In this thesis, the monitoring of the immune system in balance and during active responses (by flow cytometry) plays a central role. In chapter 2.1 and 2.2, we investigated the optimal sample... Show moreIn this thesis, the monitoring of the immune system in balance and during active responses (by flow cytometry) plays a central role. In chapter 2.1 and 2.2, we investigated the optimal sample logistics for high-dimensional flow cytometry in clinical trials. In chapter 3.1-3.4, we monitored the longitidinal kinetics of circulating immune cells in humans after vaccination or bacterial challenge against Bordetella pertussis. Lastly, in chapter 4, we investigated the immune system in humans carrying a genetic variant of PLCG2 'p.P522R', which is associated with increased longevity and reduced chance of developing dementia. Show less
Upon infection or vaccination, naïve CD8+ T cells clonally expand and differentiate into antigen-specific effector cell populations, which turn into phenotypically diverse memory CD8+ T cell... Show moreUpon infection or vaccination, naïve CD8+ T cells clonally expand and differentiate into antigen-specific effector cell populations, which turn into phenotypically diverse memory CD8+ T cell subsets that differ in their cytokine polyfunctionality, cytolytic capacity and homing properties. The circulating memory CD8+ T cell pool can be largely classified in two major subsets: effector memory T (TEM) and central memory T (TCM) cells. Contrary to the classical view that lymphocytes continuously recirculate, tissue-resident memory T (TRM) cells have been discovered as cells with the unique ability to reside in tissues with limited recirculation through the blood. Antigen-specific CD8+ TRM cells are induced upon antigen encounter and localize to many different tissues, including barrier tissues, where they play a crucial role in protection against infectious and malignant disease. Within these major circulating and resident T cell subsets a variety of phenotypes and functions exists. However, the development of the heterogeneous populations of memory T cells is not fully understood. In this thesis we dissect the development and heterogeneity of antigen-specific circulating and tissue-resident CD8+ T cells upon vaccination and infection, study their protective capacity in infectious and malignant disease and use this information to improve vaccination and immunotherapeutic strategies. Show less
This thesis describes two studies on the efficacy and safety of vaccinations in patients stable autoimmune myasthenia gravis, one with tetanus revaccination and one with influenza vaccination. Of... Show moreThis thesis describes two studies on the efficacy and safety of vaccinations in patients stable autoimmune myasthenia gravis, one with tetanus revaccination and one with influenza vaccination. Of both vaccinations, the humoral response and clinical parameters of the disease are described. For tetanus revaccination, also the cellular response is described. Furthermore, the validation of a disease specific quality of life questionnaire is described. Show less
This thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and... Show moreThis thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and atherosclerosis. For each of these treatment strategies, the liposomal formulation was tailored to obtain the desired therapeutic effect. Chapter 2 reviews some of the most important physicochemical properties (size, shape, and rigidity) that determine the immunological effects of liposomes and other nanoparticles. In chapter 3 we present a detailed study on the effect of the rigidity of anionic liposomes, as measured by atomic force microscopy, on antigen-specific regulatory T-cell (Treg) responses. In chapter 4, we show that our optimized anionic liposomes can induce potent antigen-specific Treg responses, and can be used to delay atherosclerosis progression in a mouse model. Chapter 5 also focuses on liposomal treatment of atherosclerosis, but here targeted liposomes were prepared to successfully deliver a small molecule to foam cells in atherosclerotic plaques. In Chapter 6, we used cationic liposomes in combination with an adjuvant for cancer immunotherapy in mice. Finally, we summarize the overall findings in chapter 7 and discuss perspectives of using liposomes for vaccination and targeted drug delivery. Show less
Different aspects of respiratory tract infection have been studied. Automatic syndromic surveillance for early detection of infections is feasible. Peak in ILI in hospitals is most flu seasons... Show moreDifferent aspects of respiratory tract infection have been studied. Automatic syndromic surveillance for early detection of infections is feasible. Peak in ILI in hospitals is most flu seasons before rise in cases in primary care. Influenza vaccination can safely be given to oncology patients who use checkpoint inhibitors. we were unable to demonstrate an attenuation of immune response in patients treated with non-lytic rifampicin for pneumococcal pneumonia. Cardiac surgery during influenza season is a risk factor for postoperative ARDS. Show less
