RESUMO
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.
Assuntos
Vacina contra Brucelose/imunologia , Brucella/imunologia , Brucelose/prevenção & controle , Polissacarídeos/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Brucelose/diagnóstico , Brucelose/imunologia , Brucelose/transmissão , Bovinos , Reações Cruzadas/imunologia , Epitopos , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Camundongos , Oligossacarídeos/síntese química , Oligossacarídeos/imunologia , Polissacarídeos/síntese química , Polissacarídeos Bacterianos/síntese química , Polissacarídeos Bacterianos/imunologia , Soroalbumina Bovina/síntese química , Soroalbumina Bovina/imunologia , Toxoide Tetânico/síntese química , Toxoide Tetânico/imunologia , Vacinas Conjugadas/imunologiaRESUMO
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.
Assuntos
Células Dendríticas/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Vacinas Conjugadas/imunologia , Vacinas Sintéticas/imunologia , Animais , Formação de Anticorpos/fisiologia , Candida albicans/crescimento & desenvolvimento , Química Click/métodos , Humanos , Toxoide Tetânico/imunologia , beta-Glucanas/químicaRESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Brucella/imunologia , Glicoconjugados/síntese química , Glicoconjugados/imunologia , Epitopos/imunologia , Glicoconjugados/químicaRESUMO
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.
Assuntos
Dissacarídeos/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Toxina Shiga II/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Cristalografia por Raios X , Dissacarídeos/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Toxina Shiga II/antagonistas & inibidores , Toxina Shiga II/metabolismo , Análise de Sobrevida , Toxemia/prevenção & controleRESUMO
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.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Brucella abortus/imunologia , Brucella melitensis/imunologia , Brucelose Bovina/diagnóstico , Testes Sorológicos/métodos , Animais , Bovinos , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Oligossacarídeos/síntese química , Oligossacarídeos/imunologiaRESUMO
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.
Assuntos
Clostridioides difficile , Simulação por Computador , Bibliotecas de Moléculas Pequenas/metabolismo , Toxinas Biológicas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Metabolismo dos Carboidratos , Técnicas de Química Combinatória , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Toxinas Biológicas/químicaRESUMO
In a previous attempt to generate a protective vaccine against Candida albicans, a ß-mannan tetanus toxoid conjugate showed poor immunogenicity in mice. To improve the specific activation toward the fungal pathogen, we aimed to target Dectin-1, a pattern-recognition receptor expressed on monocytes, macrophages, and dendritic cells. Laminarin, a ß-glucan ligand of Dectin-1, was incorporated into the original ß-mannan tetanus toxoid conjugate providing a tricomponent conjugate vaccine. A macrophage cell line expressing Dectin-1 was employed to show binding and activation of Dectin-1 signal transduction pathway by the ß-glucan-containing vaccine. Ligand binding to Dectin-1 resulted in the following: 1) activation of Src family kinases and Syk revealed by their recruitment and phosphorylation in the vicinity of bound conjugate and 2) translocation of NF-κB to the nucleus. Treatment of immature bone marrow-derived dendritic cells (BMDCs) with tricomponent or control vaccine confirmed that the ß-glucan-containing vaccine exerted its enhanced activity by virtue of dendritic cell targeting and uptake. Immature primary cells stimulated by the tricomponent vaccine, but not the ß-mannan tetanus toxoid vaccine, showed activation of BMDCs. Moreover, treated BMDCs secreted increased levels of several cytokines, including TGF-ß and IL-6, which are known activators of Th17 cells. Immunization of mice with the novel type of vaccine resulted in improved immune response manifested by high titers of Ab recognizing C. albicans ß-mannan Ag. Vaccine containing laminarin also affected distribution of IgG subclasses, showing that vaccine targeting to Dectin-1 receptor can benefit from augmentation and immunomodulation of the immune response.
