The chemical and structural heterogeneity of toxoid vaccines makes their analysis challenging. However, detailed insights on a molecular level can be obtained by mass spectrometry. Our initial... Show moreThe chemical and structural heterogeneity of toxoid vaccines makes their analysis challenging. However, detailed insights on a molecular level can be obtained by mass spectrometry. Our initial focus was the identification of formaldehyde-induced modifications in diphtheria toxin, which is described in Chapter 2. Subsequently, the methods described in Chapter 2 were applied to study what effects formaldehyde-induced modifications on model proteins have on their susceptibility to enzymatic proteolysis (Chapter 3). During the analysis of these model proteins, unknown formaldehyde-induced modifications were observed. The structural elucidation of these modifications, the discovery of a new type of crosslinks and various other subsequent reaction products are described in Chapter 4. The degradomics analysis described in Chapter 3 was applied to tetanus toxoids to distinguish heat-denaturated toxoids from their original state (Chapter 5). In order to reduce the analysis time and further improve the degradomics approach, an optimized strategy using Tandem Mass Tag multiplexing for the relative quantification of peptides was developed for the analysis of diphtheria toxoids (Chapter 6). Finally, Chapter 7 provides a brief discussion on the results presented in this thesis and offers some perspectives on implementation of the findings for toxoid vaccine development, quality control and further research. Show less
Worldwide, more than a billion people are infected with helminths. These worm infections are chronic in nature and can lead to considerable morbidity. Immunologically these infections are... Show moreWorldwide, more than a billion people are infected with helminths. These worm infections are chronic in nature and can lead to considerable morbidity. Immunologically these infections are interesting; chronic helminth infections are characterized by skewing towards a T helper 2 type response as well as regulatory responses. The latter is thought to prevent strong immune responses against parasitic worms, allowing their long-term survival and restricting pathology. This regulatory network is thought to also temper responses to non-helminth antigens, like allergens or self antigens, possibly leading to lower prevalence of allergies and autoimmune diseases in subjects that are chronically infected with helminths. Thus, helminths may bear molecules that have potential therapeutic action against allergies and possibly other inflammatory diseases. However, on the other side of the coin, this would predict that helminth infected subjects might not respond strongly to third party antigens like vaccines. Helminth derived molecules have been identified that induce T helper 2 and regulatory responses via modulation of dendritic cells and some appear to do so via TLR signaling. New insights into these pathways could be useful to antagonize suppression and hence boost vaccine efficacy or to optimize suppression induced by helminth derived molecules and control inflammatory diseases. Show less
