RESUMEN
Bacterial glycoconjugate vaccines have a major role in preventing microbial infections. Immunogenic bacterial glycans, such as O-antigen polysaccharides, can be recombinantly expressed and combined with specific carrier proteins to produce effective vaccines. O-Antigen polysaccharides are typically polydisperse, and carrier proteins can have multiple glycosylation sites. Consequently, recombinant glycoconjugate vaccines have a high structural heterogeneity, making their characterization challenging. Since development and quality control processes rely on such characterization, novel strategies are needed for faster and informative analysis. Here, we present a novel approach employing minimal sample preparation and ultrahigh-resolution mass spectrometry analysis for protein terminal sequencing and characterization of the oligosaccharide repeat units of bacterial glycoconjugate vaccines. Three glycoconjugate vaccine candidates, obtained from the bioconjugation of the O-antigen polysaccharides from E. coli serotypes O2, O6A, and O25B with the genetically detoxified exotoxin A from Pseudomonas aeruginosa, were analyzed by MALDI-in-source decay (ISD) FT-ICR MS. Protein and glycan ISD fragment ions were selectively detected using 1,5-diaminonaphtalene and a 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxybenzoic acid mixture (super-DHB) as a MALDI matrix, respectively. The analysis of protein fragments required the absence of salts in the samples, while the presence of salt was key for the detection of sodiated glycan fragments. MS/MS analysis of O-antigen ISD fragments allowed for the detection of specific repeat unit signatures. The developed strategy requires minute sample amounts, avoids the use of chemical derivatizations, and comes with minimal hands-on time allowing for fast corroboration of key structural features of bacterial glycoconjugate vaccines during early- and late-stage development.
Asunto(s)
Vacunas contra Escherichia coli , Escherichia coli/metabolismo , Vacunas contra Escherichia coli/metabolismo , Antígenos O , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en TándemRESUMEN
Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.
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Proteínas Portadoras/química , Análisis por Micromatrices/métodos , Polisacáridos/química , Polisacáridos/inmunología , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Células CHO , Cricetulus , Glicómica , Humanos , Sistema Inmunológico , Lectinas , Oligosacáridos , Polisacáridos/clasificación , Unión Proteica , Proteínas Recombinantes , Especificidad de la EspecieRESUMEN
As a major player of the innate immune system, surfactant protein D (SP-D) recognizes and promotes elimination of various pathogens such as Gram-negative bacteria. SP-D binds to l-glycero-d-manno-heptose (Hep), a constituent of the partially conserved lipopolysaccharide (LPS) inner core of many Gram-negative bacteria. Binding and affinity of trimeric human SP-D to Hep in distinct LPS inner core glycans differing in linkages and adjacent residues was elucidated using glycan array and surface plasmon resonance measurements that were compared to in silico interaction studies. The combination of in vitro assays using defined glycans and molecular docking and dynamic simulation approaches provides insights into the interaction of trimeric SP-D with those glycan ligands. Trimeric SP-D wildtype recognized larger LPS inner core oligosaccharides with slightly enhanced affinity than smaller compounds suggesting the involvement of stabilizing secondary interactions. A trimeric human SP-D mutant D324N+D325N+R343K resembling rat SP-D bound to various LPS inner core structures in a similar pattern as observed for the wildtype but with higher affinity. The selective mutation of SP-D promotes targeting of LPS inner core oligosaccharides on Gram-negative bacteria to develop novel therapeutic agents.
Asunto(s)
Lipopolisacáridos/química , Proteína D Asociada a Surfactante Pulmonar/química , Sustitución de Aminoácidos , Cristalografía por Rayos X , Humanos , Cinética , Simulación del Acoplamiento Molecular , Unión ProteicaRESUMEN
Self-assembling peptides can be used to create tunable higher-order structures for the multivalent presentation of a variety of ligands. We describe a novel, fiber-forming coiled-coil-based peptide that assembles to display, simultaneously, carbohydrate and peptide ligands recognized by biomacromolecules. Preassembly decoration of the scaffold with a diphtheria toxin peptide epitope or a mannose motif did not interfere with self-assembly of the nanostructure. The resulting multivalent display led to tighter binding by antidiphtheria toxin antibodies and mannose-specific carbohydrate binding proteins, respectively. The potential of this self-assembling peptide to display ligands in bioanalytical assays is illustrated by its decoration with a disaccharide glycotope from the Leishmania parasite. Carbohydrate-specific antibodies produced in response to a Leishmania infection are detected more sensitively in human and canine sera due to the multivalent presentation on the self-assembled scaffold. Thus, nanofibers based on coiled-coil peptides are a powerful tool for the development of bioassays and diagnostics.
