Alpha-gal syndrome: when treatment of hypovolemic shock can lead to anaphylaxis.

Delayed anaphylaxis after ingestion of red meat because of galactose-alpha-1,3-galactose (alpha-gal) syndrome has increased in recent years. The mechanism involves an immunoglobulin E reaction to alpha-gal, a molecule found in mammalian meat, dairy products, medications and excipients containing mammalian-derived components, and tick salivary glycans. Sensitization occurs due to the bite of a lone star tick and the transmission of alpha-gal molecules into person's bloodstream. We describe a case of alpha-gal syndrome with severe food, drug, and perioperative allergy in which anaphylaxis with hypovolemic shock occurred immediately after an emergency surgical procedure, when a gelatin-containing drug was injected. This case study confirms that the clinical manifestations of alpha-gal syndrome could be different depending on the route of administration, with immediate reactions if an alpha-gal-containing drug is injected and delayed type allergic manifestations occurring several hours after oral intake. The purpose of this report is to highlight the importance of risk communication in case of exposure to medical products and surgical procedures of patients with alpha-gal syndrome and to encourage drug manufacturers to indicate clearly the origin of excipients in product literature.

Genetic and structural basis of the human anti-α-galactosyl antibody response.

Humans lack the capacity to produce the Galα1-3Galß1-4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.

Dissection of the anti-Candida albicans mannan immune response using synthetic oligomannosides reveals unique properties of ß-1,2 mannotriose protective epitopes.

Candida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and ß anomery complemented with a synthetic ß-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or ß linkages, excepted for MAb B6.1 (protective epitope) reacting with ß-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients' sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing ß- and α-Man epitopes and detection of antibodies against ß-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α- and ß-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/ß ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

COVID-19 in the Developing World: Is the Immune Response to α-Gal an Overlooked Factor Mitigating the Severity of Infection?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which has affected millions of people worldwide. Considerably lower prevalence and fatality rates resulting from COVID-19 are reported in Africa and Asia than in the industrialized world. In this Viewpoint, we discuss the possibility that this intriguing phenomenon could be, among other factors, due to protective immunity of the oligosaccharide galactose-α-1,3-galactose (α-Gal). The α-Gal immunity induced by gut microbiota that express the same glycan modification may prevent COVID-19 through the activation of different mechanisms involved in SARS-CoV-2 neutralization and the downregulation of the inflammatory response in the lungs of infected patients.

Amplifying immunogenicity of prospective Covid-19 vaccines by glycoengineering the coronavirus glycan-shield to present α-gal epitopes.

The many carbohydrate chains on Covid-19 coronavirus SARS-CoV-2 and its S-protein form a glycan-shield that masks antigenic peptides and decreases uptake of inactivated virus or S-protein vaccines by APC. Studies on inactivated influenza virus and recombinant gp120 of HIV vaccines indicate that glycoengineering of glycan-shields to present α-gal epitopes (Galα1-3Galß1-4GlcNAc-R) enables harnessing of the natural anti-Gal antibody for amplifying vaccine efficacy, as evaluated in mice producing anti-Gal. The α-gal epitope is the ligand for the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. Upon administration of vaccines presenting α-gal epitopes, anti-Gal binds to these epitopes at the vaccination site and forms immune complexes with the vaccines. These immune complexes are targeted for extensive uptake by APC as a result of binding of the Fc portion of immunocomplexed anti-Gal to Fc receptors on APC. This anti-Gal mediated effective uptake of vaccines by APC results in 10-200-fold higher anti-viral immune response and in 8-fold higher survival rate following challenge with a lethal dose of live influenza virus, than same vaccines lacking α-gal epitopes. It is suggested that glycoengineering of carbohydrate chains on the glycan-shield of inactivated SARS-CoV-2 or on S-protein vaccines, for presenting α-gal epitopes, will have similar amplifying effects on vaccine efficacy. α-Gal epitope synthesis on coronavirus vaccines can be achieved with recombinant α1,3galactosyltransferase, replication of the virus in cells with high α1,3galactosyltransferase activity as a result of stable transfection of cells with several copies of the α1,3galactosyltransferase gene (GGTA1), or by transduction of host cells with replication defective adenovirus containing this gene. In addition, recombinant S-protein presenting multiple α-gal epitopes on the glycan-shield may be produced in glycoengineered yeast or bacteria expression systems containing the corresponding glycosyltransferases. Prospective Covid-19 vaccines presenting α-gal epitopes may provide better protection than vaccines lacking this epitope because of increased uptake by APC.

