Subramanian Govindan

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.

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. Fully characterized conjugates and batch-to-batch comparisons are the key to eventually create completely defined conjugates. A variety of glycoconjugates consisting of CRM197 and synthetic oligosaccharide epitopes was characterised using mass spectrometry techniques. The primary structure was assessed by combining intact protein MALDI-TOF-MS, LC-MALDI-TOF-MS middle-down and LC-ESI-MS bottom-up approaches. The middle-down approach on CNBr cleaved glycopeptides provided almost complete sequence coverage, facilitating rapid batch-to-batch comparisons, resolving glycan loading and identification of side products. Regions close to the N- and C-termini were most efficiently conjugated.

Semisynthetic glycoconjugate vaccine candidate against Streptococcus pneumoniae serotype 5

Significance Each year, Streptococcus pneumoniae infections cause millions of deaths worldwide. The capsular polysaccharide (CPS) based glycoconjugate vaccine Prevnar13 prevents serious illness caused by 13 serotypes. S. pneumoniae serotype 5 (ST-5) is included in the vaccine; however, it suffers from production problems due to modifications or degradation during isolation and conjugation. A medicinal chemistry approach helped to understand the structural features of ST-5 CPS and design a stable semisynthetic oligosaccharide-based vaccine candidate. Oligosaccharide leads for immunological evaluations in vivo were identified employing glycan microarrays. The stable monovalent ST-5 oligosaccharide glycoconjugate vaccine candidate showed a superior immune response in rabbits when compared with the ST-5 CPS present in the multivalent vaccine Prevnar13. Glycoconjugate vaccines based on isolated capsular polysaccharide (CPS) save millions of lives annually by preventing invasive pneumococcal disease caused by Streptococcus pneumoniae. Some components of the S. pneumoniae glycoconjugate vaccine Prevnar13 that contains CPS antigens from 13 serotypes undergo modifications or degradation during isolation and conjugation, resulting in production problems and lower efficacy. We illustrate how stable, synthetic oligosaccharide analogs of labile CPS induce a specific protective immune response against native CPS using S. pneumoniae serotype 5 (ST-5), a problematic CPS component of Prevnar13. The rare aminosugar l-PneuNAc and a branched l-FucNAc present in the natural repeating unit (RU) are essential for antibody recognition and avidity. The epitope responsible for specificity differs from the part of the antigen that is stabilized by chemical modification. Glycoconjugates containing stable, monovalent synthetic oligosaccharide analogs of ST-5 CPS RU induced long-term memory and protective immune responses in rabbits superior to those elicited by the ST-5 CPS component in multivalent Prevnar13.

Total synthesis of a Streptococcus pneumoniae serotype 12F CPS repeating unit hexasaccharide

The Gram-positive bacterium Streptococcus pneumoniae causes severe disease globally. Vaccines that prevent S. pneumoniae infections induce antibodies against epitopes within the bacterial capsular polysaccharide (CPS). A better immunological understanding of the epitopes that protect from bacterial infection requires defined oligosaccharides obtained by total synthesis. The key to the synthesis of the S. pneumoniae serotype 12F CPS hexasaccharide repeating unit that is not contained in currently used glycoconjugate vaccines is the assembly of the trisaccharide β-D-GalpNAc-(1→4)-[α-D-Glcp-(1→3)]-β-D-ManpNAcA, in which the branching points are equipped with orthogonal protecting groups. A linear approach relying on the sequential assembly of monosaccharide building blocks proved superior to a convergent [3 + 3] strategy that was not successful due to steric constraints. The synthetic hexasaccharide is the starting point for further immunological investigations.