RESUMEN
Influenza viruses escape immunity owing to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. We found that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 bound and disengaged CD19 from its chaperone CD81, permitting CD19 to translocate to the B cell receptor complex to trigger signaling. Moreover, Gb3 regulated major histocompatibility complex class II expression to increase diversity of T follicular helper and GC B cells reactive with subdominant epitopes. In influenza infection, elevating Gb3, either endogenously or exogenously, promoted broadly reactive antibody responses and cross-protection. These data demonstrate that Gb3 determines the affinity and breadth of B cell immunity and has potential as a vaccine adjuvant.
Asunto(s)
Anticuerpos Antivirales , Linfocitos B , Centro Germinal , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Trihexosilceramidas , Formación de Anticuerpos , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Centro Germinal/efectos de los fármacos , Centro Germinal/inmunología , Trihexosilceramidas/metabolismo , Trihexosilceramidas/farmacología , Animales , Ratones , Ratones Noqueados , Humanos , Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunologíaRESUMEN
Influenza viruses escape immunity due to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. Here, we demonstrate that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 binds and disengages CD19 from its chaperone CD81 for subsequent translocation to the B cell receptor (BCR) complex to trigger signaling. Abundance of Gb3 amplifies the PI3-kinase/Akt/Foxo1 pathway to drive affinity maturation. Moreover, this lipid regulates MHC-II expression to increase diversity of T follicular helper (Tfh) and GC B cells reactive with subdominant epitopes. In influenza infection, Gb3 promotes broadly reactive antibody responses and cross-protection. Thus, we show that Gb3 determines affinity as well as breadth in B cell immunity and propose this lipid as novel vaccine adjuvant against viral infection. One Sentence Summary: Gb3 abundance on GC B cells selects antibodies with high affinity and broad epitope reactivities, which are cross-protective against heterologous influenza infection.
RESUMEN
Brucellosis is a global disease and the world's most prevalent zoonosis. All cases in livestock and most cases in humans are caused by members of the genus Brucella that possess a surface O-polysaccharide (OPS) comprised of a rare monosaccharide 4-deoxy-4-formamido-D-mannopyranose assembled with α1,2 and α1,3 linkages. The OPS of the bacterium is the basis for serodiagnostic tests for brucellosis. Bacteria that also contain the same rare monosaccharide can induce antibodies that cross-react in serological tests. In previous work we established that synthetic oligosaccharides, representing elements of the Brucella A and M polysaccharide structures, were excellent antigens to explore the antibody response in the context of infection, immunisation and cross reaction. These studies suggested the existence of antibodies that are specific to the tip of the Brucella OPS. Sera from naturally and experimentally Brucella abortus-infected cattle as well as from cattle experimentally infected with the cross-reactive bacterium Yersinia enterocolitica O:9 and field sera that cross react in conventional serological assays were studied here with an expanded panel of synthetic antigens. The addition of chemical features to synthetic antigens that block antibody binding to the tip of the OPS dramatically reduced their polyclonal antibody binding capability providing conclusive evidence that the OPS tip (non-reducing end) is a potent epitope. Selected short oligosaccharides, including those that were exclusively α1,2 linked, also demonstrated superior specificity when evaluated with cross reactive sera compared to native smooth lipopolysaccharide (sLPS) antigen and capped native OPS. This surprising discovery suggests that the OPS tip epitope, even though common to both Brucella and Y. enterocolitica O:9, has more specific diagnostic properties than the linear portion of the native antigens. This finding opens the way to the development of improved serological tests for brucellosis.