Using a variety of anti-malaria tools has resulted in a steady decline of malaria in several endemic countries worldwide. An effective vaccine will be critical to halt malaria or even succeed to... Show moreUsing a variety of anti-malaria tools has resulted in a steady decline of malaria in several endemic countries worldwide. An effective vaccine will be critical to halt malaria or even succeed to final eradication. In that perspective, we studied the potential of whole sporozoite immunization by bites of P. falciparum infected mosquitoes under chemoprophylaxis (CPS). In this thesis we further explored this CPS model and assessed different immunizing doses, type of chemoprophylaxis and immunological determinants of disease and protection. We found a clear dose dependent efficacy, independent of type of chemoprophylaxis, found CD107a and CD8 T cells producing granzyme B related to protective immunity. In the field many genetically different strains circulate and a future vaccine should be able to cover multiple strains. We re-challenged volunteers with a different strain and found modest heterologously protection.We retrospectively assessed the parasitological dynamics and adverse events using a positive qPCR rather than thick smear and found reduced the clinical symptoms of malaria for volunteers after challenge.Successful malaria eradication will be more likely to be achieved with a multi-disciplinary approach. Additionally, sufficient and continuous funds will proof to be of tremendous necessity. Show less
Immunotherapy of cancer has established itself in recent years as a promising novel approach to treat cancer patients. One of the experimental approaches is based on therapeutic vaccination. We... Show moreImmunotherapy of cancer has established itself in recent years as a promising novel approach to treat cancer patients. One of the experimental approaches is based on therapeutic vaccination. We have previously developed vaccines consisting of synthetic long peptides (SLP) which successfully eradicated premalignant lesions in 50% of patients. To further improve these vaccines, a Toll-like receptor ligand (TLR-L) was conjugated to SLP which enables targeting of the SLP to relevant antigen-presenting cells while concomitantly activating these cells. In fact, the research described in this thesis shows that TLR-L SLP conjugates induce enhanced antitumor immunity. Furthermore, optimization of the TLR-L led to even furher improved antitumor responses in mice. Using human cancer patient-derived lymph node cells, we show that lymph node-derived T cells are favorably activated by the TLR-L SLP conjugates. Finally, we combine multiple innate immune stimulatory agonists (TLR2-L and NOD2-L) in one molecule to establish synergistic immune activation. Overall, the research described in this thesis demonstrates the potency of TLR-L SLP conjugates as cancer vaccines, which could strongly contribute to the treatment of cancer patients. Show less
The generally poor prognosis of patients with epithelial ovarian cancer patients treated with curative intent, calls for additional treatment modalities and possible success might lie in a... Show moreThe generally poor prognosis of patients with epithelial ovarian cancer patients treated with curative intent, calls for additional treatment modalities and possible success might lie in a combination of chemotherapy and immunotherapy. This thesis focuses on the interaction of chemotherapy with the immune system and describes new combined chemo-immunotherapy treatment strategies. This thesis has explored new strategies, immune-modulation of the IL-6 pathway and a vaccine against p53, to enhance immune surveillance and to disable tumour immune evasion in ovarian cancer patients. The future challenge for immunotherapy against ovarian cancer is a tailored combinatorial approach to test the rationale of potentially synergistic therapies that can induce efficient antitumour immunity and prolong patients’ survival. Show less
Clinical responses to immunotherapy can be complete and sustained; however, compared to responses to conventional cancer therapies such as chemotherapy they are often delayed. It has become clear... Show more Clinical responses to immunotherapy can be complete and sustained; however, compared to responses to conventional cancer therapies such as chemotherapy they are often delayed. It has become clear that when different cancer therapies are combined they can potentially synergize. Understanding how individual cancer therapeutics work enables the development of the most optimal combinations of cancer therapies. In this thesis, we have used interesting combinations of chemotherapy, cancer vaccines and immune checkpoint blockade and obtained insights in the mechanisms involved in these combinations. For example, we combined chemotherapy with vaccination and observed that none of the tested chemotherapeutics had a negative effect on vaccination, while only a few displayed synergy with vaccination in tumor eradication. We found that vaccine-induced T cells produce Tumor Necrosis Factor-α which could sensitize tumor cells for chemotherapy-induced cell death. Furthermore, we have shown that different cancer therapies modulate intratumoral myeloid cells in a unique manner which can critically affect the therapeutic efficacy of the therapy. Understanding how different therapies affect the local tumor environment will help to improve clinical responses and decrease toxicity in patients. Show less
