Anti-glycan Antibodies Research Articles

Contemporary review of IgA nephropathy.

IgA nephropathy (IgAN) is considered the most common primary glomerulonephritis worldwide with a predilection for Asian-Pacific populations and relative rarity in those of African descent. Perhaps 20%-50% of patients progress to kidney failure. The pathogenesis is incompletely understood. Mesangial deposition of immune complexes containing galactose-deficient IgA1 complexed with anti-glycan IgG or IgA antibodies results in mesangial cell activation and proliferation, inflammatory cell recruitment, complement activation, and podocyte damage. Diagnosis requires a biopsy interpreted by the Oxford criteria. Additional pathologic features include podocytopathy, thrombotic microangiopathy, and C4d staining. Biomarkers predicting adverse outcomes include proteinuria, reduced GFR, hypertension, and pathology. Acceptable surrogate endpoints for therapeutic trials include ongoing proteinuria and rate of eGFR decline. The significance of persisting hematuria remains uncertain. The mainstay of therapy is supportive, consisting of lifestyle modifications, renin-angiotensin inhibition (if hypertensive or proteinuric), sodium-glucose-transporter 2 inhibition (if GFR reduced or proteinuric), and endothelin-receptor antagonism (if proteinuric). Immunosuppression should be considered for those at high risk after maximal supportive care. Corticosteroids are controversial with the most positive results observed in Chinese. They carry a high risk of serious side effects. Similarly, mycophenolate may be most effective in Chinese. Other immunosuppressants are of uncertain benefit. Tonsillectomy appears efficacious in Japanese. Active areas of investigation include B-cell inhibition with agents targeting the survival factors BAFF and APRIL and complement inhibition with agents targeting the alternate pathway (Factors B and D), the lectin pathway (MASP-2), and the common pathway (C3 and C5). Hopefully soon, the who and the how of immunosuppression will be clarified, and kidney failure can be forestalled.

O-glycosylation of IgA1 and the pathogenesis of an autoimmune disease IgA nephropathy.

IgA nephropathy is a kidney disease characterized by deposition of immune complexes containing abnormally O-glycosylated IgA1 in the glomeruli. Specifically, some O-glycans are missing galactose that is normally β1,3-linked to N-acetylgalactosamine of the core 1 glycans. These galactose-deficient IgA1 glycoforms are produced by IgA1-secreting cells due to a dysregulated expression and activity of several glycosyltransferases. Galactose-deficient IgA1 in the circulation of patients with IgA nephropathy is bound by IgG autoantibodies and the resultant immune complexes can contain additional proteins, such as complement C3. These complexes, if not removed from the circulation, can enter the glomerular mesangium, activate the resident mesangial cells, and induce glomerular injury. In this review, we briefly summarize clinical and pathological features of IgA nephropathy, review normal and aberrant IgA1 O-glycosylation pathways, and discuss the origins and potential significance of natural anti-glycan antibodies, namely those recognizing N-acetylgalactosamine. We also discuss the features of autoantibodies specific for galactose-deficient IgA1 and the characteristics of pathogenic immune complexes containing IgA1 and IgG. In IgA nephropathy, kidneys are injured by IgA1-containing immune complexes as innocent bystanders. Most patients with IgA nephropathy progress to kidney failure and require dialysis or transplantation. Moreover, most patients after transplantation experience a recurrent disease. Thus, a better understanding of the pathogenetic mechanisms is needed to develop new disease-specific treatments.

Synthesis of propargyl glycosides of Streptococcus pneumoniae serotypes 6A and 6B for glycoconjugate vaccines

We developed a method for making immune responses to bacterial glycans T cell-dependent, which involves attachment of short, synthetic glycans to a virus-like nanoparticle (VLP). This strategy enhances immune responses to glycans by facilitating cognate T cell help of B cells, leading to antibody class switching and affinity maturation yielding high-affinity, anti-glycan antibodies. This method requires synthesis of bacterial glycans as propargyl glycosides for covalent attachment to VLPs, and the resulting short linker between the VLP and glycan is important for optimal T cell receptor recognition. In this work, glycans that are part of the capsular polysaccharides (CPS) produced by Streptococcus pneumoniae serotypes Sp6A and Sp6B were synthesized as disaccharides and trisaccharides. The optimal glycan epitope for antibody binding to the CPS from these serotypes is unknown, and differing “frames” of disaccharides and trisaccharides were prepared to elucidate the optimal antigen for antibody binding.

