Robert M. F. van der Put

Glycoconjugate vaccines have proven their worth in the protection and prevention of infectious diseases. The introduction of the Haemophilus influenzae type b vaccine is the prime example, followed by other glycoconjugate vaccines. Glycoconjugate vaccines consist of two components: the carrier protein and the carbohydrate antigen. Current carrier proteins are tetanus toxoid, diphtheria toxoid, CRM197, Haemophilus protein D and the outer membrane protein complex of serogroup B meningococcus. Carbohydrate antigens have been produced mainly by extraction and purification from the original host. However, current efforts show great advances in the development of synthetically produced oligosaccharides and bioconjugation. This review evaluates the advances of glycoconjugate vaccines in the last five years. We focus on developments regarding both new carriers and antigens. Innovative developments regarding carriers are outer membrane vesicles, glycoengineered proteins, new carrier proteins, virus-like particles, protein nanocages and peptides. With regard to conjugated antigens, we describe recent developments in the field of antimicrobial resistance (AMR) and ESKAPE pathogens.

With the ongoing development of conjugate vaccines battling infectious diseases, there is a need for novel carriers. Although tetanus toxoid and CRM197 belong to the traditional carrier proteins, outer-membrane vesicles (OMVs) are an excellent alternative: in addition to their size, OMVs have self-adjuvanting properties due to the presence of genetically detoxified lipopolysaccharide (LPS) and are therefore ideal as a vaccine component or antigen carrier. An essential aspect of their development for vaccine products is characterization of OMVs with respect to size and purity. We report on the development of a field-flow fractionation multiangle light-scattering (FFF-MALS) method for such characterization. Here, we introduced NIST-traceable particle-size standards and BSA as a model protein to verify the precision of the size and purity analysis of the OMVs. We executed a validation program according to the principles provided in the ICH Guidelines Q2 (R1) to assess the quality attributes of the results obtained by FFF-MALS analysis. All validation characteristics showed excellent results with coefficients of variation between 0.4 and 7.32%. Estimation of limits of detection for hydrodynamic radius and particle concentration revealed that as little as 1 μg OMV still yielded accurate results. With the validated method, we further characterized a full downstream purification process of our proprietary OMV. This was followed by the evaluation of other purified OMVs from different bacterial origin. Finally, functionalizing OMVs with N-γ-(maleimidobutyryl)oxysuccinimide-ester (GMBS), generating ready-to-conjugate OMVs, did not affect the structural integrity of the OMVs and as such, they could be evaluated with the validated FFF-MALS method.

Shigella, the causative agent of shigellosis, is among the main causes of diarrheal diseases with still a high morbidity in low-income countries. Relying on chemical synthesis, we implemented a multidisciplinary strategy to design SF2a-TT15, an original glycoconjugate vaccine candidate targeting Shigella flexneri 2a (SF2a). Whereas the SF2a O-antigen features nonstoichiometric O-acetylation, SF2a-TT15 is made of a synthetic 15mer oligosaccharide, corresponding to three non-O-acetylated repeats, linked at its reducing end to tetanus toxoid by means of a thiol-maleimide spacer. We report on the scale-up feasibility under GMP conditions of a high yielding bioconjugation process established to ensure a reproducible and controllable glycan/protein ratio. Preclinical and clinical batches complying with specifications from ICH guidelines, WHO recommendations for polysaccharide conjugate vaccines, and (non)compendial tests were produced. The obtained SF2a-TT15 vaccine candidate passed all toxicity-related criteria, was immunogenic in rabbits, and elicited bactericidal antibodies in mice. Remarkably, the induced IgG antibodies recognized a large panel of SF2a circulating strains. These preclinical data have paved the way forward to the first-in-human study for SF2a-TT15, demonstrating safety and immunogenicity. This contribution discloses the yet unreported feasibility of the GMP synthesis of conjugate vaccines featuring a unique homogeneous synthetic glycan hapten fine-tuned to protect against an infectious disease.

A gradient method has been devised for the rapid analysis of alkaline hydrolyzates of Haemophilus influenzae type b (Hib) capsular polysaccharide‐based vaccines by high‐performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD). As compared with published procedures, peak shape and sensitivity were significantly improved with this approach, analysis time was short and there was little interference from impurities. The limits of detection and quantification were established with a purified reference polysaccharide. We propose this method as a practical alternative for the analysis of minute amounts of Hib polysaccharide, which can be lower than with the conventional approaches. Copyright © 2013 John Wiley & Sons, Ltd.