RESUMEN
Glycoconjugates prepared from the capsular polysaccharide of several pathogenic bacteria and carrier proteins, such as CRM 197 or tetanus toxoid, have been one of the most successful public health measures to be implemented in the last quarter century. A crucial element in the success of conjugate vaccines has been the recruitment of T-cell help and systematic induction of a secondary immune response. The seminal discovery, that degraded polysaccharide fragments with attached peptide are presented to the T-cell receptor of carbohydrate specific T-cells by MHC-II molecules that bind to the peptide component of degraded vaccine, suggests potentially novel designs for conjugate vaccines. A fully synthetic conjugate vaccine was constructed from a 1,2-linked ß-mannose trisaccharide conjugated to a T-cell peptide, previously shown to afford protection against Candida albicans. This combined B- and T-cell epitope was synthesized with a C-terminal azidolysine residue for subsequent conjugation by click chemistry. Four copies of a ß-1,3 linked hexaglucan dendritic cell epitope were conjugated to an asymmetric dendrimer bearing an alkyne terminated tether. Click chemistry of these two components created a conjugate vaccine that induced antibodies to all three epitopes of the fully synthetic construct.
Asunto(s)
Células Dendríticas/inmunología , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Vacunas Conjugadas/inmunología , Vacunas Sintéticas/inmunología , Animales , Formación de Anticuerpos/fisiología , Candida albicans/crecimiento & desarrollo , Química Clic/métodos , Humanos , Toxoide Tetánico/inmunología , beta-Glucanos/químicaRESUMEN
Brucellosis is a serious zoonotic bacterial disease that is ranked by the World Health Organization among the top seven "neglected zoonoses" that threaten human health and cause poverty. It is a costly, highly contagious disease that affects ruminants, cattle, sheep, goats, and other productive animals such as pigs. Symptoms include abortions, infertility, decreased milk production, weight loss, and lameness. Brucellosis is also the most common bacterial disease that is transmitted from animals to humans, with approximately 500â¯000 new human cases each year. Detection and slaughter of infected animals is required to eradicate the disease, as vaccination alone is currently insufficient. However, as the most protective vaccines compromise serodiagnosis, this creates policy dilemmas, and these often result in the failure of eradication and control programs. Detection of antibodies to the Brucella bacterial cell wall O-polysaccharide (OPS) component of smooth lipopolysaccharide is used in diagnosis of this disease, and the same molecule contributes important protective efficacy to currently deployed veterinary whole-cell vaccines. This has set up a long-standing paradox that while Brucella OPS confers protective efficacy to vaccines, its presence results in similar antibody profiles in infected and vaccinated animals. Consequently, differentiation of infected from vaccinated animals (DIVA) is not possible, and this limits efforts to combat the disease. Recent clarification of the chemical structure of Brucella OPS as a block copolymer of two oligosaccharide sequences has provided an opportunity to utilize unique oligosaccharides only available via chemical synthesis in serodiagnostic tests for the disease. These oligosaccharides show excellent sensitivity and specificity compared with the native polymer used in current commercial tests and have the added advantage of assisting discrimination between brucellosis and infections caused by several bacteria with OPS that share some structural features with those of Brucella. During synthesis and immunochemical evaluation of these synthetic antigens, it became apparent that an opportunity existed to create a polysaccharide-protein conjugate vaccine that would not create antibodies that give false positive results in diagnostic tests for infection. This objective was reduced to practice, and immunization of mice showed that antibodies to the Brucella A antigen could be developed without reacting in a diagnostic test based on the M antigen. A conjugate vaccine of this type could readily be developed for use in humans and animals. However, as chemical methods advance and modern methods of bacterial engineering mature, it is expected that the principles elucidated by these studies could be applied to the development of an inexpensive and cost-effective vaccine to combat endemic brucellosis in animals.