Reduction in Xenogeneic Epitopes on Porcine Endothelial Cells by Periodate Oxidation.

Patterns of humoral immune responses represent a major hurdle in terms of pig-to-human xenotransplantation approaches. The best-known xenogeneic glycan antigens present in pigs are the αGal (Galili antigen) and the non-human sialic acid Neu5Gc. As there are further differences between porcine and human cellular surface glycosylation, a much broader range of glycan epitopes with xeno-reactive relevance can be anticipated. Therefore, we set out to chemically modify porcine cellular surface glycans in a global approach by applying sodium periodate (NaIO4) oxidation. Porcine endothelial cells were exposed to oxidation with 1 to 5 mM NaIO4 for different time periods at 37 °C or 4 °C and under static or dynamic conditions. The impact on cellular survival was determined by applying live/dead assays. Oxidation of αGal-epitopes was assessed by fluorescence microscopy-based quantification of isolectin-B4 (IL-B4) staining. Overall immunogenicity of porcine cells was determined by human serum antibody binding. Treatment of porcine endothelial cells and tissues with NaIO4 led to reduced binding of the αGal-specific IL-B4 and/or human serum antibodies. NaIO4 was revealed to be cytotoxic when performed at elevated temperatures and for a prolonged time. However, by applying 2 mM NaIO4 for 60 min at 4 °C, a high extent of cellular viability and a relevant reduction in detectable αGal epitope were observed. No differences were detected irrespectively on whether the cells were oxidized under static or flow conditions. Glycan epitopes on living cells can be oxidized with NaIO4 while maintaining their viability. Accordingly, this strategy holds promise to prevent immune reactions mediated by preformed anti-glycan antibodies.

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Abstract 3860: Characterizing peripheral immune correlates of tumor response in breast cancer patients immunized with the P10s-PADRE vaccine

Abstract Introduction: We demonstrated that vaccination of HR+/HER2− breast cancer subjects with P10s-PADRE cancer vaccine in combination with standard-of-care chemotherapy led to generation of antibodies (Ab), activation of Natural Killer (NK) and T cells, increases in IFNγ and TNFα, and increases in TILs and stromal CD3+ T cells. The current study was conducted to assess the vaccine's efficacy, particularly focusing on tumor shrinkage and pathologic Complete Response (pCR). Additionally, it aims to explore the association of specific peripheral-blood immune responses, such as antibody levels and NK and T-cell activation, with pCR and objective reduction in primary tumor size. Methods: Blood samples collected at baseline and after treatment of at least 25 subjects were used in characterization of immune responses. Serum and plasma samples were used in ELISA, cytokine, and glycan-array assays to measure anti-peptide and anti-glycan antibodies and blood-cytokine levels. Peripheral blood mononuclear cells (PBMCs) were interrogated by RNA-seq, flow cytometry, and ELISpot assays. Results: We observed that high levels of anti-peptide and anti-GD2 antibody responses correlated with pCR. Ab response was positively correlated with differential expression of CD56, GZMA, IFNγ, NKp46, and CD8b, among which GZMA and NKp46 showed a statistically significant association. Vaccine-induced functional cellular immune responses (measured by differential expression of IFNγ, GZMA, CD8, CD69, NKp46, CD56, and PRF1) correlated with reduction of primary tumor size. We also observed substantial differences among immune responders vs non-responders in immune gene expression and in cytokine production in post-immune PBMCs after specific and polyclonal stimulation. Conclusions: The data suggest that immunization of HR+/HER2− breast cancer patients with P10s-PADRE in combination with chemotherapy leads to both humoral and cellular immune responses. While induction of a robust antibody response is a good marker for potential clinical efficacy, vaccine-associated increases of the ratio of CD8+ cells and the expression of CD69, CD8b, GZMA, IFNγ, NKp46, and PRF1 seem to serve as additional efficacy indicators. Future studies are underway to validate these findings and establish reliable prognostic biomarkers. Citation Format: Fariba Jousheghany, John P. Lee, Bernice Nounamo, Vinay Raj, Jennifer L. Faulkner, Eric Siegel, Issam Makhoul, Thomas Kieber-Emmons, Behjatolah Monzavi-Karbassi. Characterizing peripheral immune correlates of tumor response in breast cancer patients immunized with the P10s-PADRE vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3860.