Assuntos
Células Dendríticas/metabolismo , Sistemas de Liberação de Medicamentos , Lectinas Tipo C/administração & dosagem , Toxoide Tetânico/administração & dosagem , Toxoide Tetânico/imunologia , beta-Glucanas/metabolismo , Animais , Sítios de Ligação/imunologia , Linhagem Celular , Células Dendríticas/imunologia , Sistemas de Liberação de Medicamentos/métodos , Epitopos/imunologia , Epitopos/metabolismo , Glucanos , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Toxoide Tetânico/metabolismo , Trissacarídeos/administração & dosagem , Trissacarídeos/imunologia , Trissacarídeos/metabolismo , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/metabolismo , beta-Glucanas/imunologiaRESUMO
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.
Assuntos
Antígenos de Bactérias/imunologia , Brucella/imunologia , Brucelose/diagnóstico , Dissacarídeos/química , Glicoconjugados/química , Glicoconjugados/imunologia , Polissacarídeos Bacterianos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/química , Vacinas Bacterianas/imunologia , Brucella/isolamento & purificação , Brucella/fisiologia , Brucelose/sangue , Configuração de Carboidratos , Bovinos , Glicoconjugados/síntese química , Humanos , Camundongos , Modelos Moleculares , Polissacarídeos Bacterianos/químicaRESUMO
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.
Assuntos
Anticorpos/análise , Especificidade de Anticorpos , Haptenos/imunologia , Oligossacarídeos/imunologia , Peptídeos/imunologia , Pirrolidinas/imunologia , Tensoativos/química , Compostos de Vinila/imunologia , Adsorção , Anticorpos/imunologia , Candida albicans/química , Candida albicans/imunologia , Química Click , Ensaio de Imunoadsorção Enzimática , Reutilização de Equipamento , Haptenos/química , Conformação Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/imunologia , Oligossacarídeos/química , Peptídeos/química , Polímeros/química , Pirrolidinas/química , Compostos de Vinila/químicaRESUMO
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.
Assuntos
Proteína gp120 do Envelope de HIV/química , Manose/análogos & derivados , Manose/química , Polissacarídeos/química , Polissacarídeos/síntese química , Modelos Moleculares , Estrutura MolecularRESUMO
Bacterial capsular polysaccharides have been used as effective vaccines for adults but infants and seniors respond poorly to these immunogens because pure polysaccharides are unable to activate T-cells resulting in antibodies of low affinity and poor immunological memory. These deficiencies are addressed by conjugate vaccines composed of bacterial polysaccharide covalently attached to protein carriers such as tetanus or diphtheria toxoids. These vaccines activate T-cells and have been hugely effective in reducing the incidence of dangerous infectious diseases such as bacterial meningitis in infants and adults. The methods of conjugation often produce conjugate vaccines that contain polysaccharides with several cross links to one or more protein carrier molecules. Synthesis of large oligosaccharides derivatized to achieve single site attachment to protein has established the minimal size of an oligosaccharide that is required to produce protective antibody. Several examples are described where such oligosaccharides range in size from 8 to 16 monosaccharides. A more limited set of examples show that protective antibodies may be elicited by conjugate vaccines composed of tri and tetrasaccharide epitopes. One example is the ß-mannan present in the phosphomannan glycoprotein of Candida albicans. Reverse engineering a protective antibody that recognizes this oligosaccharide revealed that a disaccharide was the minimal epitope. Further epitope mapping by functional group modification established that the internal mannose residue of a disaccharide was involved in the most important antibody-disaccharide interactions. NMR binding studies in combination with homology modeling of the bound ß-mannan antigen suggested an optimum oligosaccharide for inclusion in a conjugate vaccine. Several conjugate vaccines were developed to test these conclusions. Immunization of mice or rabbits with conjugates containing disaccharide or trisaccharide conjugated to immunogenic proteins followed by live challenge experiments showed that the vaccines reduced fungal burden. The results of these and other studies suggest new approaches to novel synthetic conjugate vaccines.