Asunto(s)
Vacuna contra la Brucelosis/inmunología , Brucella/inmunología , Brucelosis/prevención & control , Polisacáridos/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/metabolismo , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Brucelosis/diagnóstico , Brucelosis/inmunología , Brucelosis/transmisión , Bovinos , Reacciones Cruzadas/inmunología , Epítopos , Humanos , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Ratones , Oligosacáridos/síntesis química , Oligosacáridos/inmunología , Polisacáridos/síntesis química , Polisacáridos Bacterianos/síntesis química , Polisacáridos Bacterianos/inmunología , Albúmina Sérica Bovina/síntesis química , Albúmina Sérica Bovina/inmunología , Toxoide Tetánico/síntesis química , Toxoide Tetánico/inmunología , Vacunas Conjugadas/inmunologíaRESUMEN
Using amphiphilic cyclodextrin as a scaffold, the first class of PK-glycoconjugates capable of high avidity binding to both Stx1 and Stx2 toxins in solid-phase assay formats is reported. The generated glycomicroarray effectively mimics the plasma membrane surface while discriminating binding of the two Stx toxins, with unprecedented affinity to Stx2.
Asunto(s)
Ciclodextrinas/química , Toxina Shiga I/química , Tensoactivos/química , Trisacáridos/química , Modelos Moleculares , Estructura MolecularRESUMEN
Brucellosis is diagnosed by detection of antibodies in the blood of animals and humans that are specific for two carbohydrate antigens, termed A and M, which are present concurrently in a single cell wall O-polysaccharide. Animal brucellosis vaccines contain these antigenic determinants, and consequently infected and vaccinated animals cannot be differentiated as both groups produce A and M specific antibodies. We hypothesized that chemical synthesis of a pure A vaccine would offer unique identification of infected animals by a synthetic M diagnostic antigen that would not react with antibodies generated by this vaccine. Two forms of the A antigen, a hexasaccharide and a heptasaccharide conjugated to tetanus toxoid via reducing and nonreducing terminal sugars, were synthesized and used as lead vaccine candidates. Mouse antibody profiles to these immunogens showed that to avoid reaction with diagnostic M antigen it was essential to maximize the induction of anti-A antibodies that bind internal oligosaccharide sequences and minimize production of antibodies directed toward the terminal nonreducing monosaccharide. This objective was achieved by conjugation of Brucella O-polysaccharide to tetanus toxoid via its periodate oxidized terminal nonreducing monosaccharide, thereby destroying terminal epitopes and focusing the antibody response on internal A epitopes. This establishes the method to resolve the decades-long challenge of how to create effective brucellosis vaccines without compromising diagnosis of infected animals.
RESUMEN
The dominant cell wall antigen of Brucella bacteria is the O-polysaccharide component of the smooth lipopolysaccharide. Infection by various Brucella biovars causes abortions and infertility in a wide range of domestic and wild animals and debilitating disease in humans. Diagnosis relies on the detection of antibodies to the A and M antigens expressed in the O-polysaccharide. This molecule is a homopolymer of the rare monosaccharide, 4-formamido-4,6-dideoxy-d-mannopyranose (Rha4NFo). The A epitope is created by a uniform α1,2 linked internal polymeric sequence capped by a distinct tetrasaccharide sequence defining the M antigen. Unique oligosaccharides only available by chemical synthesis and conjugated via reducing and non-reducing residues to bovine serum albumin have revealed the structural basis of the fine specificity that allows the discrimination of these closely related A and M epitopes. All three M specific monoclonal antibodies (mAbs) are inferred to possess groove type binding sites open at each end, and recognize an α1,3 linked Rha4NFo disaccharide as a part of a trisaccharide epitope, which in two mAbs includes the terminal Rha4NFo residue. The binding site of one of these antibodies is sufficiently large to engage up to six Rha4NFo residues and involves weak recognition of α1,2 linked Rha4NFo residues. The third mAb binds an internal trisaccharide epitope of the M tetrasaccharide. Two A specific mAbs also possess groove type binding sites that accommodate six and four α1,2 linked Rha4NFo residues.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Brucella/inmunología , Glicoconjugados/síntesis química , Glicoconjugados/inmunología , Epítopos/inmunología , Glicoconjugados/químicaRESUMEN
Conjugation of synthetic oligosaccharides and native polysaccharides to proteins is an important tool in glycobiology to create vaccines and antigens to screen lectins, toxins, and antibodies. A novel approach to potentiate and profile the immune response to vaccines involves targeting antigens directly to dendritic cells (DCs), the key cells engaged in the immunization process. Inclusion of a carbohydrate ligand recognized by C-type lectins expressed on their cell surface ensures targeting of vaccines to DCs and improved immunological responses. Here we describe a strategy that permits three sequential orthogonal conjugation reactions to prepare glycoconjugates and apply them to the synthesis of a conjugate vaccine that is targeted for uptake by DCs. The carrier protein is treated with an azo-transfer reagent to convert accessible amino groups to azide and then amide bond formation via reaction with carboxylic acid side chains is used to attach amino tether groups of a ligand to the protein. Azide-alkyne Huisgen cycloaddition conjugation, "click chemistry" is used to attach a second ligand equipped with a propargyl group or an analogous terminal alkyne, and following reduction of protein azide groups back to amine, these amino acid side chains can be subjected to amide formation such as reaction with succinimide esters or homobifunctional coupling reagents such as dialkyl squarate.