Preface: The content of this thesis is based on research that was conducted at the travel and vaccination clinic at Leiden University Medical Centre (LUMC). This clinic provides pre-travel care to... Show morePreface: The content of this thesis is based on research that was conducted at the travel and vaccination clinic at Leiden University Medical Centre (LUMC). This clinic provides pre-travel care to the general population, and to special groups of travellers, such as patients who use immunosuppressants or who have chronic diseases. The clinic is closely connected to the department of Infectious Diseases at LUMC. The setting of a travel clinic within an academic medical hospital, provides unique circumstances for medical research, like an experienced team of nurses, expertise regarding immunization, a constant flux of travellers and the knowledge and infrastructure that is required for research into microbiology, virology and parasitology. Examples of research that stem from this clinic are projects on immunization against malaria, yellow fever, travellers' diarrhea, poliomyelitis and hepatitis B, vaccination of immunocompromised patients, and projects on travel related acquisition of extended spectrum ß-lactamase producing Enterobacteriacae and on the utility of post-travel screening of asymptomatic travellers for parasites. Show less
The subject of this thesis is the study of the function and behavior of CD8+ T cells that have exited the circulation and entered a tissue in order to get rid of an infection. In particular, the... Show moreThe subject of this thesis is the study of the function and behavior of CD8+ T cells that have exited the circulation and entered a tissue in order to get rid of an infection. In particular, the research focus on 1- how effector CD8+ T cells can localize infected cells within a tissue. 2-how, once the infection has been cleared, effector CD8+ T cells become memory T cells that take up permanent residence in the tissue; also, how the patrolling behavior that these cells acquire in the tissue allows quick control of local secondary infections. 3-how tissue-resident memory CD8+ T cells that have re-encountered a specific antigen can induce an increased level of local immune protection against infections. Show less
This dissertation reports on the relation between the immune system, colorectal cancer and immunotherapy. In the first part, expression of HLA class I and expression of CXCL5 in colocectal cancer... Show moreThis dissertation reports on the relation between the immune system, colorectal cancer and immunotherapy. In the first part, expression of HLA class I and expression of CXCL5 in colocectal cancer was studied. Low expression of HLA class I in rectal tumors was associated with poor survival of rectal cancer patients. Low expression of CXCL5 in cancer cells was significantly associated with poor prognosis in a population of colorectal cancer patients and correlated with presence of intra-tumoral CD8+ T-cell infiltration. In the second part of this thesis we focused on induction of tumor specific T-cells. For immunotherapeutic purposes distinction should be made between microsatellite instable (MSI-H) and microsatellite stable (MSS) colorectal tumors, as MSI-H tumors express neo-antigens __foreign__ to the immune system while immunotherapy against MSS tumors depends on tumor associated __self__-antigens. We developed a methodology predicting immunogenic behavior of frameshift-mutated antigens present in MSI-H tumors that was based on accumulation and MHC class I presentation. This method can be used to develop cancer immunotherapy of patients at risk for MSI-H tumors. In the last two chapters we described safety and immunogenicity of a p53 synthetic long peptides vaccine combined with and without Interferon-alpha. Addition of IFN-_ to the p53-SLP_ vaccine significantly improved p53-specific after vaccination. Altogether this dissertation reports on the relation between the immune system, colorectal cancer and immunotherapy. This knowledge can be used to further optimize immunotherapeutic strategies to treat cancer patients. Show less
