Comprehensive characterization of bacterial glycoconjugate vaccines by liquid chromatography - mass spectrometry.

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.

Glycan and Protein Analysis of Glycoengineered Bacterial Vaccines by MALDI-in-Source Decay FT-ICR Mass Spectrometry.

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.

Combined effects of glycan chain length and linkage type on the immunogenicity of glycoconjugate vaccines.

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.

Cellular uptake of polylactide particles induces size dependent cytoskeletal remodeling in antigen presenting cells.

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.

Microbe-focused glycan array screening platform.

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.

Synthesis, Liposomal Formulation, and Immunological Evaluation of a Minimalistic Carbohydrate-α-GalCer Vaccine Candidate.

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.

Development of an Efficacious, Semisynthetic Glycoconjugate Vaccine Candidate against Serotype 1.

Infections with 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.

A semisynthetic serotype 8 glycoconjugate vaccine.

Glycoconjugate vaccines based on capsular polysaccharides (CPSs) of pathogenic bacteria such as 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 serotype 8 (ST8).

A Semi-synthetic Oligosaccharide Conjugate Vaccine Candidate Confers Protection against Streptococcus pneumoniae Serotype 3 Infection.

The identification of immunogenic glycotopes that render glycoconjugate vaccines protective is key to improving vaccine efficacy. Synthetic oligosaccharides are an attractive alternative to the heterogeneous preparations of purified polysaccharides that most marketed glycoconjugate vaccines are based on. To investigate the potency of semi-synthetic glycoconjugates, we chose the least-efficient serotype in the current pneumococcal conjugate vaccine Prevnar 13, Streptococcus pneumoniae serotype 3 (ST3).

Synthetic Lipoteichoic Acid Glycans Are Potential Vaccine Candidates to Protect from Clostridium difficile Infections.

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.

Structure binding relationship of human surfactant protein D and various lipopolysaccharide inner core structures.

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.

Multivalent display of minimal Clostridium difficile glycan epitopes mimics antigenic properties of larger glycans.

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.

Genetic characterization of an adapted pandemic 2009 H1N1 influenza virus that reveals improved replication rates in human lung epithelial cells.

The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate.

Cross Reactive Material 197 glycoconjugate vaccines contain privileged conjugation sites.

Production of glycoconjugate vaccines involves the chemical conjugation of glycans to an immunogenic carrier protein such as Cross-Reactive-Material-197 (CRM197). Instead of using glycans from natural sources recent vaccine development has been focusing on the use of synthetically defined minimal epitopes. While the glycan is structurally defined, the attachment sites on the protein are not.

Deciphering Antigenic Determinants of Streptococcus pneumoniae Serotype 4 Capsular Polysaccharide using Synthetic Oligosaccharides.

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).

Chemical Synthesis Elucidates the Immunological Importance of a Pyruvate Modification in the Capsular Polysaccharide of Streptococcus pneumoniae Serotype 4.

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 serotype 4 (ST4), a pathogen responsible for pneumococcal infections.

Generation of Monoclonal Antibodies against Defined Oligosaccharide Antigens.

Unique carbohydrate antigens are expressed on the surface of various pathogens, including bacteria, parasites, and viruses, and aberrant glycosylation is a frequent feature of cancer cells. Antibodies recognizing such carbohydrate antigens may be used for the specific detection of potentially harmful cells, immunohistochemistry, and diagnostic and therapeutic applications. The generation of specific and strongly binding antibodies against defined carbohydrate epitopes is challenging, since isolated carbohydrates often suffer from low purity, usually have limited immunogenicity, and induce antibodies of low affinity.

A Self-Assembling Peptide Scaffold for the Multivalent Presentation of Antigens.

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.

Antigenic potential of a highly conserved Neisseria meningitidis lipopolysaccharide inner core structure defined by chemical synthesis.

Neisseria meningitidis is a leading cause of bacterial meningitis worldwide. We studied the potential of synthetic lipopolysaccharide (LPS) inner core structures as broadly protective antigens against N. meningitidis.

Diagnosis of toxoplasmosis using a synthetic glycosylphosphatidylinositol glycan.

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.

Fusion and self-assembly of biodegradable polymer particles into scaffoldlike and membranelike structures at room temperature for regenerative medicine.

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.

Influences of process and formulation parameters on powder flow properties and immunogenicity of spray dried polymer particles entrapping recombinant pneumococcal surface protein A.

Particle size, antigen load and its release characteristic are the three the main attributes of polymer particles based vaccine delivery systems. The present studies focus on the formulation of spray dried polylactide microparticles entrapping pneumococcal surface protein A (PspA). Influence of process variables during polymer particle formation were optimized by using half-factorial design.

Glycan arrays as tools for infectious disease research.

Infectious diseases cause millions of deaths worldwide each year and are a major burden for economies, especially in underdeveloped countries. Glycans and their interactions with other biomolecules are involved in all major steps of infection. Glycan arrays enable the rapid and sensitive detection of those interactions and are among the most powerful techniques to study the molecular biology of infectious diseases.