Abstract 5896: Platform approach to develop antibodies specifically recognizing cancer-associated glycoforms

Abstract Introduction: Recent advances in antibody engineering have created a portfolio of highly potent therapeutic approaches like antibody-drug conjugates (ADCs), radioimmunoconjugates (RITs), chimeric antigen receptors (CARs) or bi-specifics. Due to the increased potency, these drugs are more than ever dependent on very clean targets to prevent side effects. We aim to address this major problem through our approach to develop antibodies with improved tumor specificity and reduced on-target/off-tumor binding utilizing the aberrant O-glycosylation on tumor cells. Altered glycosylation of proteins and lipids is one of the most drastic changes in cancer, giving rise to truncated or highly fucosylated and highly sialylated glycans which are almost absent on normal cells. Thus, developing antibodies against protein/carbohydrate combined epitopes (GlycoTargets) comprising these tumor-specific glycans enables highly potent therapies with reduced off-tumor toxicity. Experimental procedures: A workflow and corresponding database was established to evaluate the O-glycosylation of proteins. The first and most basic criterion is the presence of suitable O-glycosylated peptide stretches in the extracellular domain of a protein based on predictions. For proteins fulfilling this criterion cancer-relevant expression is analyzed in a second step. This is done either using publicly available data or experimentally (immunohistochemistry or protein expression data). During the final major step, the theoretical existence of suitable O-glycosylation is confirmed by our own experimental data. This includes analysis of cellularly expressed proteins by anti-glycan antibodies and mass spectrometric studies. Results summary: Case studies will be presented that highlight our workflow to identify suitable GlycoTargets based on publicly available data analysis and experimental confirmation. Additionally, the subsequent development of antibodies against the identified GlycoTargets will be shown. Conclusion: A standardized process was developed for the data collection and prioritization of potential GlycoTargets. The obtained information is subsequently used for targeted discovery of antibodies that bind to protein/carbohydrate combined glycoepitopes (GlycoTargets) offering increased tumor-specificity compared to simple protein targets. Citation Format: Patrik Kehler, Johanna Gellert, Lisa Kalfhues, Sophie Marinoff, Manon Weis, Andreas Franz, Naomi Kast, Stephanie Gurka, Marianne Morche, Evelyn Hartung, Timo Lischke, Sarah Mayer-Hain, Antje Danielczyk. Platform approach to develop antibodies specifically recognizing cancer-associated glycoforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5896.

Discordant Antigenic Properties of Soluble and Virion SARS-CoV-2 Spike Proteins.

Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2.

Prevention of experimental autoimmune encephalomyelitis by targeting 6-sulfo sialyl Lewis X glycans involved in lymphocyte homing.

Lymphocyte homing to peripheral lymph nodes (PLN) is critical for immune surveillance. However, autoimmune diseases such as multiple sclerosis (MS) can occur due to excessive immune responses in the PLN. Here we show that 6-sulfo sialyl Lewis X (6-sulfo sLex) glycans on high endothelial venules that function as ligands for l-selectin on lymphocytes play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 double-knockout mice lacking the expression of 6-sulfo sLeX glycans, the EAE symptoms and the numbers of effector Th1 and Th17 cells in the draining lymph nodes (dLN) and spinal cords (SC) were significantly reduced. To determine whether 6-sulfo sLeX could serve as a target for MS, we also examined the effects of anti-glycan monoclonal antibody (mAb) SF1 against 6-sulfo sLeX in EAE. Administration of mAb SF1 significantly reduced EAE symptoms and the numbers of antigen-specific effector T cells in the dLN and SC in association with suppression of critical genes including Il17a and Il17f that are involved in the pathogenesis of EAE. Taken together, these results suggest that 6-sulfo sLeX glycan would serve as a novel target for MS.

Serum antibody screening using glycan arrays.