Alpha-gal syndrome: challenges to understanding sensitization and clinical reactions to alpha-gal.

INTRODUCTION: The α-Gal syndrome (AGS) is a type of allergy characterized by an IgE antibody response against the carbohydrate Galα1-3Galß1-4GlcNAc-R (α-Gal). Tick bites are recognized as the most important cause of anti-α-Gal IgE antibody increase in humans. Several risk factors have been associated with the development of AGS, but their integration into a standardized disease diagnosis has proven challenging. AREAS COVERED: Herein we discuss the current AGS diagnosis based on anti-α-Gal IgE titers and propose an algorithm that considers all co-factors in the clinical history of α-Gal-sensitized patients to be incorporated into the AGS diagnosis. The need for identification of host-derived gene markers and tick-derived proteins for the diagnosis of the AGS is also discussed. EXPERT OPINION: The current AGS diagnosis based on anti-α-Gal IgE titers has limitations because not all patients sensitized to α-Gal and with anti-α-Gal IgE antibodies higher than the cutoff (0.35 IU/ml) develop anaphylaxis to mammalian meat and AGS. The basophil activation test proposed to differentiate between patients with AGS and asymptomatic α-Gal sensitization cannot be easily implemented as a generalized clinical test. In coming years, the algorithm proposed here could be used in a mobile application for easier AGS diagnosis in the clinical practice.

Allergic Reactions and Immunity in Response to Tick Salivary Biogenic Substances and Red Meat Consumption in the Zebrafish Model.

Ticks are arthropod ectoparasite vectors of pathogens and the cause of allergic reactions affecting human health worldwide. In humans, tick bites can induce high levels of immunoglobulin E antibodies against the carbohydrate Galα1-3Galß1-(3)4GlcNAc-R (α-Gal) present in glycoproteins and glycolipids from tick saliva that mediate anaphylactic reactions known as the alpha-Gal syndrome (AGS) or red meat allergy. In this study, a new animal model was developed using zebrafish for the study of allergic reactions and the immune mechanisms in response to tick salivary biogenic substances and red meat consumption. The results showed allergic hemorrhagic anaphylactic-type reactions and abnormal behavior patterns likely in response to tick salivary toxic and anticoagulant biogenic compounds different from α-Gal. However, the results showed that only zebrafish previously exposed to tick saliva developed allergic reactions to red meat consumption with rapid desensitization and tolerance. These allergic reactions were associated with tissue-specific Toll-like receptor-mediated responses in types 1 and 2 T helper cells (TH1 and TH2) with a possible role for basophils in response to tick saliva. These results support previously proposed immune mechanisms triggering the AGS and provided evidence for new mechanisms also potentially involved in the AGS. These results support the use of the zebrafish animal model for the study of the AGS and other tick-borne allergies.

Incidence and Management of Olaratumab Infusion-Related Reactions.

PURPOSE: Olaratumab is a human monoclonal immunoglobulin G1 antibody against platelet-derived growth factor receptor-α. We report the nature and frequency of infusion-related reactions (IRRs) with olaratumab in clinical trials and postmarketing reports. METHODS: Data from patients exposed to olaratumab across nine clinical trials were reviewed for IRRs. Blood samples were also analyzed for pre-existing immunoglobulin E anti-galactose-α-1,3-galactose (anti-α-Gal) antibodies. RESULTS: In the clinical trials, IRRs were identified in 70 of 485 patients (14.4%). The most frequent symptoms included flushing, fever or chills, and dyspnea. For 68 of 70 patients (97.1%), the first IRR occurred during the first two cycles of treatment. Grade 3 or worse IRRs were reported in 11 patients (2.3%), all during the first infusion and usually within 15 minutes of the start of the infusion. One IRR-related fatality (0.2%) occurred in a nonpremedicated patient with grade 3 or worse cardiac comorbidities. There was an association between grade 3 or worse IRRs and pre-existing anti-α-Gal antibodies, with a trend toward higher IRR rates in US geographies known to have a higher prevalence of anti-α-Gal antibodies. IRRs in postmarketing reports were consistent in nature and severity with those in the clinical trials. CONCLUSION: Premedication with corticosteroids and antihistamines should occur in all patients before olaratumab infusion, as indicated in labels in the United States and the European Union. Patients receiving olaratumab should be monitored for IRRs in a setting where resuscitation equipment is available for the treatment of IRRs.