Microneedle-based transcutaneous immunisation is an appealing alternative to the classical manner of injecting vaccines by intramuscular or subcutaneous route. Importantly, as a consequence of the... Show moreMicroneedle-based transcutaneous immunisation is an appealing alternative to the classical manner of injecting vaccines by intramuscular or subcutaneous route. Importantly, as a consequence of the fact that the skin is in direct contact with the environment and should protect the body against pathogens, it contains more antigen presenting cells, such as dendritic cells than the muscles or subcutaneous tissue and thereby offers the possibility to induce a more effective immune response. The combination of microneedles and adjuvanted subunit vaccines may offer effective vaccination whereas ensuring patient safety and vaccine application in a painless manner. The principal aim of this thesis was to design subunit vaccine formulations that can be combined with microneedles for transcutaneous immunisation. The approaches described in this thesis have generated new insights into the main requirements for transcutaneous immunisation. Microneedles definitively have the potential to be an excellent utensil for the delivery of vaccines into the skin. However, the skin is a very elastic organ and the actual conduits formed by microneedle pre-treatment will be considerably smaller than the diameter of the microneedles. Therefore, a small antigen-adjuvant entity is the preferred formulation, as it will be transported efficiently through the microneedle conduits while it retains the co-delivery of antigen and adjuvant. Show less
The research described in this thesis investigates the effect of viral infection on the risk of rejection. It was previously thought that a viral specific T-cell receptor could not crossreact... Show moreThe research described in this thesis investigates the effect of viral infection on the risk of rejection. It was previously thought that a viral specific T-cell receptor could not crossreact against allogeneic HLA molecules, however here it is proven that this is not only possible but is also common. The research performed in this thesis has important clinical implications for solid organ (kidney) and bone marrow transplantation. Viral infection commonly generates alloreactive T-cells, and may therefore be responsible for the failure of tolerance inducing regimens in primates and humans. It is shown that vaccination can also induce HLA-specific alloreactive T-cell responses and it is therefore recommended that vaccines be given to transplantation candidates at least three months prior to expected transplantation date. It is also shown that alloreactive T-cells can be tissue specific. In the final chapter of the thesis thesis it is shown that allogeneic cell therapy could conversely be useful to elicit a viral specific T-cell response in patients with infections, such as HIV. Show less
In conclusion, this thesis describes different clinical aspects on the use of anti-TNF. Initial combination treatment with infliximab and methotrexate seems very promising in early RA and may even... Show moreIn conclusion, this thesis describes different clinical aspects on the use of anti-TNF. Initial combination treatment with infliximab and methotrexate seems very promising in early RA and may even alter the course of the disease. In patients with intolerance or inadequate response to infliximab, a second TNF-blocking agent can be successful, although, the introduction of new biologicals such as rituximab and abatacept provides other therapeutic options. Further research is needed to answer the question which drugs are indicated after failing a first TNF-blocking agent, or even as initial therapy. The use of intra-articular infliximab is inferior to intra-articular depomedrol in chronic and/or recurrent gonartrtitis and this expensive treatment should not have benefit of the doubt in these patients. In contrast, the use of infliximab in rheumatoid vasculitis seems promising in patients who have contra-indication for the recommended immunosupression. Influenza- and pneumococcal vaccinations result in less and lower antibody titers during treatment with anti-TNF, however, antibodies were formed and it remains unclear whether the lower laboratory outcomes are clinically relevant. To date, most guidelines recommend vaccination in patients using immunosuppressive therapy. During the use of anti-TNF neurological symptoms comparable to those of multiple sclerosis (MS) have been described and MR changes have been detected. The use of anti-TNF should be considered as contra-indicated in patients with signs and symptoms compatible with MS. Show less