Assuntos
Antifúngicos/química , Glicoconjugados/química , Vacinas Conjugadas/química , Animais , Anticorpos Monoclonais/imunologia , Antifúngicos/imunologia , Candidíase/prevenção & controle , Desenho de Fármacos , Glicoconjugados/síntese química , Glicoconjugados/uso terapêutico , Glicopeptídeos/química , Glicopeptídeos/uso terapêutico , Humanos , Toxoide Tetânico/química , Vacinação , Vacinas Conjugadas/uso terapêuticoRESUMO
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.
Assuntos
Inibidores Enzimáticos/química , Toxina Shiga II/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Inibidores Enzimáticos/metabolismo , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Ligantes , Ligação Proteica , Toxina Shiga II/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
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.
Assuntos
Anticorpos Antivirais , Linfócitos B , Centro Germinativo , Infecções por Orthomyxoviridae , Orthomyxoviridae , Triexosilceramidas , Formação de Anticorpos , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/imunologia , Triexosilceramidas/metabolismo , Triexosilceramidas/farmacologia , Animais , Camundongos , Camundongos Knockout , Humanos , Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologiaRESUMO
A self-consistent model of ß-mannan oligosaccharides bound to a monoclonal antibody, C3.1, that protects mice against Candida albicans has been developed through chemical mapping, NMR spectroscopic, and computational studies. This antibody optimally binds di- and trisaccharide epitopes, whereas larger oligomers bind with affinities that markedly decrease with increasing chain length. The (1â2)-ß-linked di-, tri-, and tetramannosides bind in helical conformations similar to the solution global minimum. Antibody recognition of the di- and trisaccharide is primarily dependent on the mannose unit at the reducing end, with the hydrophobic face of this sugar being tightly bound. Recognition of a tetrasaccharide involves a frameshift in the ligand interaction, shown by strong binding of the sugar adjacent to the reducing end. We show that frameshifting may also be deliberately induced by chemical modifications. Molecular recognition patterns similar to that of mAb C3.1, determined by saturation transfer difference-NMR, were also observed in polyclonal sera from rabbits immunized with a trisaccharide glycoconjugate. The latter observation points to the importance of internal residues as immunodominant epitopes in (1â2)-ß-mannans and to the viability of a glycoconjugate vaccine composed of a minimal length oligosaccharide hapten.
Assuntos
Anticorpos Monoclonais/imunologia , Candida albicans/metabolismo , Mananas/imunologia , Sequência de Carboidratos , Simulação por Computador , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Mananas/química , Mananas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
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.
RESUMO
Diarrhea-associated hemolytic uremic syndrome (D+HUS) is the most common cause of acute renal failure among children. Renal damage in D+HUS is caused by Shiga toxin (Stx), which is elaborated by Shigella dysenteriae and certain strains of Escherichia coli, in North America principally E coli O157:H7. Recent studies demonstrate that Stx also induces von Willebrand factor (VWF) secretion by human endothelial cells and causes thrombotic thrombocytopenic purpura, a disease with similarities to D+HUS, in Adamts13(-/-) mice. Stx occurs in 2 variants, Stx1 and Stx2, each of which is composed of 1 catalytically active A subunit that is responsible for cytotoxicity, and 5 identical B subunits that mediate binding to cell-surface globo-triaosylceramide. We now report that B subunits from Stx1 or Stx2 can stimulate the acute secretion of VWF in the absence of the cytotoxic A subunit. This rapid effect requires binding and clustering of globotriaosylceramide, and depends on plasma membrane cholesterol and caveolin-1 but not clathrin. Furthermore, similar to Stx2 holotoxin, the isolated Stx2B subunits induce thrombotic microangiopathy in Adamts13(-/-) mice. These results demonstrate the existence of a novel Stx B-induced lipid raft-dependent signaling pathway in endothelial cells that may be responsible for some of the biological effects attributed previously to the cytotoxic Stx A subunit.
