Matthias A. Oberli

A Possible Oligosaccharide-Conjugate Vaccine Candidate for Clostridium difficile Is Antigenic and Immunogenic

Nosocomial infections with the Gram-positive pathogen Clostridium difficile pose a major risk for hospitalized patients and result in significant costs to health care systems. Here, we present the chemical synthesis of a PS-II hapten of a cell wall polysaccharide of hypervirulent ribotype 027 of C. difficile. Mice were immunized with a conjugate consisting of the synthetic hexasaccharide and the diphtheria toxoid variant CRM(197). The immunogenicity of the glycan repeating unit was demonstrated by the presence of specific IgG antibodies in the serum of immunized mice. Murine monoclonal antibodies interact with the synthetic hexasaccharide, as determined by microarray analysis. Finally, we found that specific IgA antibodies in the stool of hospital patients infected with C. difficile recognize the synthetic PS-II hexasaccharide hapten.

Continuous-Flow Synthesis of Biaryls Enabled by Multistep Solid-Handling in a Lithiation/Borylation/Suzuki–Miyaura Cross-Coupling Sequence†

Continuous-flow methods have gained considerable interest over the last decade since they offer several advantages over traditional batch manufacturing processes. Recently, the scope of continuous-flow processes has expanded to include multistep synthetic transformations, which are attractive in that they can result in less waste due to fewer purification steps and less manipulation of compounds. Yet, the development of multistep continuous-flow syntheses remains a particularly difficult challenge due to increased complexity as compared to single step processes. Flow-rate synergy, solvent compatibility, and the effect of by-products and impurities must be considered and optimized in downstream reactions. In addition, a major challenge for the development of multistep syntheses in continuous flow is the handling of the solids, which usually leads to irreversible clogging. Although ultrasonication has been used to address this problem in one-step continuous-flow methodologies, to the best of our knowledge, no examples of multistep continuous-flow methods including a solid-forming reaction have been disclosed. Palladium-catalyzed C C bond-forming reactions serve as useful methods in the synthesis of functionalized materials and biologically active compounds. The Suzuki–Miyaura coupling reaction (SMC) can be regarded as one of the most important reactions for these bond-forming processes. In general, organoboron reagents are prepared via lithium or magnesium organometallic compounds in a two-step process. Given the significance of biaryls in the pharmaceutical industry, we anticipated that the preparation of a boronate reagent, immediately followed by a Suzuki– Miyaura cross-coupling reaction in one single streamlined process would be of great interest for the chemical community. Herein, we report the three-step synthesis of biaryls from the lithiation of aryl halides/heteroarenes, followed by borylation and Suzuki–Miyaura coupling under continuousflow conditions. Notably, this process was made possible through efficient handling of solids under multistep conditions with the aid of acoustic irradiation (Scheme 1). After the completion of our work, the one-pot preparation of magnesium di(hetero)aryland magnesium dialkenylboronates for Suzuki–Miyaura coupling reactions was reported by Knochel et al.

Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer Immunotherapy

The induction of a strong cytotoxic T cell response is an important prerequisite for successful immunotherapy against many viral diseases and tumors. Nucleotide vaccines, including mRNA vaccines with their intracellular antigen synthesis, have been shown to be potent activators of a cytotoxic immune response. The intracellular delivery of mRNA vaccines to the cytosol of antigen presenting immune cells is still not sufficiently well understood. Here, we report on the development of a lipid nanoparticle formulation for the delivery of mRNA vaccines to induce a cytotoxic CD 8 T cell response. We show transfection of dendritic cells, macrophages, and neutrophils. The efficacy of the vaccine was tested in an aggressive B16F10 melanoma model. We found a strong CD 8 T cell activation after a single immunization. Treatment of B16F10 melanoma tumors with lipid nanoparticles containing mRNA coding for the tumor-associated antigens gp100 and TRP2 resulted in tumor shrinkage and extended the overall survival of the treated mice. The immune response can be further increased by the incorporation of the adjuvant LPS. In conclusion, the lipid nanoparticle formulation presented here is a promising vector for mRNA vaccine delivery, one that is capable of inducing a strong cytotoxic T cell response. Further optimization, including the incorporation of different adjuvants, will likely enhance the potency of the vaccine.

Immunological evaluation of a synthetic Clostridium difficile oligosaccharide conjugate vaccine candidate and identification of a minimal epitope.

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-(1→3)-Glc as a minimal epitope. A CRM197-Rha-(1→3)-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-(1→3)-Glc disaccharide are promising novel vaccine candidates against C. difficile that are currently in preclinical evaluation.

A General Method for Suzuki-Miyaura Coupling Reactions Using Lithium Triisopropyl Borates

Conditions for the Suzuki-Miyaura coupling of lithium triisopropyl borates are reported, as well as a procedure for a one-pot lithiation, borylation, and subsequent Suzuki-Miyaura coupling of various heterocycles with aryl halides. These borate species are much more stable toward protodeboronation than the corresponding boronic acids and can conveniently be stored on benchtop at room temperature.

Molecular analysis of Carbohydrate−Antibody interactions : case study using a Bacillus anthracis Tetrasaccharide

The process for selecting potent and effective carbohydrate antigens is not well-established. A combination of synthetic glycan microarray screening, surface plasmon resonance analysis, and saturation transfer difference NMR spectroscopy was used to dissect the antibody-binding surface of a carbohydrate antigen, revealing crucial binding elements with atomic-level detail. This analysis takes the first step toward uncovering the rules for structure-based design of carbohydrate antigens.

In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.

Synthesis of a hexasaccharide repeating unit from Bacillus anthracis vegetative cell walls.

The first synthesis of hexasaccharide 1 representing a repeat unit of a polysaccharide specific to the vegetative cell wall of Bacillus anthracis is reported. The synthetic hexasaccharide is equipped with an n-pentenyl handle at the reducing terminus to allow for further functionalization. Key transformations during the synthesis are the conversion of a glucose into a mannosazide residue, a (2+2) coupling, followed by double alpha-galactosylation to furnish the hexasaccharide, and global deprotection under Birch conditions.

Determination of Carbohydrate-Binding Preferences of Human Galectins with Carbohydrate Microarrays

Galectins are a class of carbohydrate‐binding proteins named for their galactose‐binding preference and are involved in a host of processes ranging from homeostasis of organisms to immune responses. As a first step towards correlating the carbohydrate‐binding preferences of the different galectins with their biological functions, we determined carbohydrate recognition fine‐specificities of galectins with the aid of carbohydrate microarrays. A focused set of oligosaccharides considered relevant to galectins was prepared by chemical synthesis. Structure–activity relationships for galectin–sugar interactions were determined, and these helped in the establishment of redundant and specific galectin actions by comparison of binding preferences. Distinct glycosylations on the basic lactosyl motifs proved to be key to galectin binding regulation—and therefore galectin action—as either high‐affinity ligands are produced or binding is blocked. High‐affinity ligands such as the blood group antigens that presumably mediate particular functions were identified.

Glycan arrays containing synthetic Clostridium difficile lipoteichoic acid oligomers as tools toward a carbohydrate vaccine.

Clostridium difficile is a leading cause of severe nosocomial infections. Cell-surface carbohydrate antigens are promising vaccine candidates. Here we report the first total synthesis of oligomers of the lipoteichoic acid antigen repeating unit. Synthetic glycan microarrays revealed anti-glycan antibodies in the blood of patients that help to define epitopes for vaccine development.