Asunto(s)
Aminoácidos/química , Azidas/química , Glicoconjugados/química , Vacunas/química , Alquinos/química , Amidas/química , Ácidos Carboxílicos/química , Química Clic/métodos , Reacción de Cicloadición/métodos , Células Dendríticas , Indicadores y Reactivos/química , Lectinas Tipo C/química , Ligandos , Oligosacáridos/química , Polisacáridos/química , Proteínas/químicaRESUMEN
The C epitope of Brucella O-polysaccharide (O-PS) has so far lacked definitive structural identity. Revised structures for this antigen revealed a unique capping perosamine tetrasaccharide consisting of a sequence of 1,2:1,3:1,2 interresidue linkages. Here, using synthetic oligosaccharide glycoconjugates, the α-1,3 linkage of the O-PS is shown to be an integral structural requirement of this epitope. Although A-dominant strains possess only one or two copies of the capping tetrasaccharide, this creates a unique pentasaccharide antigenic determinant with the linkage sequence 1,2:1,3:1,2:1,2 that is always present in major pathogenic Brucella species.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Brucella/inmunología , Epítopos/química , Antígenos O/química , Polisacáridos Bacterianos/química , Animales , Epítopos/inmunología , Humanos , Modelos Moleculares , Estructura Molecular , Antígenos O/inmunología , Polisacáridos Bacterianos/inmunologíaRESUMEN
Transformation of 3-O-benzyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose into 2,4,6-tri-O-benzoyl-3-O-benzyl glucopyranosyl imidate proceeded efficiently via crystalline benzyl and per-benzoylated derivatives. This imidate glycosylated di-O-isopropylidene-α-D-glucofuranose in high yield and glycosylation of the disaccharide after removal of the 3'-O-benzyl ether afforded the ß1,3 linked trisaccharide in excellent yield. Di- and trisaccharides imidates were readily prepared from the furanose terminated glycosylation products but both were unreactive in glycosylation reaction with the debenzylated di- and trisaccharide alcohols. The 3'-O-benzyl perbenzoylated disaccharide pyranose derivative could be selectively debenzoylated and converted to the corresponding perbenzoylated 4,6:4',6'-di-O-benzylidene derivative. Lewis acid catalyzed glycosidation gave the selectively protected disaccharide ethylthioglycoside in good overall yield. Glycosidation of this thioglycoside donor with 5-methoxycarbonylpentanol gave the disaccharide tether glycoside and after catalytic removal of benzyl ether the resulting disaccharide alcohol was glycosylated by the thioglycoside in a 2+2 reaction to yield a tetrasaccharide. Repetition of selective deprotection of the terminal 3-O-benzyl ether followed by glycosylation by the disaccharide thioglycoside gave a protected hexasaccharide. Hydrogenolysis of this hexasaccharide followed by transesterification and second hydrogenolysis to remove a residual benzyl group gave the target hexasaccharide glycoside 1 as a Dectin-1 ligand functionalized to permit covalent attachment to glycoconjugate vaccines and thereby facilitate improved antigen processing by dendritic cells.