Confirmed Hypoallergenicity of a Novel Whey-Based Extensively Hydrolyzed Infant Formula Containing Two Human Milk Oligosaccharides.

BACKGROUND: We sought to determine whether an extensively hydrolyzed formula (EHF) supplemented with two human milk oligosaccharides (HMO) was tolerated by infants with cow's milk protein allergy (CMPA). METHODS: A whey-based EHF (Test formula) containing 2'fucosyl-lactose (2'FL) and lacto-N-neotetraose (LNnT) was assessed for clinical hypoallergenicity and safety. The Control formula was a currently marketed EHF without HMO. Children with CMPA, aged 2 months to 4 years, were assessed by double-blind, placebo-controlled food challenges (DBPCFC) to both formulas, in randomized order. If both DBPCFC were negative, subjects participated in a one-week, open food challenge (OFC) with the Test formula. Symptoms and adverse events were recorded. Hypoallergenicity was accepted if at least 90% (with 95% confidence intervals) of subjects tolerated the Test formula. RESULTS: Of the 82 children with CMPA that were screened, 67 (intention-to-treat [ITT] cohort-mean age 24.5 ± 13.6 months; range 2-57; 45 [67.2%] male) were randomized to receive either the Test or the Control formula during the first DBPCFC. Of these, 64 children completed at least one DBPCFC (modified intention-to-treat [mITT] cohort). Three children were excluded due to protocol deviations (per protocol [PP] cohort; n = 61). There was one allergic reaction to the Test, and one to the Control formula. On the mITT analysis, 63 out of 64 (98.4%; 95% CI lower bound 92.8%), and on the PP analysis 60 out of 61 (98.4%; 95% CI lower bound 92.5%) participants tolerated the Test formula, confirming hypoallergenicity. CONCLUSION: The whey-based EHF supplemented with 2'FL and LNnT met the clinical hypoallergenicity criteria and can be recommended for the management of CMPA in infants and young children.

The alpha-Gal syndrome: new insights into the tick-host conflict and cooperation.

This primer focuses on a recently diagnosed tick-borne allergic disease known as the alpha-Gal syndrome (AGS). Tick bites induce in humans high levels of IgE antibodies against the carbohydrate Galα1-3Galß1-(3)4GlcNAc-R (α-Gal) present on tick salivary glycoproteins and tissues of non-catarrhine mammals, leading to the AGS in some individuals. This immune response evolved as a conflict and cooperation between ticks and human hosts including their gut microbiota. The conflict is characterized by the AGS that mediate delayed anaphylaxis to red meat consumption and certain drugs such as cetuximab, and immediate anaphylaxis to tick bites. The cooperation is supported by the capacity of anti-α-Gal IgM and IgG antibody response to protect against pathogens with α-Gal on their surface. Despite the growing diagnosis of AGS in all world continents, many questions remain to be elucidated on the tick proteins and immune mechanisms triggering this syndrome, and the protective response against pathogen infection elicited by anti-α-Gal antibodies. The answer to these questions will provide information for the evaluation of risks, diagnosis and prevention of the AGS, and the possibility of using the carbohydrate α-Gal to develop vaccines for the control of major infectious diseases.

Design of a mimotope-peptide based double epitope vaccine against disseminated candidiasis.

Hematogenously disseminated candidiasis in humans is the third leading cause of nosocomial bloodstream infections in the US. There is no FDA approved antifungal vaccine or prophylactic/therapeutic antibody for use in humans. We first reported novel synthetic peptide and glycopeptide vaccines against Candida albicans cell surface epitopes that protect mice against disseminated candidiasis. We showed that antibodies specific for the peptide Fba (derived from C. albicans cell surface protein fructose bisphosphate aldolase) or for C. albicans cell surface glycan epitope ß-1, 2-mannotriose [ß-(Man)3]) are both protective. This is an important step forward in vaccine design against disseminated candidiasis in humans. However, given the complexity of oligosaccharide synthesis, in this study we performed a new strategy for use of peptide mimotopes that structurally mimic the protective glycan epitope ß-(Man)3 as surrogate immunogens that substitute for the glycan part of glycopeptide [ß-(Man)3-Fba] vaccine. All five selected mimotopes are immunogenic in mice and three mimotopes were able to induce protection in mice against disseminated candidiasis. Furthermore, immunization with three mimotope-peptide conjugate vaccines was also able to induce specific antibody responses, and importantly, protection against disseminated candidiasis in mice. Therefore, our new design of a mimotope-peptide based double epitope vaccine against candidiasis is a potential vaccine candidate that is economical to produce, highly efficacious and safe for use in humans.