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Presentación de Antígeno , Carbohidratos/inmunología , Péptidos/química , Péptidos/metabolismo , Animales , Antígenos de Protozoos/metabolismo , Carbohidratos/química , Perros , Humanos , Leishmania/inmunología , Modelos Moleculares , Nanofibras/química , Estructura Secundaria de ProteínaRESUMEN
Carbohydrate modifications are believed to strongly affect the immunogenicity of glycans. Capsular polysaccharides (CPS) from bacterial pathogens are frequently equipped with a pyruvate that can be placed across the 4,6-, 3,4-, or 2,3-positions. A trans-2,3-linked pyruvate is present on the CPS of the Gram-positive bacterium Streptococcus pneumoniae serotypeâ 4 (ST4), a pathogen responsible for pneumococcal infections. To assess the immunological importance of this modification within the CPS repeating unit, the first total synthesis of the glycan was carried out. Glycan microarrays containing a series of synthetic antigens demonstrated how antibodies raised against natural ST4â CPS specifically recognize the pyruvate within the context of the tetrasaccharide repeating unit. The pyruvate modification is a key motif for designing minimal synthetic carbohydrate vaccines for ST4.
Asunto(s)
Polisacáridos Bacterianos/síntesis química , Ácido Pirúvico/química , Streptococcus pneumoniae/química , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunología , Ácido Pirúvico/inmunología , Serogrupo , Streptococcus pneumoniae/inmunologíaRESUMEN
We report here a novel surfactant mediated fusion of polylactide particles into scaffoldlike structures at room temperature. In the presence of ethanol, evenly spread surfactant coated polylactide particles fused immediately into membranelike structures. Polymer scaffolds of the desired shape and size could be fabricated from polylactide particles using this fusion process. Desorption of surfactant molecules from the surface of the particles during ethanol treatment and the degree of solubility of the polymer in alcohol were found to be the main reasons for the fusion of particles into a scaffold at room temperature. TGA and DSC studies of the polylactide particles showed that the particles were stable at room temperature, and FTIR studies showed that there was no change in characteristics of the polymer after the fusion of particles into a scaffold-type structure. These scaffolds supported three-dimensional growth of animal cells in vitro and release model protein in a sustained manner for a long period of time. In an experimental animal wound model, the polylactide membranes showed faster wound closure, indicating its use as a passive dressing material. This polymer particle fusion process thus provides a novel method of scaffold fabrication for various biomedical applications.
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Polímeros/química , Medicina Regenerativa/métodos , Implantes Absorbibles , Animales , Línea Celular Tumoral , Células Cultivadas , Humanos , Células MCF-7 , Melanoma Experimental , Tamaño de la Partícula , Poliésteres/química , Ratas , Ratas Wistar , Solubilidad , Tensoactivos/química , TemperaturaRESUMEN
Bacterial capsular polysaccharides are components of many modern vaccines, but they are weakly immunogenic. Herein, we describe the delivery of pneumococcal capsular polysaccharide serotype-1 (PCP-1) in polylactide polymeric particles to enhance its immunogenicity. Immunization with PCP-1-entrapped particles elicited long-term memory antibody responses from a single intramuscular injection. PCP-1-entrapped nanoparticles (NPs) elicited significantly higher anti-PCP-1 IgG responses than that observed with soluble and microparticles (MPs) formulations. Delivering PCP-1 and pneumococcal proteins in same particles did not improve the IgG response. The sera of animals immunized with PCP-1-entrapped particles promoted efficient opsonophagocytosis of pneumococci by macrophages. Single-dose immunization with PCP-1-entrapped particles conferred a long-term serotype-specific protection against lethal pneumococcal challenge. The higher immunogenicity of PCP-1 nanoparticles showed correlation with enhanced uptake by antigen-presenting cells. The results highlight the potential of polymeric nanoparticles as an efficient means of presenting polysaccharide antigens to the immune system.