Humans and other animals produce a diverse collection of antibodies, many of which bind to carbohydrate chains, referred to as glycans. These anti-glycan antibodies are a critical part of our immune systems' defenses. Whether induced by vaccination or natural exposure to a pathogen, anti-glycan antibodies can provide protection against infections and cancers. Alternatively, when an immune response goes awry, antibodies that recognize self-glycans can mediate autoimmune diseases. In any case, serum anti-glycan antibodies provide a rich source of information about a patient's overall health, vaccination history, and disease status. Glycan microarrays provide a high-throughput platform to rapidly interrogate serum anti-glycan antibodies and identify new biomarkers for a variety of conditions. In addition, glycan microarrays enable detailed analysis of the immune system's response to vaccines and other treatments. Herein we review applications of glycan microarray technology for serum anti-glycan antibody profiling.

Understanding hypergammaglobulinemia in experimental or natural visceral leishmaniasis.

Nonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of proteins, albumin and globulins was determined through the biuret and bromocresol green reaction, where the HGG was better expressed through the albumin/globulin (A/G) ratio. HGG was associated with a higher concentration of specific anti-glycan antibodies (BSA-G)/promastigote soluble extract (PSE) and the presence of circulating immune complexes (IC) by dissociative enzyme-linked immunoassay (ELISA). The study found monovalent IC in 37.9% (PSE) and 50% (BSA-G) of sera from infected hamsters, with increased frequency as the disease progressed. HGG was found in >60% of the samples in dogs with VL, associated with higher levels of specific immunoglobulin (Ig)A and IgM, but not IgG, determined using the PSE and BSA-G ELISA. HGG was associated with the presence of monovalent IC in 58.9% (PSE) and 63.4% (BSA-G) positive dog samples. HGG may result not only from the nonspecific activation of B cells, with greater production of specific and nonspecific antibodies, but also due to lower IgG excretion due to the presence of soluble monovalent IC. HGG correlates to the progression of VL and may be a marker for manifested disease.

Glycan-specific molecularly imprinted polymers towards cancer diagnostics: merits, applications, and future perspectives.

Aberrant glycans are a hallmark of cancer states. Notably, emerging evidence has demonstrated that the diagnosis of cancers with tumour-specific glycan patterns holds great potential to address unmet medical needs, especially in improving diagnostic sensitivity and selectivity. However, despite vast glycans having been identified as potent markers, glycan-based diagnostic methods remain largely limited in clinical practice. There are several reasons that prevent them from reaching the market, and the lack of anti-glycan antibodies is one of the most challenging hurdles. With the increasing need for accelerating the translational process, numerous efforts have been made to find antibody alternatives, such as lectins, boronic acids and aptamers. However, issues concerning affinity, selectivity, stability and versatility are yet to be fully addressed. Molecularly imprinted polymers (MIPs), synthetic antibody mimics with tailored cavities for target molecules, hold the potential to revolutionize this dismal progress. MIPs can bind a wide range of glycan markers, even those without specific antibodies. This capacity effectively broadens the clinical applicability of glycan-based diagnostics. Additionally, glycoform-resolved diagnosis can also be achieved through customization of MIPs, allowing for more precise diagnostic applications. In this review, we intent to introduce the current status of glycans as potential biomarkers and critically evaluate the challenges that hinder the development of in vitro diagnostic assays, with a particular focus on glycan-specific recognition entities. Moreover, we highlight the key role of MIPs in this area and provide examples of their successful use. Finally, we conclude the review with the remaining challenges, future outlook, and emerging opportunities.

Prevention of Collagen-Induced Arthritis by an Anti-Glycan Monoclonal Antibody Reactive with 6-Sulfo Sialyl Lewis x in DBA/1 Mice.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial tissue inflammation, substantially impacting the quality of life of patients. The interaction between L-selectin and its glycoprotein ligands modified with 6-sulfo sialyl Lewis x (6-sulfo sLex) is known to mediate lymphocyte homing to initiate immune responses. Thus, this process could be a potential therapeutic target for RA. Herein, we explored the preventive effects of an anti-6-sulfo sLex monoclonal antibody (mAb), SF1, on collagen-induced arthritis (CIA) in DBA/1 mice. Mice were administered SF1 from day 21 postfirst immunization with type II collagen (CII), and the effects of SF1 on both clinical and histopathological disease progression evoked by the second immunization were examined. SF1 significantly suppressed clinical features and histological levels associated with arthritis severity. Enzyme-linked immunosorbent assay consistently indicated that SF1 inhibited the production of CII-specific IgG2a. Based on the reverse transcription-quantitative PCR analysis, SF1 suppressed the expression of interferon-γ, a T helper 1 cytokine, as well as that of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, in draining lymph nodes. Collectively, these results indicate that SF1, an anti-sulfated glycan mAb, could be beneficial in preventing CIA in mice and may afford as a novel agent to treat RA.