Development of α-Gal-Antibody Conjugates to Increase Immune Response by Recruiting Natural Antibodies.

Cancer treatment with antibodies (Abs) is one of the most successful therapeutic strategies for obtaining high selectivity. In this study, α-gal-Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α-gal and anti-gal Abs. The potency of the α-gal-Ab conjugates depended on the amount of α-gal conjugated to the antibody: the larger the amount of α-gal introduced, the higher the level of cytotoxicity observed. The conjugation of antibodies with an α-gal dendrimer allowed the introduction of large amounts of α-gal to the Ab, without loss of affinity for the target cell. The method described here will enable the re-development of Abs to improve their potency.

Total Synthesis of the Trisaccharide Antigen of the Campylobacter jejuni RM1221 Capsular Polysaccharide via de Novo Synthesis of the 6-Deoxy-d- manno-heptose Building Blocks.

A de novo approach utilizing the d-proline-catalyzed and LDA-promoted aldol reactions as key steps for the preparation of differentiated-protected 6-deoxy-d- manno-heptose building blocks was developed. PPh3AuBAr4F-catalyzed glycosylation with the 6-deoxy-d- manno-heptosyl o-hexynylbenzoate as donor was demonstrated as a direct and practical method for the stereoselective synthesis of the ß-linked 6-deoxy-d- manno-heptoside as the major product. Coupling of the 6-deoxy-α-d- manno-heptosyl H-phosphonate with the 3-hydroxyl disaccharide acceptor based on H-phosphonate chemistry was described for the construction of the trisaccharide skeleton with the acid-labile phosphodiester linkage. Finally, first total synthesis of the unique trisaccharide antigen of the capsular polysaccharide of Campylobacter jejuni RM1221 that belongs to HS:53 serotype complex was accomplished for further evaluation as vaccine candidate against C. jejuni RM1221 infection.

Synthesis and biological evaluation of a trisaccharide repeating unit derivative of Streptococcus pneumoniae 19A capsular polysaccharide.

Streptococcus pneumoniae (SP) is a common human pathogen associated with a broad spectrum of diseases and it is still a leading cause of mortality and morbidity worldwide, especially in children. Moreover, SP is increasingly associated with drug resistance. Vaccination against the pathogen may thus represent an important strategy to overcome its threats to human health. In this context, revealing the molecular determinants of SP immunoreactivity may be relevant for the development of novel molecules with therapeutic perspectives as vaccine components. Serogroup 19 comprises the immune-cross reactive types 19F, 19A, 19B and 19C and it accounts for a high percentage of invasive pneumococcal diseases, mainly caused by serotypes 19F and 19A. Herein, we report the synthesis and biological evaluation of an aminopropyl derivative of the trisaccharide repeating unit of SP 19A. We compare two different synthetic strategies, based on different disconnections between the three monosaccharides which make up the final trisaccharide, to define the best approach for the preparation of the trisaccharide. Synthetic accessibility to the trisaccharide repeating unit lays the basis for the development of more complex biopolymer as well as saccharide conjugates. We also evaluate the binding affinity of the trisaccharide for anti-19A and anti-19F sera and discuss the relationship between the chemical properties of the trisaccharide unit and biological activity.

Human Milk Oligosaccharides: 2'-Fucosyllactose (2'-FL) and Lacto-N-Neotetraose (LNnT) in Infant Formula.