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Nanopartículas/administración & dosificación , Proteínas Opsoninas/metabolismo , Fagocitosis/fisiología , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/administración & dosificación , Polímeros/química , Polisacáridos Bacterianos/administración & dosificación , Polisacáridos Bacterianos/inmunología , Animales , Formación de Anticuerpos/inmunología , Células Presentadoras de Antígenos/inmunología , Células Cultivadas , Femenino , Inmunización , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/microbiología , Vacunas Neumococicas/inmunologíaRESUMEN
Around 2 billion people worldwide are infected with the apicomplexan parasite Toxoplasma gondii which induces a variety of medical conditions. For example, primary infection during pregnancy can result in fetal death or mental retardation of the child. Diagnosis of acute infections in pregnant women is challenging but crucially important as the drugs used to treat T.â gondii infections are potentially harmful to the unborn child. Better, faster, more reliable, and cheaper means of diagnosis by using defined antigens for accurate serological tests are highly desirable. Synthetic pathogen-specific glycosylphosphatidylinositol (GPI) glycan antigens are diagnostic markers and have been used to distinguish between toxoplasmosis disease states using human sera.
Asunto(s)
Glicosilfosfatidilinositoles , Polisacáridos/química , Toxoplasmosis/diagnóstico , Secuencia de Carbohidratos , Glicosilfosfatidilinositoles/química , Humanos , Datos de Secuencia MolecularRESUMEN
Bacterial pathogens can cause a broad range of infections with detrimental effects on health. Vaccine development is essential as multi-drug resistance in bacterial infections is a rising concern. Recombinantly produced proteins carrying O-antigen glycosylation are promising glycoconjugate vaccine candidates to prevent bacterial infections. However, methods for their comprehensive structural characterization are lacking. Here, we present a bottom-up approach for their site-specific characterization, detecting N-glycopeptides by nano reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS). Glycopeptide analyses revealed information on partial site-occupancy and site-specific glycosylation heterogeneity and helped corroborate the polysaccharide structures and their modifications. Bottom-up analysis was complemented by intact glycoprotein analysis using nano RP-LC-MS allowing the fast visualization of the polysaccharide distribution in the intact glycoconjugate. At the glycopeptide level, the model glycoconjugates analyzed showed different repeat unit (RU) distributions that spanned from 1 to 21 RUs attached to each of the different glycosylation sites. Interestingly, the intact glycoprotein analysis displayed a RU distribution ranging from 1 to 28 RUs, showing the predominant species when the different glycopeptide distributions are combined in the intact glycoconjugate. The complete workflow based on LC-MS measurements allows detailed and comprehensive analysis of the glycosylation state of glycoconjugate vaccines.
Asunto(s)
Vacunas Bacterianas , Glicoconjugados , Glicopéptidos , Glicoconjugados/química , Glicoconjugados/inmunología , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/química , Glicosilación , Glicopéptidos/química , Glicopéptidos/análisis , Espectrometría de Masas/métodos , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunología , Cromatografía Liquida/métodos , Cromatografía de Fase Inversa/métodosRESUMEN
T lymphocytes and myeloid cells express the immunoglobulin-like glycoprotein cluster of differentiation (CD)101, notably in the gut. Here, we investigated the cell-specific functions of CD101 during dextran sulfate sodium (DSS)-induced colitis and Salmonella enterica Typhimurium infection. Similar to conventional CD101-/- mice, animals with a regulatory T cell-specific Cd101 deletion developed more severe intestinal pathology than littermate controls in both models. While the accumulation of T helper 1 cytokines in a CD101-deficient environment entertained DSS-induced colitis, it impeded the replication of Salmonella as revealed by studying CD101-/- x interferon-g-/- mice. Moreover, CD101-expressing neutrophils were capable to restrain Salmonella infection in vitro and in vivo. Both cell-intrinsic and -extrinsic mechanisms of CD101 contributed to the control of bacterial growth and spreading. The CD101-dependent containment of Salmonella infection required the expression of Irg-1 and Nox2 and the production of itaconate and reactive oxygen species. The level of intestinal microbial antigens in the sera of inflammatory bowel disease patients correlated inversely with the expression of CD101 on myeloid cells, which is in line with the suppression of CD101 seen in mice following DSS application or Salmonella infection. Thus, depending on the experimental or clinical setting, CD101 helps to limit inflammatory insults or bacterial infections due to cell type-specific modulation of metabolic, immune-regulatory, and anti-microbial pathways.
