This thesis describes the design, synthesis, and immunological evaluation of varying (neo)antigenic peptide conjugates containing either a TLR2 or a TLR7 agonist. Chapter 2 discusses the synthesis... Show moreThis thesis describes the design, synthesis, and immunological evaluation of varying (neo)antigenic peptide conjugates containing either a TLR2 or a TLR7 agonist. Chapter 2 discusses the synthesis of a library of UPam functionalized (neo)antigenic peptides. Chapter 3 discusses the optimization of UPam and investigates the current paradigms surrounding UPam. Chapter 4 presents a new simplified agonist for TLR2, namely mini-UPam, which is then conjugated to some neoantigenic peptides and evaluated on its immunological properties. Chapter 5 discusses the design and synthesis of TLR2 and TLR7 dual functionalized antigenic peptides. Show less
The research described in this Thesis was aimed at designing and synthesizing nature-inspired compounds as part of TB vaccine discovery. A variety of synthetic analogues of mycobacterial cell wall... Show moreThe research described in this Thesis was aimed at designing and synthesizing nature-inspired compounds as part of TB vaccine discovery. A variety of synthetic analogues of mycobacterial cell wall components, from peptide and glycolipid antigens to glycolipid PAMPs has been accessed. Evaluation of the immune stimulatory activity of the novel compounds in combination with preliminary immunization studies in vivo, suggested the potential of selected synthetic conjugates as single molecule vaccines against TB. Further research is needed to verify the efficacy of these vaccine modalities. Show less
The function of TLRs in innate immunity has aroused worldwide attention soon after its discovery. Because of the broad functions of TLR2 in innate immunity, the drive for the development of TLR2... Show moreThe function of TLRs in innate immunity has aroused worldwide attention soon after its discovery. Because of the broad functions of TLR2 in innate immunity, the drive for the development of TLR2-targeted vaccines or therapeutic treatments has accelerated in the last decades. However, its dual role in both activation and suppression of innate immune responses makes it very difficult to use the available results from basic research for the development of clinical trials. In addition, it is still not clear what is the function of TLR2 in regulating phagocytic cell migration. Therefore, we aimed to determine the function of TLR2 in mycobacterial infection and explore its role in regulating phagocytic cell migration in inflammatory tissue by using a zebrafish larval model in this thesis. We showed that infection of a tlr2 mutant in zebrafish larvae leads to a higher mycobacterial burden, accompanied by a lower number of granulomas and increased extracellular bacterial growth. Through a tail fin wounding and tail fin infection zebrafish model, we demonstrated that tlr2 is involved in modulating leukocyte migration. This thesis provides a better understanding of the functions of TLR2 in innate immune responses to infection and tissue wounding. Show less
Lachmandas, E.; Beigier-Bompadre, M.; Cheng, S.C.; Kumar, V.; Laarhoven, A. van; Wang, X.H.; ... ; Netea, M.G. 2016
The mammalian immune system protects, amongst others, against invading pathogens and consists of an innate and adaptive component. The innate system is the first line of defense in which pattern... Show moreThe mammalian immune system protects, amongst others, against invading pathogens and consists of an innate and adaptive component. The innate system is the first line of defense in which pattern recognition receptors, like TLR2, NOD1 and NOD2 receptors, detect pathogen associated molecular patterns (PAMPS) that are specific for pathogens. PAMPs exhibit a broad structural variety and the exact molecular structures of ligands that bind to the corresponding PRRs are mostly unknown. The research presented in this Thesis is directed to the design, synthesis and immunological evaluation of new NOD1, NOD2 and TLR2 ligands as well as conjugates in which these ligands are covalently bound to an antigenic OVA-derived peptide. The designed NLR and TLR ligand-antigen conjugates contribute to the insight of the processes of the mammalian immune system at molecular level. Further elucidation of pattern recognition receptor acti vation and antigen presentation with these conjugates may eventually result in synthetic vaccine modalities. Show less
Human fitness is critically reliant on the immune system to provide protection against pathogens. We argue that a pro-inflammatory response is crucial for defense against pathogens and that it is... Show moreHuman fitness is critically reliant on the immune system to provide protection against pathogens. We argue that a pro-inflammatory response is crucial for defense against pathogens and that it is very likely that infectious pressure has resulted in selective survival for genetic variants encoding for higher pro-inflammatory responsiveness. In industrialized populations many chronic diseases have been associated with an imbalance in pro- and anti-inflammatory responsiveness. We argue that from an evolutionary perspective, these chronic diseases in later life could be explained by genetic adaptations to survive a harsh environment. In order to study the role of the innate immune response in life-history regulation in a pathogen-rich environment, we set out a study in Northern Ghana. In a population living under adverse conditions we studied the role of the inflammatory response in survival and fertility. In Chapter 1 a general introduction was given on the research hypotheses and an overview of aims and description of the study population and methods. The general frame-work