Asunto(s)
Oligosacáridos/síntesis química , beta-Glucanos/química , beta-Glucanos/farmacología , Animales , Glicosilación , Humanos , Lectinas Tipo C/metabolismo , Estructura Molecular , Oligosacáridos/química , Oligosacáridos/farmacología , Vacunas Conjugadas/químicaRESUMEN
Members of the genus Brucella have cell wall characteristics of Gram-negative bacteria, which in the most significant species includes O-polysaccharide (OPS). Serology is the most cost-effective means of detecting brucellosis, as infection with smooth strains of Brucella leads to the induction of high antibody titers against the OPS, an unbranched homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranosyl residues (D-Rha4NFo) that are variably α(1â2)- and α(1â3)-linked. Six d-Rha4NFo homo-oligosaccharides were synthesized, each containing a single α(1â3) link but with a varied number of α(1â2) links. After conjugation to bovine serum albumin (BSA), glycoconjugates 1 to 6 were used to develop individual indirect enzyme-linked immunosorbent assays (iELISAs). The diagnostic capabilities of these antigens were applied to panels of cattle serum samples, including those falsely positive in conventional assays, and the results were compared with those of the complement fixation test (CFT), serum agglutination test (SAT), fluorescent polarization assay (FPA), smooth lipopolysaccharide (sLPS) iELISA, and competitive enzyme-linked immunosorbent assay (cELISA) methods. Results from field serum samples demonstrated that all of the synthetic antigens had excellent diagnostic capabilities. Assays developed with the α(1â3)-linked disaccharide conjugate 1 were the best at resolving false-positive serological results. This was supported by the results from serum samples derived from experimentally infected cattle. Data from synthetic trisaccharide antigens 2 and 3 and tetrasaccharide antigen 4 identified an OPS epitope equally common to all Brucella abortus and Brucella melitensis strains but unique to Brucella. Synthetic oligosaccharide conjugates function as effective surrogates for naturally derived antigens. The creation of discrete OPS epitope antigens reveals not only the previously untapped diagnostic potential within this key diagnostic structure but also holds significance for the design of brucellosis vaccines and diagnostics that enable the differentiation of infected from vaccinated animals.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Brucella abortus/inmunología , Brucella melitensis/inmunología , Brucelosis Bovina/diagnóstico , Pruebas Serológicas/métodos , Animales , Bovinos , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/inmunología , Oligosacáridos/síntesis química , Oligosacáridos/inmunologíaRESUMEN
A focused library of virtual heterobifunctional ligands was generated in silico and a set of ligands with recombined fragments was synthesized and evaluated for binding to Clostridium difficile toxins. The position of the trisaccharide fragment was used as a reference for filtering docked poses during virtual screening to match the trisaccharide ligand in a crystal structure. The peptoid, a diversity fragment probing the protein surface area adjacent to a known binding site, was generated by a multi-component Ugi reaction. Our approach combines modular fragment-based design with in silico screening of synthetically feasible compounds and lays the groundwork for future efforts in development of composite bifunctional ligands for large clostridial toxins.