RESUMEN
Clostridium difficile is the cause of emerging nosocomial infections that result in abundant morbidity and mortality worldwide. Thus, the development of a vaccine to kill the bacteria to prevent this disease is highly desirable. Several recently identified bacterial surface glycans, such as PS-I and PS-II, are promising vaccine candidates to preclude C. difficile infection. To circumvent difficulties with the generation of natural PS-I due to its low expression levels in bacterial cultures, improved chemical synthesis protocols for the pentasaccharide repeating unit of PS-I and oligosaccharide substructures were utilized to produce large quantities of well-defined PS-I related glycans. The analysis of stool and serum samples obtained from C. difficile patients using glycan microarrays of synthetic oligosaccharide epitopes revealed humoral immune responses to the PS-I related glycan epitopes. Two different vaccine candidates were evaluated in the mouse model. A synthetic PS-I repeating unit CRM197 conjugate was immunogenic in mice and induced immunoglobulin class switching as well as affinity maturation. Microarray screening employing PS-I repeating unit substructures revealed the disaccharide Rha-(1â3)-Glc as a minimal epitope. A CRM197-Rha-(1â3)-Glc disaccharide conjugate was able to elicit antibodies recognizing the C. difficile PS-I pentasaccharide. We herein demonstrate that glycan microarrays exposing defined oligosaccharide epitopes help to determine the minimal immunogenic epitopes of complex oligosaccharide antigens. The synthetic PS-I pentasaccharide repeating unit as well as the Rha-(1â3)-Glc disaccharide are promising novel vaccine candidates against C. difficile that are currently in preclinical evaluation.
Asunto(s)
Vacunas Bacterianas/inmunología , Clostridioides difficile/química , Enterocolitis Seudomembranosa/terapia , Epítopos/inmunología , Oligosacáridos/inmunología , Vacunas Sintéticas/inmunología , Vacunas Bacterianas/química , Conformación de Carbohidratos , Clostridioides difficile/inmunología , Enterocolitis Seudomembranosa/inmunología , Datos de Secuencia Molecular , Oligosacáridos/química , Vacunas Sintéticas/químicaRESUMEN
The development and use of antibacterial glycoconjugate vaccines have significantly reduced the occurrence of potentially fatal childhood and adult diseases such as bacteremia, bacterial meningitis, and pneumonia. In these vaccines, the covalent linkage of bacterial glycans to carrier proteins augments the immunogenicity of saccharide antigens by triggering T cell-dependent B cell responses, leading to high-affinity antibodies and durable protection. Licensed glycoconjugate vaccines either contain long-chain bacterial polysaccharides, medium-sized oligosaccharides, or short synthetic glycans. Here, we discuss factors that affect the glycan chain length in vaccines and review the available literature discussing the impact of glycan chain length on vaccine efficacy. Furthermore, we evaluate the available clinical data on licensed glycoconjugate vaccine preparations with varying chain lengths against two bacterial pathogens, Haemophilus influenzae type b and Neisseria meningitidis group C, regarding a possible correlation of glycan chain length with their efficacy. We find that long-chain glycans cross-linked to carrier proteins and medium-sized oligosaccharides end-linked to carriers both achieve high immunogenicity and efficacy. However, end-linked glycoconjugates that contain long untethered stretches of native glycan chains may induce hyporesponsiveness by T cell-independent activation of B cells, while cross-linked medium-sized oligosaccharides may suffer from suboptimal saccharide epitope accessibility.
RESUMEN
Phagocytosis of particulate vaccine delivery systems is a critical immune mechanism involved in antigen capture and processing by macrophages and dendritic cells. The internalization and degradation of the particles involve a complex sequence of events. This process coordinates lipids, signaling proteins, and the cytoskeleton. Dynamic changes in the actin cytoskeleton are essential for phagocytosis and antigen presentation. Knowledge regarding the correlation of surface properties, attached ligand density and geometric size of particles with the efficiency of phagocytosis may facilitate their design and application. To investigate this, polylactide biodegradable particles with different diameters (2-4 µm and 200-300 nm) were exposed to murine macrophages and dendritic cells and the effect of size on a series of cellular responses was evaluated. Cellular uptake studies using microscopy and flow cytometry showed size dependent internalization of particles, with nanoparticles accumulating in cells at a faster rate. The particles induced homoaggregation of cells and also showed cytoskeletal remodeling that could be inhibited by cytochalasin-D. Scanning electron microscopy images showed the time dependent formation of phagocytic cups and invaginations that promote particle uptake. The particles were observed to co-localized with the endo-lysosomal compartments after phagocyotosis. In our experiments, particle mediated immunoactivation, antigen processing and cytokine secretion have shown a good correlation with the uptake process. These findings would allow a better understanding of the process of particle uptake and may be instrumental in the rational design of optimal vaccine delivery systems.
