown to be an efficient catalyst for one-pot three-enzyme synthesis of sialyl Tn (STn) antigens and derivatives containing natural and non-natural sialic acid forms.

Human xeno-autoantibodies against a non-human sialic acid serve as novel serum biomarkers and immunotherapeutics in cancer.

Human carcinomas can metabolically incorporate and present the dietary non-human sialic acid Neu5Gc, which differs from the human sialic acid N-acetylneuraminic acid (Neu5Ac) by 1 oxygen atom. Tumor-associated Neu5Gc can interact with low levels of circulating anti-Neu5Gc antibodies, thereby facilitating tumor progression via chronic inflammation in a human-like Neu5Gc-deficient mouse model. Here we show that human anti-Neu5Gc antibodies can be affinity-purified in substantial amounts from clinically approved intravenous IgG (IVIG) and used at higher concentrations to suppress growth of the same Neu5Gc-expressing tumors. Hypothesizing that this polyclonal spectrum of human anti-Neu5Gc antibodies also includes potential cancer biomarkers, we then characterize them in cancer and noncancer patients' sera, using a novel sialoglycan microarray presenting multiple Neu5Gc-glycans and control Neu5Ac-glycans. Antibodies against Neu5Gcα2-6GalNAcα1-O-Ser/Thr (GcSTn) were found to be more prominent in patients with carcinomas than with other diseases. This unusual epitope arises from dietary Neu5Gc incorporation into the carcinoma marker Sialyl-Tn, and is the first example of such a novel mechanism for biomarker generation. Finally, human serum or purified antibodies rich in anti-GcSTn-reactivity kill GcSTn-expressing human tumors via complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity. Such xeno-autoantibodies and xeno-autoantigens have potential for novel diagnostics, prognostics, and therapeutics in human carcinomas.

Sialidase substrate specificity studies using chemoenzymatically synthesized sialosides containing C5-modified sialic acids.

para-Nitrophenol-tagged sialyl galactosides containing sialic acid derivatives in which the C5 hydroxyl group of sialic acids was systematically substituted with a hydrogen, a fluorine, a methoxyl or an azido group were successfully synthesized using an efficient chemoenzymatic approach. These compounds were used as valuable probes in high-throughput screening assays to study the importance of the C5 hydroxyl group of sialic acid in the recognition and the cleavage of sialoside substrates by bacterial sialidases.

Recent progress in chemical and chemoenzymatic synthesis of carbohydrates.

The important roles that carbohydrates play in biological processes and their potential application in diagnosis, therapeutics, and vaccine development have made them attractive synthetic targets. Despite ongoing challenges, tremendous progresses have been made in recent years for the synthesis of carbohydrates. The chemical glycosylation methods have become more sophisticated and the synthesis of oligosaccharides has become more predictable. Simplified one-pot glycosylation strategy and automated synthesis are increasingly used to obtain biologically important glycans. On the other hand, chemoenzymatic synthesis continues to be a powerful alternative for obtaining complex carbohydrates. This review highlights recent progress in chemical and chemoenzymatic synthesis of carbohydrates with a particular focus on the methods developed for the synthesis of oligosaccharides, polysaccharides, glycolipids, and glycosylated natural products.

Parallel chemoenzymatic synthesis of sialosides containing a C5-diversified sialic acid.

A convenient chemoenzymatic strategy for synthesizing sialosides containing a C5-diversified sialic acid was developed. The alpha2,3- and alpha2,6-linked sialosides containing a 5-azido neuraminic acid synthesized by a highly efficient one-pot three-enzyme approach were converted to C5''-amino sialosides, which were used as common intermediates for chemical parallel synthesis to quickly generate a series of sialosides containing various sialic acid forms.

Chemoenzymatic synthesis of a new class of macrocyclic oligosaccharides.

A novel and highly efficient chemoenzymatic method has been developed for the preparation of structurally defined macrocyclic oligosaccharides of varied sizes. This method involves chemical or chemoenzymatic synthesis of oligosaccharides containing a galactose at the nonreducing end and a propargyl group at the reducing end as sialyltransferase acceptors. Introducing an azido-containing sialic acid to the nonreducing end of the galactosides through a sialyltransferase-catalyzed enzymatic reaction followed by copper(I)-catalyzed Huisgen's 1,3-dipolar cycloaddition of alkyne and azide provides size-defined macrocyclic carbohydrates. The produced negatively charged macrocycles have high solubility in water and interact with hydrophobic small molecules in a size-dependent manner.