Asunto(s)
Clostridioides difficile , Simulación por Computador , Bibliotecas de Moléculas Pequeñas/metabolismo , Toxinas Biológicas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Metabolismo de los Hidratos de Carbono , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Toxinas Biológicas/químicaRESUMEN
Selective strategies for the construction of novel three component glycoconjugate vaccines presenting Candida albicans cell wall glycan (ß-1,2 mannoside) and polypeptide fragments on a tetanus toxoid carrier are described. The first of two conjugation strategies employed peptides bearing an N-terminal thiopropionyl residue for conjugation to a trisaccharide equipped with an acrylate linker and a C-terminal S-acetyl thioglycolyl moiety for subsequent linking of neoglycopeptide to bromoacetylated tetanus toxoid. Michael addition of acrylate trisaccharides to peptide thiol under mildly basic conditions gave a mixture of N- and C- terminal glyco-peptide thioethers. An adaptation of this strategy coordinated S-acyl protection with anticipated thioester exchange equilibria. This furnished a single chemically defined fully synthetic neoglycopeptide conjugate that could be anchored to a tetanus toxoid carrier and avoids the introduction of exogenous antigenic groups. The second strategy retained the N-terminal thiopropionyl residue but replaced the C-terminal S-acetate functionality with an azido group that allowed efficient, selective formation of neoglycopeptide thioethers and subsequent conjugation of these with propargylated tetanus toxoid, but introduced potentially antigenic triazole linkages.
Asunto(s)
Epítopos de Linfocito T/inmunología , Vacunas Fúngicas/química , Vacunas Fúngicas/síntesis química , Glicopéptidos/química , Manósidos/química , Toxoide Tetánico/química , Toxoide Tetánico/síntesis química , Acilación , Candida albicans/inmunología , Técnicas de Química Sintética , Compuestos de Sulfhidrilo/química , Trisacáridos/química , Vacunas Conjugadas/químicaRESUMEN
The cell wall O-polysaccharides of pathogenic Brucella species are homopolymers of the rare sugar 4,6-dideoxy-4-formamido-α-D-mannopyranose. Despite the apparent simplicity of the polysaccharide it appears to be a "block copolymer" composed of A and M polysaccharide sequences expressed as a single molecule. The simultaneous presence of both in the cell wall has complicated the understanding of the molecular recognition of these antigens by antibodies present in the serum of infected animals and humans and by monoclonal antibodies. Since presumptive diagnosis of brucellosis, a serious disease in domestic livestock, wild animals, and humans, is based on detection of these antibodies it is important to separate the two antigenic epitopes, one of which is also found in other bacteria. Chemical synthesis provides the only means to achieve this outcome. A series of six oligosaccharides from di to hexasaccharides 1-6 were synthesized and conjugated to proteins to provide glycoconjugate antigens and conjugate vaccines. These chemically defined antigens identified the M antigenic determinant and provided a structural basis for understanding the fine specificity of monoclonal and polyclonal antibodies that bind the M antigen. This resulted in the discovery of a disaccharide that shows considerable potential as an unambiguous diagnostic antigen for detecting brucellosis in humans and animals and two hexasaccharide conjugate vaccine candidates that produce high levels of O-polysaccharide specific antibodies in mice.
Asunto(s)
Antígenos Bacterianos/inmunología , Brucella/inmunología , Brucelosis/diagnóstico , Disacáridos/química , Glicoconjugados/química , Glicoconjugados/inmunología , Polisacáridos Bacterianos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/química , Vacunas Bacterianas/inmunología , Brucella/aislamiento & purificación , Brucella/fisiología , Brucelosis/sangre , Conformación de Carbohidratos , Bovinos , Glicoconjugados/síntesis química , Humanos , Ratones , Modelos Moleculares , Polisacáridos Bacterianos/químicaRESUMEN
Copovidone, a copolymer of vinyl acetate and N-vinyl-2-pyrrolidone, was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and after deacetylation the polymer was functionalized by introduction of amino, azide, and alkyne pendant groups to allow attachment of glycans and peptide. Candida albicans ß-mannan trisaccharides 1 and 2 and M. tuberculosis arabinan hexasaccharide 3 with appropriate tethers were conjugated to the polymers by squarate or click chemistry. C. albicans T-cell peptide 4 bearing a C-terminal ε-azidolysine was also conjugated to copovidone by click chemistry. The resulting conjugates provide convenient non-protein-based antigens that are readily adsorbed on ELISA plates, and display excellent characteristics for assay of antibody binding to the haptenic group of interest. Copovidone and BSA glycoconjugates exhibited similar adsorption characteristics when used to coat ELISA plates, and both conjugates were optimal when used as coating solutions at low nanogram/mL concentrations. Provided that the copovidone conjugated glycan is stable to acid, assay plates can be easily processed for reuse at least three times without detectable variation or degradation in ELISA readout.