Asunto(s)
Citoesqueleto , Fagocitosis , Animales , Células Presentadoras de Antígenos , Ratones , Tamaño de la Partícula , PoliésteresAsunto(s)
Anticuerpos Antibacterianos , Lipopolisacáridos , Peste/diagnóstico , Yersinia pestis/química , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/química , Técnica del Anticuerpo Fluorescente , Humanos , Límite de Detección , Lipopolisacáridos/química , Ratones , Estructura Molecular , Análisis por Matrices de ProteínasRESUMEN
Infections with Streptococcus pneumoniae are a major health burden. Glycoconjugate vaccines based on capsular polysaccharides (CPSs) successfully protect from infection, but not all pneumococcal serotypes are covered with equal potency. Marketed glycoconjugate vaccines induce low levels of functional antibodies against the highly invasive serotype 1 (ST1), presumably due to the obscuring of protective epitopes during chemical activation and conjugation to carrier proteins. Synthetic oligosaccharide antigens can be designed to carry linkers for site-selective protein conjugation while keeping protective epitopes intact. Here, we developed an efficacious semisynthetic ST1 glycoconjugate vaccine candidate. A panel of synthetic oligosaccharides served to reveal a critical role of the rare aminosugar, 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose (d-AAT), for ST1 immune recognition. A monovalent ST1 trisaccharide carrying d-AAT at the nonreducing end induced a strong antibacterial immune response in rabbits and outperformed the ST1 component of the multivalent blockbuster vaccine Prevenar 13, paving the way for a more efficacious vaccine.
RESUMEN
Fully synthetic glycan-based vaccines hold great potential as preventive and therapeutic vaccines against infectious diseases as well as cancer. Here, we present a two-component platform based on the facile conjugation of carbohydrate antigens to α-galactosylceramide (α-GalCer) to yield fully synthetic vaccine candidates. Formulation of the cancer-associated Tn antigen glycolipid model vaccine candidate into liposomes of different sizes and subsequent immunization of mice generated specific, high-affinity antibodies against the carbohydrate antigen with characteristics of T cell-dependent immunity. Liposome formulation elicited more reproducible glycan immunity than a conventional glycoconjugate vaccine bearing the same glycan antigen did. Further evaluation of the immune response revealed that the size of the liposomes influenced the glycan antibody responses toward either a cellular (Th1) or a humoral (Th2) immune phenotype. The glycolipid vaccine platform affords strong and robust antiglycan antibody responses in vivo without the need for an external adjuvant.
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Galactosilceramidas/química , Galactosilceramidas/inmunología , Liposomas/química , Animales , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Técnicas de Química Sintética , Composición de Medicamentos , Femenino , Galactosilceramidas/síntesis química , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Inmunización , Ratones Endogámicos C57BL , FenotipoRESUMEN
Glycoconjugate vaccines based on capsular polysaccharides (CPSs) of pathogenic bacteria such as Streptococcus pneumoniae successfully protect from disease but suffer from incomplete coverage, are troublesome to manufacture from isolated CPSs, and lack efficacy against certain serotypes. Defined, synthetic oligosaccharides are an attractive alternative to isolated CPSs but require the identification of immunogenic and protective oligosaccharide antigens. We describe a medicinal chemistry strategy based on a combination of automated glycan assembly (AGA), glycan microarray-based monoclonal antibody (mAb) reverse engineering, and immunological evaluation in vivo to uncover a protective glycan epitope (glycotope) for S. pneumoniae serotype 8 (ST8). All four tetrasaccharide frameshifts of ST8 CPS were prepared by AGA and used in glycan microarray experiments to identify the glycotopes recognized by antibodies against ST8. One tetrasaccharide frameshift that was preferentially recognized by a protective, CPS-directed mAb was conjugated to the carrier protein CRM197. Immunization of mice with this semisynthetic glycoconjugate followed by generation and characterization of a protective mAb identified protective and nonprotective glycotopes. Immunization of rabbits with semisynthetic ST8 glycoconjugates containing protective glycotopes induced an antibacterial immune response. Coformulation of ST8 glycoconjugates with the marketed 13-valent glycoconjugate vaccine Prevnar 13 yielded a potent 14-valent S. pneumoniae vaccine. Our strategy presents a facile approach to develop efficient semisynthetic glycoconjugate vaccines.