of the study was explained in depth in Chapter 2. Here we hypothesized that human life-history regulation in our evolutionary past, or under adverse conditions can largely be explained by selections that operated in the innate immune response. We proposed that fertility is associated with an anti-inflammatory response, whereas survival in a pathogen-rich environment is dependent on a strong pro-inflammatory response. We hypothesize that populations living under adverse conditions have been selected for a pro-inflammatory innate immune response. Also we argue that fitness in itself is a conflict between pro- and anti-inflammatory responsiveness where concessions have to be made to allow reproduction as well as defense. Furthermore we argue that evolutionary programming of the inflammatory response might underlie age-related diseases as observed in populations living under affluent conditions. It is arbitrary how to measure an innate immune response ex vivo, that reflects a general response mode irrespective the type of pathogen involved. In Chapter 3 a method is described to test innate tendency of immune activation. The assay is also validated. Given the fact that bacteria and other pathogens normally use several TLRs together to induce an immune response, we argue that mixed stimulation of both TLR2 and TLR4 receptors gives a broader view of an immune response than with the usual assay on a single TLR-agonist. Compared to variability of cytokine production in the Netherlands, we show that ex vivo IL10 production is comparable. Therefore we suggest that in Ghana IL10 is highly genetically regulated. TNF_, on the other hand, is more prone to variation in general, but especially in Ghana and might be more dependent on environmental modulation. In Chapter 4 we compared age-related cytokine production in adverse and affluent conditions. When measured cross-sectionally, IL10 production decreases with age in the Netherlands and in Ghana. TNF_ production decreases with age in the Netherlands, but remained equal over all age-categories in Ghana or, dependent on the stimulation, increased with age. We conclude that the decline in innate cytokine responses is an intrinsic ageing phenomenon, while pathogen exposure and/or selective survival may drive pro-inflammatory responses under adverse living conditions. As TLR2 and TLR4 are important recognition receptors for a large set of pathogens, it might be that variation in these receptors results in different induction of innate immune responses and selective survival. In Chapter 5 we report that at the end of the TLR4 gene there was variation that associated with higher ex vivo LPS-induced IL10 production. None of the variants in TLR2 or TLR4 however were associated with P. falciparum infection or survival. As the prevalence of malaria was high in this area, we conclude that it is likely that in contrast to other studies, these genetic variants do not play a role in disease state and outcome of infection. Another pathogen receptor is PTX3. It is not only involved in recognition of pathogens, but also in the formation of the extracellular matrix of the oocyte. Therefore it might be crucial for female fertility. In Chapter 6 we assess whether genetic variation in PTX3 production is associated with life-time reproductive success. We found genetic variants in PTX3 that associated with higher PTX3 production capacity ex vivo and increased fertility and vice versa. We found no evidence for selective survival of genetic variants. We conclude that PTX3 is important for human fertility, whereas no concessions were made with regard to survival. In Chapter 7 we asses the role of IL10 in survival. We report on genetic variation in the IL10 gene that associated with lower IL10 and higher TNF_ production. Carriers of these genetic variants had a higher survival chance when living under adverse conditions. However, survival chances of these variants decreased when people had access to clean drinking water. We conclude to have found evidence that adverse environmental conditions favor selection for a pro-inflammatory response pattern. Show less
May, Linda; Bodegom, David van; Frolich, Marijke; Lieshout, Lisette van; Slagboom, P Eline; Westendorp, Rudi GJ and Kuningas, Maris 2010
Toll-like receptors (TLRs) are involved in the induction of an adequate immune response on infection. We hypothesized that genetic variation in TLR4 and TLR2 genes could influence this response and... Show moreToll-like receptors (TLRs) are involved in the induction of an adequate immune response on infection. We hypothesized that genetic variation in TLR4 and TLR2 genes could influence this response and lead to variability in cytokine production and survival. We tested this hypothesis in 4292 participants who were followed up for all-cause mortality for 6 years and live under adverse environmental conditions in the Upper-East region of Ghana, where malaria is endemic. In 605 participants, tumor necrosis factor-a and interleukin-10 (IL10) production, after stimulation with lipopolysaccharide and zymosan, was measured. In addition, 34 single-nucleotide polymorphisms (SNPs) in TLR4 and 12 SNPs in TLR2 were genotyped and tested for association with cytokine production, malaria infection and mortality. In this comprehensive gene-wide approach, we identified novel SNPs in the TLR4 gene that influence cytokine production. From the analyzed SNPs, rs7860896 associated the strongest with IL10 production (P¼0.0005). None of the SNPs in this study associated with malaria or overall mortality risks. In conclusion, we demonstrate that genetic variation within the TLR4 gene influences cytokine production capacity, but in an endemic area does not influence the susceptibility to malaria infection or mortality. Show less
