Benjamin Schumann

Chemical Biology Approaches to Designing Defined Carbohydrate Vaccines

Carbohydrate antigens have shown promise as important targets for developing effective vaccines and pathogen detection strategies. Modifying purified microbial glycans through synthetic routes or completely synthesizing antigenic motifs are attractive options to advance carbohydrate vaccine development. However, limited knowledge on structure-property correlates hampers the discovery of immunoprotective carbohydrate epitopes. Recent advancements in tools for glycan modification, high-throughput screening of biological samples, and 3D structural analysis may facilitate antigen discovery process. This review focuses on advances that accelerate carbohydrate-based vaccine development and various technologies that are driving these efforts. Herein we provide a critical overview of approaches and resources available for rational design of better carbohydrate antigens. Structurally defined and fully synthetic oligosaccharides, designed based on molecular understanding of antigen-antibody interactions, offer a promising alternative for developing future carbohydrate vaccines.

A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine

Automated glycan assembly enabled antibody reverse engineering to develop a semisynthetic carbohydrate–based vaccine against the highly virulent Streptococcus pneumoniae serotype 8. Pruning out nonprotective glycotopes Pediatric vaccines targeting bacterial capsular polysaccharides are more effective for certain types of bugs than others, and the manufacturing process as well as immunodominance of different glycan epitopes (glycotopes) can lead to a mixed immune response that does not protect against disease. To directly identify glycotopes that induce a protective response, Schumann et al. combined antibody reverse engineering with automated glycan assembly using Streptococcus pneumoniae serotype 8 as a proof of concept. Promising glycotopes conjugated to a carrier protein induced protective antibodies in mice and were also immunogenic in rabbits. When combined with a commercially available pneumococcal vaccine, these glycoconjugates were able to boost the opsonophagocytic bacterial killing ability of sera from immunized rabbits. This approach leveraging semisynthetic glycoconjugates could lead to the design of more effective bacterial vaccines. 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.

Nucleophile-Directed Stereocontrol Over Glycosylations Using Geminal-Difluorinated Nucleophiles

The glycosylation reaction is the key transformation in oligosaccharide synthesis, but it is still difficult to control in many cases. Stereocontrol during cis-glycosidic linkage formation relies almost exclusively on tuning the glycosylating agent or the reaction conditions. Herein, we use nucleophile-directed stereocontrol to manipulate the stereoselectivity of glycosylation reactions. Placing two fluorine atoms in close proximity to the hydroxy group of an aliphatic amino alcohol lowers the oxygen nucleophilicity and reverses the stereoselectivity of glycosylations to preferentially form the desired cis-glycosides with a broad set of substrates. This concept was applied to the design of a cis-selective linker for automated glycan assembly. Fluorination of an amino alcohol linker does not impair glycan immobilization and lectin binding as illustrated by glycan microarray experiments. These fluorinated linkers enable the facile generation of α-terminating synthetic glycans for the formation of glycoconjugates.

CHAPTER 3:Carbohydrate Vaccines

Carbohydrates started to catch the interest of immunologists in the early 20th century. Vaccines based on bacterial polysaccharides were proven effective to prevent infections, but were overshadowed by antibiotics for a long time. When antibiotic resistances emerged, polysaccharide vaccines rapidly gained importance. However, infants were not protected from infectious disease by vaccines consisting of pure polysaccharides. Protein conjugates of bacterial saccharides were introduced to overcome this challenge. Glycoconjugate vaccines induce immunological memory against pathogens, even in infants, and have become standard in childhood vaccination schedules. Despite the effectiveness of glycoconjugate vaccines, isolation of natural pathogen-derived glycans is often tedious, and numerous measures of quality control have to be taken. Synthetic oligosaccharides facilitate glycoconjugate vaccine manufacturing and serve as tools for detailed mechanistic studies on vaccine immunology. Chapter 3 provides insight into the production, mechanisms of action, and future directions of carbohydrate-based vaccines.

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

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.