Asunto(s)
Glicoconjugados/inmunología , Vacunas Neumococicas/inmunología , Serogrupo , Streptococcus pneumoniae/inmunología , Vacunas Conjugadas/inmunología , Animales , Antibacterianos/farmacología , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Cápsulas Bacterianas/metabolismo , Femenino , Mutación del Sistema de Lectura/genética , Glicoconjugados/química , Glicómica , Células HL-60 , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Polisacáridos/metabolismo , ConejosRESUMEN
Streptococcus pneumoniae is a major cause of mortality and morbidity worldwide. More than 90 S. pneumoniae serotypes are distinguished based on the structure of their primary targets to the human immune system, the capsular polysaccharides (CPSs). The CPS of the prevalent serotype 4 (ST4) is composed of tetrasaccharide repeating units and is included in existing pneumococcal vaccines. Still, the structural antigenic determinants that are essential for protective immunity, including the role of the rare and labile cyclic trans-(2,3) pyruvate ketal modification, remain largely unknown. Molecular insights will support the design of synthetic subunit oligosaccharide vaccines. Here, we identified the key antigenic determinants of ST4 CPS with the help of pyruvated and nonpyruvated synthetic repeating unit glycans. Glycan arrays revealed oligosaccharide antigens recognized by antibodies in the human reference serum. Selected depyruvated ST4 oligosaccharides were used to formulate neoglycoconjugates and immunologically evaluated in mice. These oligosaccharides were highly immunogenic, but the resulting antiglycan antibodies showed only limited binding to the natural CPS present on the bacterial surface. Glycan array and surface plasmon resonance analysis of murine polyclonal serum antibodies as well as monoclonal antibodies revealed that terminal sugars are important in directing the immune responses. The pyruvate modification on the oligosaccharide is needed for cross-reactivity with the native CPS. These findings are an important step toward the design of oligosaccharide-based vaccines against S. pneumoniae ST4.
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Cápsulas Bacterianas/inmunología , Epítopos/inmunología , Infecciones Neumocócicas/microbiología , Polisacáridos Bacterianos/inmunología , Serogrupo , Streptococcus pneumoniae/inmunología , Animales , Cápsulas Bacterianas/química , Secuencia de Carbohidratos , Epítopos/química , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Infecciones Neumocócicas/inmunología , Polisacáridos Bacterianos/química , Streptococcus pneumoniae/químicaRESUMEN
Synthetic cell-surface glycans are promising vaccine candidates against Clostridium difficile. The complexity of large, highly antigenic and immunogenic glycans is a synthetic challenge. Less complex antigens providing similar immune responses are desirable for vaccine development. Based on molecular-level glycan-antibody interaction analyses, we here demonstrate that the C. difficile surface polysaccharide-I (PS-I) can be resembled by multivalent display of minimal disaccharide epitopes on a synthetic scaffold that does not participate in binding. We show that antibody avidity as a measure of antigenicity increases by about five orders of magnitude when disaccharides are compared with constructs containing five disaccharides. The synthetic, pentavalent vaccine candidate containing a peptide T-cell epitope elicits weak but highly specific antibody responses to larger PS-I glycans in mice. This study highlights the potential of multivalently displaying small oligosaccharides to achieve antigenicity characteristic of larger glycans. The approach may result in more cost-efficient carbohydrate vaccines with reduced synthetic effort.
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Clostridioides difficile/inmunología , Disacáridos/inmunología , Epítopos de Linfocito T/inmunología , Polisacáridos/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Formación de Anticuerpos/inmunología , Antígenos Bacterianos/inmunología , Western Blotting , Clostridioides difficile/fisiología , Disacáridos/química , Disacáridos/metabolismo , Enterocolitis Seudomembranosa/sangre , Enterocolitis Seudomembranosa/inmunología , Enterocolitis Seudomembranosa/microbiología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/metabolismo , Femenino , Glicoconjugados/química , Glicoconjugados/inmunología , Glicoconjugados/metabolismo , Interacciones Huésped-Patógeno/inmunología , Inmunización/métodos , Ratones Endogámicos C57BL , Estructura Molecular , Polisacáridos/química , Polisacáridos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
Infections with Clostridium difficile increasingly cause morbidity and mortality worldwide. Bacterial surface glycans including lipoteichoic acid (LTA) were identified as auspicious vaccine antigens to prevent colonization. Here, we report on the potential of synthetic LTA glycans as vaccine candidates. We identified LTA-specific antibodies in the blood of C. difficile patients. Therefore, we evaluated the immunogenicity of a semi-synthetic LTA-CRM197 glycoconjugate. The conjugate elicited LTA-specific antibodies in mice that recognized natural LTA epitopes on the surface of C. difficile bacteria and inhibited intestinal colonization of C. difficile in mice in vivo. Our findings underscore the promise of synthetic LTA glycans as C. difficile vaccine candidates.