β-Propiolactone is often applied for inactivation of viruses and preparation of viral vaccines. However, the exact nature of the reactions of β-propiolactone with viral components is largely unknown. The purpose of the current study was to elucidate the chemical modifications occurring on nucleotides and amino acid residues caused by β-propiolactone. Therefore, a set of nucleobase analogues was treated with β-propiolactone, and reaction products were identified and quantified. NMR revealed at least one modification in either deoxyguanosine, deoxyadenosine, or cytidine after treatment with β-propiolactone. However, no reaction products were found from thymidine and uracil. The most reactive sides of the nucleobase analogues and nucleosides were identified by NMR. Furthermore, a series of synthetic peptides was used to determine the conversion of reactive amino acid residues by liquid chromatography-mass spectrometry. β-Propiolactone was shown to react with nine different amino acid residues. The most reactive residues are cysteine, methionine, and histidine and, to a lesser degree, aspartic acid, glutamic acid, tyrosine, lysine, serine, and threonine. Remarkably, cystine residues (disulfide groups) do not react with β-propiolactone. In addition, no reaction was observed for β-propiolactone with asparagine, glutamine, and tryptophan residues. β-Propiolactone modifies proteins to a larger extent than expected from current literature. In conclusion, the study determined the reactivity of β-propiolactone with nucleobase analogues, nucleosides, and amino acid residues and elucidated the chemical structures of the reaction products. The study provides detailed knowledge on the chemistry of β-propiolactone inactivation of viruses.

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.

Diphtheria toxoid is produced by detoxification of diphtheria toxin with formaldehyde. This study was performed to elucidate the chemical nature and location of formaldehyde-induced modifications in diphtheria toxoid. Diphtheria toxin was chemically modified using 4 different reactions with the following reagents: (1) formaldehyde and NaCNBH3, (2) formaldehyde, (3) formaldehyde and NaCNBH3 followed by formaldehyde and glycine, and (4) formaldehyde and glycine. The modifications were studied by SDS-PAGE, primary amino group determination, and liquid chromatography-electrospray mass spectrometry of chymotryptic digests. Reaction 1 resulted in quantitative dimethylation of all lysine residues. Reaction 2 caused intramolecular cross-links, including the NAD+-binding cavity and the receptor-binding site. Moreover, A fragments and B fragments were cross-linked by formaldehyde on part of the diphtheria toxoid molecules. Reaction 3 resulted in formaldehyde-glycine attachments, including in shielded areas of the protein. The detoxification reaction typically used for vaccine preparation (reaction 4) resulted in a combination of intramolecular cross-links and formaldehyde-glycine attachments. Both the NAD+-binding cavity and the receptor-binding site of diphtheria toxin were chemically modified. Although CD4+ T-cell epitopes were affected to some extent, one universal CD4+ T-cell epitope remained almost completely unaltered by the treatment with formaldehyde and glycine.

Aluminium phosphate is a commonly used adjuvant consisting of heterogeneously sized aggregates up to several micrometers. However, aluminium phosphate nanoparticles may exhibit an improved adjuvant effect. In this study, nanoparticles were made by sonication of commercially available aluminium phosphate adjuvant, resulting in particles with a size (Z-average diameter) between 200-300 nm and a point of zero charge of 4.5. To prevent reaggregation, which occurred within 14 days, a screening of excipients was performed to identify stabilisers effective under physiological conditions (pH 7.4, 290 mOsm). The amino acids threonine, asparagine, and L-alanyl-L-1-aminoethylphosphonic acid (LAPA) stabilised sonicated aluminium phosphate. Particle sizes remained stable between 400-600 nm at 37 °C during 106 days. Contrarily, arginine induced strong reaggregation to a particle size larger than 1000 nm. The stability of aluminium phosphate nanoparticles was strongly affected by the pH. Aggregation mainly occurred below pH 7. The adsorption capacity, a potentially relevant parameter for adjuvants, was slightly reduced in the presence of asparagine, when using a model antigen (lysozyme). LAPA, arginine, threonine and aspartic acid reduced protein adsorption significantly. The adjuvant effect of aluminium phosphate nanoparticles was studied by immunisation of mice with diphtheria toxoid adjuvanted with the aluminium phosphate nanoparticles. The presence of LAPA, threonine, aspartic acid or asparagine did not alter diphtheria toxoid-specific antibody or toxin-neutralising antibody titres. Arginine increased diphtheria toxoid-specific antibody titres but not toxin-neutralising antibody titres. In conclusion, aluminium phosphate nanoparticles were stabilised by particular amino acids and induced an adjuvant effect comparable to that of aluminium phosphate microparticles.