Mannose antibody levels in gastric cancer patients (literature review and clinical and experimental study)

Introduction. Glycans play an important role in the pathogenesis of malignant neoplasms, including stomach cancer. In recent years, the attention of many researchers has been drawn to mannose (Man) – hexose, which is an indispensable component of all N-chains of glycoproteins involved in both normal physiological and pathological processes.Aim. To investigate the role of innate immunity factors and ways to influence them through mannose and mannose-containing glycans in gastric cancer patients. Materials and methods. Data on the role of mannose – one of the key monosaccharides in the formation of glycoprotein N-chains – and its binding receptors (mannose receptor, mannose-binding lectin, antibodies) in gastric cancer since 2006 are presented. Levels of anti-glycan antibodies in blood serum samples of 235 gastric cancer patients and 76 healthy donors were evaluated using a glycochip.Results. It has been shown that the level of IgM-class antibodies to Manβ – the core part of N-glycans – in gastric cancer patients is significantly lower compared to the donor group, regardless of age (p = 0.0001). To assess the effect of age on the levels of antiglycan antibodies, patients were divided into two subgroups – before and after 45 years. In the group under 45 years of age, significant differences in the levels of antiglycan antibodies to Manβ persisted, while significant differences in the levels of antiglycan antibodies to Manβ1-4GlcNAcβ were not observed. when comparing groups of patients and donors older than 45 years, the levels of antibodies to Manβ and Manβ1-4GlcNAcβ were significantly higher only in donors. Conclusion. Deficiency of humoral immunity may be one of the key factors in the initiation and progression of carcinogenesis in humans. In our work, in patients with stomach cancer, we revealed a deficiency of antiglycan antibodies to Manβ and to Manβ1-4GlcNAcβ – core fragments of N-chains of glycoproteins, and the deficiency increased with age. The results of the study are a promising platform for further research aimed not only at studying the role of anti-mannose antibodies, but also at developing approaches to adoptive immunoprophylaxis.

Serum components influence antibody reactivity to glycan and DNA antigens

We previously generated three types of anti-glycan monoclonal IgM antibodies that react with certain structures on the glycans of glycosphingolipids and glycoproteins. As the nucleotide sequences for the variable regions of these IgM antibodies showed homology with those of anti-DNA antibodies deposited in public databases, we analyzed the reactivity of the anti-glycan IgM antibodies to DNA by ELISA. We found that anti-α2,6-sialyl LacNAc IgM in the supernatant of a hybridoma culture cross-reacted with DNA, and after purification of the IgM by zirconia column chromatography, the highly purified IgM showed increased cross-reactivity to DNA. As most of the contaminating bovine serum proteins in the culture supernatant were removed by the purification process, it is likely that a part of the removed components influences antibody reactivity to DNA. Purified anti-DNA antibodies prepared from lupus model NZB/W F1 and MRL/lpr mouse sera and normal human serum were then analyzed, and similar results showing increased reactivity to DNA were obtained. Furthermore, ELISA using these purified antibodies and various carbohydrate antigens showed that the antigen-binding specificity of these antibodies was altered by the purification process from serum-containing antibody preparations. Our results indicate that mammalian serum contains components that strongly influence antibody reactivity to carbohydrate antigens, including DNA.