In an epoch where every generation travels more frequently and at longer distances than the previous generation, with a mean increase of 30 million travellers per year from 1995 until today,... Show moreIn an epoch where every generation travels more frequently and at longer distances than the previous generation, with a mean increase of 30 million travellers per year from 1995 until today, physicians throughout the world are confronted with new diseases. In absolute numbers, this implies that each year, roughly 4 million travellers appeal to specialised health care, either abroad or at home, because of systemic febrile illness, diarrhoea or dermatologic disorders. During the last decades, travel medicine has evolved into a distinct discipline of Infectious Diseases, eventhough transmission of infectious agents into vulnerable populations through travel has been well know for centuries. Improvement of of protection against travel-related diseases can be achieved through knowledge on the following topics; 1. Epidemiology of travel-related diseases, 2. Morbidity and mortality of these illnesses in specific groups of travellers, 3. Adherence to travel health precautions, 4. Responsivity against vaccination, and 5. Availability of preventive measures, such as vaccines. The research described in this thesis addresses these various topics. Show less
Atherosclerosis, the predominantly underlying pathology of cardiovascular events, is the consequence of lipid deposition in the arterial wall, mostly as consequence of high levels of serum... Show moreAtherosclerosis, the predominantly underlying pathology of cardiovascular events, is the consequence of lipid deposition in the arterial wall, mostly as consequence of high levels of serum cholesterol. Treatment of atherosclerosis is mainly focused at the reduction of cholesterol levels by lipid lowering medication, such as statins. Despite the use of statins and prophylactic treatments, such as a reduction in blood pressure and a reduction in risk factors to prevent atherosclerosis, cardiovascular disease is still the major cause of death in the Western world.1-3 As the struggle against atherosclerosis continues and its prevalence is increasing in the world, it is pivotal to find new targets for implementing new strategies against atherosclerosis. Inflammation is considered to be a major component in the process of atherosclerosis and is involved in initiation, progression and destabilization of the atherosclerotic lesion. Although the beneficial effects of statins on atherosclerosis may partly be ascribed to their anti-inflammatory properties, relatively little is known about the exact mechanism and contribution of different inflammatory cells and products in atherosclerosis. The aim of this thesis was to further elucidate the contribution of various components of the inflammatory response in atherosclerosis and thereby finding new intervention points to reduce the incidence and the consequences of atherosclerosis. 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
This thesis describes clinical and immunological aspects of immunoablative therapy followed by reinfusion of T-cell depleted autologous stem cells in patients with progressive refractory Juvenile... Show moreThis thesis describes clinical and immunological aspects of immunoablative therapy followed by reinfusion of T-cell depleted autologous stem cells in patients with progressive refractory Juvenile Idiopathic Arthritis (JIA). After an intensive immunoablative therapy in order to eradicate auto-agressive T cells, autologous hematopoietic stem cells of bone marrow, purged of potentially autoreactive mature T lymphocytes, were reinfused as rescue to reduce the aplastic phase after autologous stem cell transplantation (ASCT). Chapter 1 addresses the background and rationale leading to this study. Chapter 2 describes the safety, efficacy, complications and long term clinical outcome after ASCT in 22 patients with JIA, who were refractory to conventional medication. In chapter 3 the efficacy and safety of ASCT in 34 JIA patients transplanted in 9 different centers in Europe were evaluated. In chapter 4 the immunological effects of the conditioning on cellular infiltrates and expression of cytokines in the synovial tissue of two JIA patients were studied before and 6 months after ASCT. The subject of chapter 5 of this thesis is macrophage activation syndrome in systemic JIA patients. The actual effect of conditioning in vivo and graft manipulation ex vivo on the intended elimination of the adaptive (auto)immunological memory after ASCT was studied in 19 JIA/systemic lupus erythematodes (SLE) and 10 multiple sclerosis (MS) patients (chapter 7). In order to obtain reference values for anti-rabies specific humoral and cellular immune responses after a single and booster vaccination, we conducted a study in 18 healthy controls as described in chapter 6. Show less
This thesis introduces a novel T cell vaccination method that uses a tattoo machine to inject DNA in the skin of the vaccinee. In comparison to other experimental vaccination methods DNA tattooing... Show moreThis thesis introduces a novel T cell vaccination method that uses a tattoo machine to inject DNA in the skin of the vaccinee. In comparison to other experimental vaccination methods DNA tattooing is very strong: besides small laboratory animals also large animals mount strong T cell responses upon tattoo DNA vaccination. Show less
