mAb 1H8 rather than 1F1 was used as it binds ST8 CPS better and IgG are more relevant to the outcome of vaccinations than IgM antibodies

mAb 1H8 rather than 1F1 was used as it binds ST8 CPS better and IgG are more relevant to the outcome of vaccinations than IgM antibodies. Fig. S16. Immune response against semisynthetic ST8 glycoconjugates in rabbits. Fig. S17. Adsorption of ST8 glycoconjugates to Prevnar 13, as assessed by flow cytometry. Fig. S18. Effect of co-formulation of semisynthetic ST8 glycoconjugates with Prevnar 13 on the immune response against several pneumococcal CPSs. Table S1. (+)-CBI-CDPI1 Sequences of automated assembly of protected ST8 CPS-related tetrasaccharide frameshifts. Table S2. Anti-polysaccharide IgG endpoint titers of rabbits immunized with Prevnar 13 alone or co-formulated with semisynthetic ST8 glycoconjugates. NIHMS859486-supplement-supplemental.pdf (14M) GUID:?33A8EE02-C29B-4C43-9C3C-AF2CD7FDA92D Abstract 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 (+)-CBI-CDPI1 identification of immunogenic and protective oligosaccharide antigens. (+)-CBI-CDPI1 Here, 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 to uncover a protective glycan epitope (glycotope) for 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 non-protective glycotopes. Immunization of rabbits with semisynthetic ST8 glycoconjugates containing protective glycotopes induced an antibacterial immune response. Co-formulation of ST8 glycoconjugates with the marketed 13-valent glycoconjugate vaccine Prevnar 13 yielded a potent 14-valent vaccine. Our strategy presents a facile approach to develop efficient semisynthetic glycoconjugate vaccines. Introduction Most marketed vaccines against encapsulated pathogenic bacteria, such as and serotypes. More immunogenic and efficacious pneumococcal glycoconjugate vaccines such as Prevnar 13 are based on isolated CPSs that are chemically conjugated to carrier proteins. Immunization with vaccines based on isolated polysaccharides generates a polyclonal immune response that may be directed towards multiple glycotopes. Each of these glycotopes may be either protective (+)-CBI-CDPI1 (when antibodies that recognize the sequence protect from disease) or non-protective. Reaction to impurities present in the isolate, to neo-epitopes introduced (+)-CBI-CDPI1 during isolation and conjugation or to immunodominant glycotopes as part of an immune evasion strategy by the pathogen may cause a non-protective response (2C5). Furthermore, the abundance of co-isolated impurities in crude polysaccharide preparations hampers the manufacture of polysaccharide-based vaccines, and a number of quality control steps is needed to characterize isolates (1). Chemically defined and free from cell-derived contaminants, synthetic oligosaccharides are a powerful alternative as vaccine antigens. Synthetic oligosaccharides can be designed based on protective glycotopes so that conjugation to a carrier protein yields efficaceous semisynthetic vaccines. However, the design of oligosaccharide antigens requires information on the identity of protective glycotopes within a CPS. The conventional iterative vaccine development approach relies on the synthesis of antigens and immunological evaluation of glycoconjugates (6, 7). Thereby, the plethora of putative glycotopes in a polysaccharide challenges traditional chemical synthesis because a number of different structures are needed. A more recent glycotope discovery approach uses structure-based reverse engineering of antigen binding sites of protective monoclonal antibodies (mAbs) and has greatly advanced the field of glycoconjugate vaccines (1, 8). However, structure elucidation of carbohydrate-mAb interactions by classical biophysical methods such as X-ray KILLER crystallography is slow and suffers from low throughput. Two methods of chemical glycobiology are particularly suitable to address the shortcomings of glycotope discovery. First, AGA can produce complex glycans by employing an automated solid-phase oligosaccharide synthesis workflow (9, 10) and.