Automated glycan-bead coupling for high throughput, highly reproducible anti-glycan antibody analysis

Automation of diagnostic assays generally aims to increase reproducibility and throughput while decreasing human errors and hands-on time. Here, we introduce a protocol for the automated chemical conjugation of glycans to color-coded magnetic beads using the KingFisher Flex magnetic particle processor. The resulting glycan-coupled magnetic beads allow the detection of anti-glycan antibodies of different isotypes from various species. By generating anti-glycan antibody profiles, monoclonal antibodies can be screened for their specificity and cross-reactivity, while anti-glycan antibody profiles from different human body fluids can aid in predicting response to treatment or outcome of disease. This efficient, scalable protocol can also be adapted to attach proteins and other biomolecules to beads, making it useful for a wider range of applications that require bead-based laboratory methods.

Antibody-Dependent Enhancement with a Focus on SARS-CoV-2 and Anti-Glycan Antibodies.

Antibody-dependent enhancement (ADE) is a phenomenon where virus-specific antibodies paradoxically cause enhanced viral replication and/or excessive immune responses, leading to infection exacerbation, tissue damage, and multiple organ failure. ADE has been observed in many viral infections and is supposed to complicate the course of COVID-19. However, the evidence is insufficient. Since no specific laboratory markers have been described, the prediction and confirmation of ADE are very challenging. The only possible predictor is the presence of already existing (after previous infection) antibodies that can bind to viral epitopes and promote the disease enhancement. At the same time, the virus-specific antibodies are also a part of immune response against a pathogen. These opposite effects of antibodies make ADE research controversial. The assignment of immunoglobulins to ADE-associated or virus neutralizing is based on their affinity, avidity, and content in blood. However, these criteria are not clearly defined. Another debatable issue (rather terminological, but no less important) is that in most publications about ADE, all immunoglobulins produced by the immune system against pathogens are qualified as pre-existing antibodies, thus ignoring the conventional use of this term for natural antibodies produced without any stimulation by pathogens. Anti-glycan antibodies (AGA) make up a significant part of the natural immunoglobulins pool, and there is some evidence of their antiviral effect, particularly in COVID-19. AGA have been shown to be involved in ADE in bacterial infections, but their role in the development of ADE in viral infections has not been studied. This review focuses on pros and cons for AGA as an ADE trigger. We also present the results of our pilot studies, suggesting that AGAs, which bind to complex epitopes (glycan plus something else in tight proximity), may be involved in the development of the ADE phenomenon.

Allergic reactions to tick saliva components in zebrafish model

BackgroundAlpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate.MethodsProtein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA).ResultsZebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles.ConclusionsResults support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate.Graphical

Black Poplar (Populus nigra L.) Root Extracellular Trap, Structural and Molecular Remodeling in Response to Osmotic Stress.

The root extracellular trap (RET) consists of root-associated, cap-derived cells (root AC-DCs) and their mucilaginous secretions, and forms a structure around the root tip that protects against biotic and abiotic stresses. However, there is little information concerning the changes undergone by the RET during droughts, especially for tree species. Morphological and immunocytochemical approaches were used to study the RET of black poplar (Populus nigra L.) seedlings grown in vitro under optimal conditions (on agar-gelled medium) or when polyethylene glycol-mediated (PEG6000-infused agar-gelled medium) was used to mimic drought conditions through osmotic stress. Under optimal conditions, the root cap released three populations of individual AC-DC morphotypes, with a very low proportion of spherical morphotypes, and equivalent proportions of intermediate and elongated morphotypes. Immunolabeling experiments using anti-glycan antibodies specific to cell wall polysaccharide and arabinogalactan protein (AGP) epitopes revealed the presence of homogalacturonan (HG), galactan chains of rhamnogalacturonan-I (RG-I), and AGPs in root AC-DC cell walls. The data also showed the presence of xylogalacturonan (XGA), xylan, AGPs, and low levels of arabinans in the mucilage. The findings also showed that under osmotic stress conditions, both the number of AC-DCs (spherical and intermediate morphotypes) and the total quantity of mucilage per root tip increased, whereas the mucilage was devoid of the epitopes associated with the polysaccharides RG-I, XGA, xylan, and AGPs. Osmotic stress also led to reduced root growth and increased root expression of the P5CS2 gene, which is involved in proline biosynthesis and cellular osmolarity maintenance (or preservation) in aerial parts. Together, our findings show that the RET is a dynamic structure that undergoes pronounced structural and molecular remodeling, which might contribute to the survival of the root tip under osmotic conditions.