The authors reviewed the published evidence on the presence of oligosaccharides in human milk (HMO) and their benefits in in vitro and in vivo studies. The still limited data of trials evaluating the effect of mainly 2'-fucosyllactose (2'-FL) on the addition of some of HMOs to infant formula were also reviewed. PubMed was searched from January 1990 to April 2018. The amount of HMOs in mother's milk is a dynamic process as it changes over time. Many factors, such as duration of lactation, environmental, and genetic factors, influence the amount of HMOs. HMOs may support immune function development and provide protection against infectious diseases directly through the interaction of the gut epithelial cells or indirectly through the modulation of the gut microbiota, including the stimulation of the bifidobacteria. The limited clinical data suggest that the addition of HMOs to infant formula seems to be safe and well tolerated, inducing a normal growth and suggesting a trend towards health benefits. HMOs are one of the major differences between cow's milk and human milk, and available evidence indicates that these components do have a health promoting benefit. The addition of one or two of these components to infant formula is safe, and brings infant formula closer to human milk. More prospective, randomized trials in infants are need to evaluate the clinical benefit of supplementing infant formula with HMOs.

Human Milk Oligosaccharide 2'-Fucosyllactose Improves Innate and Adaptive Immunity in an Influenza-Specific Murine Vaccination Model.

Background: Human milk is uniquely suited to provide optimal nutrition and immune protection to infants. Human milk oligosaccharides are structural complex and diverse consisting of short chain and long chain oligosaccharides typically present in a 9:1 ratio. 2'-Fucosyllactose (2'FL) is one of the most prominent short chain oligosaccharides and is associated with anti-infective capacity of human milk. Aim: To determine the effect of 2'FL on vaccination responsiveness (both innate and adaptive) in a murine influenza vaccination model and elucidate mechanisms involved. Methods: A dose range of 0.25-5% (w/w) dietary 2'FL was provided to 6-week-old female C57Bl/6JOlaHsd mice 2 weeks prior primary and booster vaccination until the end of the experiment. Intradermal (i.d.) challenge was performed to measure the vaccine-specific delayed-type hypersensitivity (DTH). Antigen-specific antibody levels in serum as well as immune cell populations within several organs were evaluated using ELISA and flow cytometry, respectively. In an ex vivo restimulation assay, spleen cells were cocultured with influenza-loaded bone marrow-derived dendritic cells (BMDCs) to study the effects of 2'FL on vaccine-specific CD4+ and CD8+ T-cell proliferation and cytokine secretions. Furthermore, the direct immune regulatory effects of 2'FL were confirmed using in vitro BMDCs T-cell cocultures. Results: Dietary 2'FL significantly (p < 0.05) enhanced vaccine specific DTH responses accompanied by increased serum levels of vaccine-specific immunoglobulin (Ig) G1 and IgG2a in a dose-dependent manner. Consistently, increased activation marker (CD27) expression on splenic B-cells was detected in mice receiving 2'FL as compared to control mice. Moreover, proliferation of vaccine-specific CD4+ and CD8+ T-cells, as well as interferon-γ production after ex vivo restimulation were significantly increased in spleen cells of mice receiving 2'FL as compared to control mice, which were in line with changes detected within dendritic cell populations. Finally, we confirmed a direct effect of 2'FL on the maturation status and antigen presenting capacity of BMDCs. Conclusion: Dietary intervention with 2'FL improves both humoral and cellular immune responses to vaccination in mice, which might be attributed in part to the direct effects of 2'FL on immune cell differentiation.

Effects of kestose on gut mucosal immunity in an atopic dermatitis mouse model.

BACKGROUND: Atopic dermatitis (AD) is recently increasing among populations, but the underlying mechanisms remain controversial. Interactions between the gut microbiota and mucosal immunity are considered to be a crucial etiology. Fructooligosaccharide (FOS), prebiotics have been reported as activators of the gut microbiota. OBJECTIVE: The aim of this study was to investigate the effects of kestose, the smallest FOS and FOS on atopic dermatitis in mice. METHODS: An AD mouse model was developed by (ovalbumin) epidermal sensitization using BALB/c mice. Kestose (1%, 5%, and 10%) or FOS (5%, positive control) was orally administered throughout the study. RESULTS: In comparison with the values observed for the control AD mice, transepidermal water loss (TEWL), clinical score, and skin inflammation on histopathology were significantly decreased by the oral administration of kestose. Total IgE, thymic stromal lymphopoietin (TSLP) in skin, and IL-4 were also suppressed by this administration. In addition, the population of CD4+Foxp3+ cells in mesenteric lymph nodes (MLNs) and acetate concentrations in feces were significantly increased by kestose treatment. CONCLUSIONS: These findings suggest that kestose activates the gut immune system to induce the tolerance against allergic skin inflammations in AD.