Assuntos
Células Endoteliais/metabolismo , Metaloendopeptidases/genética , Toxinas Shiga/efeitos adversos , Toxinas Shiga/metabolismo , Shigella dysenteriae/metabolismo , Microangiopatias Trombóticas/etiologia , Fator de von Willebrand/metabolismo , Proteína ADAMTS13 , Animais , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular , Toxina da Cólera/metabolismo , Colesterol/metabolismo , Clatrina/metabolismo , Deleção de Genes , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microangiopatias Trombóticas/microbiologia , Triexosilceramidas/metabolismoRESUMO
The cell wall phosphomannan of Candida species is a complex N-linked glycoprotein with a glycan chain containing predominantly an α-linked mannose backbone with α-mannose branches. A minor ß-mannan component is attached to the branches either via a glycosidic bond (acid stable ß-mannan) or a phosphodiester bond (acid-labile ß-mannan). The α-mannan residues of the cell wall phosphomannan do not afford protective antibody, while the ß-mannan portion is a protective antigen and has become an attractive target as the key epitope of a conjugate vaccine. We report the first synthesis of a tetrasaccharide 1 consisting of a ß1,2-mannopyranosyl trisaccharide linked via a phosphodiester to methyl α-mannopyranoside. This encompasses the attachment site of the acid labile ß-mannan to the α-mannan component of the cell wall phosphomannan. The trisaccharide was formed by an iterative process to first create a ß-glucopyranoside linkage and then epimerize the C-2 center via an oxidation-reduction sequence. The phosphate diester linkage was accessed via an anomeric H-phosphonate. The binding of phosphomannan fragment 1 with the protective antibody C3.1 has been evaluated and compared with a ß-mannotrioside in hapten inhibition experiments. The observed activities are rationalized with a model for docked in the binding site of C3.1.
Assuntos
Candida albicans/química , Mananas/química , Oligonucleotídeos/química , Polissacarídeos/síntese química , Sequência de Carboidratos , Epitopos/química , Dados de Sequência Molecular , Estrutura Molecular , Polissacarídeos/químicaRESUMO
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.
RESUMO
The human neuraminidases (NEU) consist of a family of four isoforms (NEU1-NEU4). Members of this enzyme family are proposed to have important roles in health and disease through regulation of the composition of cellular sialosides. The NEU3 isoform is a membrane-associated enzyme that cleaves glycolipid substrates. However, few reports have examined the substrate specificity of the enzyme for non-natural substrates. We report here a series of 11 synthetic trisaccharides that feature modifications of the aglycone or the Neu5Ac residue of an octyl ß-sialyllactoside. The time course of substrate cleavage by NEU3 was monitored using an electrospray ionization mass spectrometry assay to obtain relative rates (k(rel)). We observed that NEU3 substrate activity was directly dependent upon the hydrophobicity of the aglycone but had no apparent requirement for features of the ceramide headgroup. We also observed that trisaccharides with incorporated azide groups in the Neu5Ac residue at either C9 or the N5-Ac position were substrates, and in the case of the N5-azidoacetyl derivative, the activity was superior to that of GM3. However, the incorporation of larger aryl groups was tolerated only at C9, but not at N5-Ac. We propose a two-site model for enzyme recognition, requiring interaction at both the Neu5Ac residue and the hydrophobic aglycone.
Assuntos
Neuraminidase/metabolismo , Sequência de Carboidratos , Membrana Celular/enzimologia , Análise de Fourier , Glicolipídeos/síntese química , Glicolipídeos/química , Glicolipídeos/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Neuraminidase/química , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
We report the synthesis and in vitro evaluation of a multivalent homing device, a polymer which contains preordered pendant groups with dual specificity, a trisaccharide moiety, which is specific for the siglec CD22, and an antibody specific hapten, nitrophenol. The device efficiently attracts antihapten IgM to the surface of human lymphoma B cells as well as to CD22-conjugated magnetic beads by mediating the formation of a ternary complex on the surface of the target.