Asunto(s)
Anticuerpos/análisis , Especificidad de Anticuerpos , Haptenos/inmunología , Oligosacáridos/inmunología , Péptidos/inmunología , Pirrolidinas/inmunología , Tensoactivos/química , Compuestos de Vinilo/inmunología , Adsorción , Anticuerpos/inmunología , Candida albicans/química , Candida albicans/inmunología , Química Clic , Ensayo de Inmunoadsorción Enzimática , Equipo Reutilizado , Haptenos/química , Conformación Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/inmunología , Oligosacáridos/química , Péptidos/química , Polímeros/química , Pirrolidinas/química , Compuestos de Vinilo/químicaRESUMEN
The oligosaccharides Man4 and Man5, substructures of the high-mannose glycans of HIV glycoprotein gp120, were synthesized with a terminal 1-thiomannopyranose residue. The anomeric thiol can be readily converted to an azidomethyl aglycone through reaction with dichloromethane and displacement with sodium azide. The resulting oligomannans were then conjugated to ubiquitin utilizing thiol alkylation or azide/alkyne reactive tethers of minimal length. By combining high efficiency conjugation reactions and a short tether, we sought to establish conjugation conditions that would permit high density clustering of oligomannans in conjugate vaccines that could produce antibodies able to bind gp120 and potentially neutralize virus. LC-UV-MS was used to separate, identify and quantify the ubiquitin glycoconjugates with differing degrees of oligomannan incorporation. Binding of the HIV protective monoclonal antibody 2G12 and concanavalin A to microtitre plates coated with glycoconjugates was measured by ELISA.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/química , Manosa/análogos & derivados , Manosa/química , Polisacáridos/química , Polisacáridos/síntesis química , Modelos Moleculares , Estructura MolecularRESUMEN
A new microtiter-plate-based method for the rapid generation and evaluation of focused compound libraries was developed and applied to screening ligand analogues for the E.â coli Shiga-like toxin Stx2a. The method is general, it mitigates the masking of intrinsic affinity gains by multivalency and enables the discovery of potential hits when starting from ligands that exhibit extremely low affinity with proteins that depend on multivalency for their function.
Asunto(s)
Inhibidores Enzimáticos/química , Toxina Shiga II/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Inhibidores Enzimáticos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Ligandos , Unión Proteica , Toxina Shiga II/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog,-GalNAc replaces the terminal-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in theB5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.
Asunto(s)
Disacáridos/química , Diseño de Fármacos , Inhibidores Enzimáticos/química , Toxina Shiga II/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cristalografía por Rayos X , Disacáridos/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Toxina Shiga II/antagonistas & inhibidores , Toxina Shiga II/metabolismo , Análisis de Supervivencia , Toxemia/prevención & controlRESUMEN
A rat monoclonal antibody 9D4 raised against the cell surface N-glycan of the parasite Trichinella spirallis protects rats against further infection. The terminal disaccharide ß-d-Tyvp(1â3)ß-d-GalNAcp (2) represents the immunodominant portion of the antigenic determinant. Chemical mapping of the antibody binding site by functional group modification employing monodeoxy and mono-O-methyl congeners identified key polar contacts and topography of the bound disaccharide. We report here a comparison of the chemical mapping studies with the antigen topography inferred from saturation transfer difference (STD) NMR experiments. During chemical mapping several congeners of compound 2 showed substantially enhanced binding. Pairing of these functional group modifications to create derivatives 6 and 7 did not show additive free energy gains and STD NMR data point to small variations in mode of binding as a probable cause. Improved syntheses of disaccharides 2-7 are reported.