Structural Insights into VLR Fine Specificity for Blood Group Carbohydrates.

High-quality reagents to study and detect glycans with high specificity for research and clinical applications are severely lacking. Here, we structurally and functionally characterize several variable lymphocyte receptor (VLR)-based antibodies from lampreys immunized with O erythrocytes that specifically recognize the blood group H-trisaccharide type II antigen. Glycan microarray analysis and biophysical data reveal that these VLRs exhibit greater specificity for H-trisaccharide compared with the plant lectin UEA-1, which is widely used in blood typing. Among these antibodies, O13 exhibits superior specificity for H-trisaccharide, the basis for which is revealed by comparative analysis of high-resolution VLR:glycan crystal structures. Using a structure-guided approach, we designed an O13 mutant with further enhanced specificity for H-trisaccharide. These insights into glycan recognition by VLRs suggest that lampreys can produce highly specific glycan antibodies, and are a valuable resource for the production of next-generation glycan reagents for biological and biomedical research and as diagnostics and therapeutics.

Glycoprotein CA19.9-specific monoclonal antibodies recognize sialic acid-independent glycotope.

A repertoire of monoclonal antibodies was generated by immunization of mice with cancer-associated glycoprotein CA19.9, and two of them were selected as optimal capture and detecting counterparts for sandwich test system for detection of CA19.9. Fine epitope specificity of the antibodies was determined using printed glycan array, enzyme-linked immunosorbent assay, and inhibitory enzyme-linked immunosorbent assay. Unexpectedly, both immunoglobulins did not bind key epitope of CA19.9 glycoprotein, tetrasaccharide SiaLeA, as well as its defucosylated form sialyl LeC (known as CA-50 epitope). The antibodies were found to have different glycan-binding profiles; however, they recognized similar glycotopes with common motif Galß1-3GlcNAcß (LeC), thus resembling specificity of human natural cancer-associated anti-LeC antibodies. We propose that cancer-specific glycopeptide epitope includes Galß1-3GlcNAcß fragment of a glycoprotein O-chain in combination with proximal hydrophobic amino acid(s) of the polypeptide chain.

Why human anti-Galα1-4Galß1-4Glc natural antibodies do not recognize the trisaccharide on erythrocyte membrane? Molecular dynamics and immunochemical investigation.

BACKGROUND: Human blood contains a big variety of natural antibodies, circulating throughout life at constant concentration. Previously, we have found natural antibodies capable of binding to trisaccharide Galα1-4Galß1-4Glc (Pk) practically in all humans. Intriguingly, the same trisaccharide is a key fragment of glycosphingolipid globotriaosylceramide (Gb3Cer) - normal component of erythrocyte and endothelial cell membrane, i.e. the antibodies and their cognate antigen coexist without any immunological reaction. AIM: To explain the inertness of human anti-Pk antibodies towards own cells. MATERIALS AND METHODS: We used a combination of immunochemical and molecular dynamics (MD) experiments. Antibodies were isolated using affinity media with Pk trisaccharide, their epitope specificity was characterized using ELISA (enzyme-linked immunosorbent assay) with a set of synthetic glycans related to Pk synthetic glycans and FACS (Fluorescence-Activated Cell Sorting) analysis of cells with inserted natural Gb3Cer and its synthetic analogue. Conformations and clustering of glycolipids immersed into a lipid bilayer were studied using MD simulations. RESULTS: Isolated specific antibodies were completely unable to bind natural Gb3Cer both inserted into cells and in artificial membrane, whereas strong interaction took place with synthetic analogue differing by the presence of a spacer between trisaccharide and lipid part. MD simulations revealed: i) although membrane-bound glycans do not form stable long-living aggregates, their transient packing is more compact in natural Gb3 as compared with the synthetic analog, ii) similar conformation of Pk glycan in composition of the glycolipids, iii) no effect on the mentioned above results when cholesterol was inserted into membrane, and iv) better accessibility of the synthetic version for interaction with proteins. CONCLUSIONS: Both immunochemical and molecular dynamics data argue that the reason of the "tolerance" of natural anti-Pk antibodies towards cell-bound Gb3Cer is the spatial inaccessibility of Pk glycotope for interaction. We can conclude that the antibodies are not related to the blood group P system.