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Letter from the Chairmen of the Supervisionary Board The year 2011 has again shown that the Biomedical Primate Research Centre is well equipped to perform its important task for science and society. Based on a strong organizational framework it has been possible to continue and expand research programmes on a number of topics which are relevant for improving our knowledge of diseases for which as yet no satisfying cure or prevention exist. Diseases with an infectious origin, diseases with anomalies in the immune or nervous system are studied in man, but often more in depth or invasive approaches have their limitations. For a better understanding studies in non-human primates are still an important option. This does not mean that the BPRC is only performing studies in these animals. Throughout the years and again well described in this annual report is the focus on exploring alternatives. An open eye for the 3Rs approach (Replacement, Refinement and Reduction) to look for future research options not or less linked to non-human primates is well demonstrated in this annual report. The cooperation with the Research Group Behavioural Biology of the University of Utrecht has been very productive in studies on individual and group behaviour and cognitive abilities of non-human primates. These activities and studies allow BPRC to be an active player in the public domain to inform the public at large and especially the younger generation on the relevant aspects of animal and in vitro studies necessary to understand cause, treatment and prevention possibilities of diseases which still form a burden to mankind and society. Examples of how to use neurofeedback training as a new non-invasive strategy to improve brain function in a neurodegenerative disease like Parkinsonism. 2 BPRC Annual Report 2011 In the malaria programme the focus is not only on P. falciparum, but also on P. vivax. Thus studying the two major human malaria parasites. The problem of HIV/Aids is far from solved. New concepts of developing a potent HIV vaccine are based on a better understanding and utilisation of the possibilities to induce relevant antiviral T- and B- cell immunity. In the field of comparative genetics studies on the major histocompatibility complex and its role in presenting peptides from intracellular origin to cytolytic CD8+ T cells add to our knowledge of the defence against viral pathogens. These are just some examples of the types of studies performed at BPRC which are relevant to health care and public health. The results of these studies are published in high ranking international scientific journals. International cooperation and sharing of data with scientists working on similar topics in another setting add to the international status of BPRC. For many years the BPRC has been active in improving its animal, laboratory and housing facilities. The last construction activities have taken place. Animals and staff are now each housed in a new improved environment. On behalf of the Supervisory Board I congratulate the director, scientific and supporting staff, all other co-workers and especially the construction team for their excellent performance. I recommend this annual report to many readers both for its scientific content and its societal relevance. Professor Joost Ruitenberg Chairman of the Supervisory Board BPRC Annual Report 2011 3 Message from the Director The scientific output of the institute is on a steady course. Nonhuman primates are still an essential partner in biomedical research. In 2011, BPRC staff members authored or co-authored The BPRC represents an unique facility covering many expertises and skills. 42 papers with an average impact factor of 4.48. In the last year, it has become evident that more and more Dutch and European partners found their way to the BPRC. It is great to hear that Average impact factor 7 6 5.63 many of these new partners chose the BPRC because the institute pays 6.10 5.35 5 4 5.14 much attention to animal welfare and plays an active role in conducting 5.13 research for alternatives on animal experimentation. 4.22 3 The construction process is almost coming to its end. All buildings are 2 1 number of publications 0 in place and necessary renovations have been finished. The last session will be devoted to cleaning up the campus and create a green and open atmosphere with trees and places were staff members can sit during Although these papers can be tracked down via special websites at lunch hours or work outside in the shade of trees, if the weather allows it. the internet, BPRC wishes to improve accessibility and consequently Although the building process is not entirely finished, it seems reasonable highlights important or special publications on its website (http://www. to expect that all the construction works will be executed within the frame bprc.nl/BPRCNL/L4/Newsstart.html). This “news page” features also of the budget that was approved and provided by the Dutch government improvements, highlights interviews and showcases several reports that in 2001. may be of interest to the general public. These events fit within the “open door policy” of the institute. This attitude is highly appreciated, in 2011 4 more than 600 people visited the facilities and wished to see with their Prof. dr. Ronald E. Bontrop own eyes which improvements were made. Director BPRC BPRC Annual Report 2011 BPRC Annual Report 2011 5 Department of Animal Science In 2011, the final phase of building activities with respect to animal housing Within the EUPRIM project, ASD is involved in a program for development of facilities was completed. All animals are now housed in new, large cages and all new methods for surveillance of bacterial and parasitic pathogens and more housing is in full compliance with the new EU Directive 2010/63.When you read detailed analysis of the presence of certain microorganisms in the airways this, the facility has received accreditation by the Association for Assessment of macaques. The results showed that many animals carried the bacterium and Accreditation of Laboratory Animal Care International (AAALAC). The Staphylococcus aureus. Further analysis showed 13 new types of which 9 completion of these new animal facilities imply significant improvement of were not related to any S. aureus strain described until now. Four types were welfare for both the animals and the animal care staff. The new cages facilitate part of a specific human clone. These findings were published in PLOS One. group housing of the animals in experimental facilities and training of these Further research related to veterinary care included the analysis of specific animals can much better be achieved in the new setting. One of the major antibiotic treatment, which is now published, and ongoing analysis of normal goals is to train the animals as optimal as possible for specific procedures to and pathological osteology in common marmosets. The Behavioural Research obtain the best results with the least possible stres. Group continued their research on prosocial behaviour in the various primate The new EU EUPRIM-NET 2 program began in January 2011. The ASD is species present at the BPRC. This group was also responsible for several involved in various parts of this network. End 2011, a joined meeting between courses on behavioural research at the BPRC for students from the University EUPRIM and the European PrimateVeterinarians (EPV) was held at the BPRC. of Utrecht. More than 30 participants from centers working with non-human primates in A large number of students received training within the ASD in 2011. Europe attended this meeting that focussed on improved veterinary techniques, This included students on animal care, behavioural research, laboratory including endoscopic procedures and the use of thermographic methods for research and veterinary research, either in specific courses or in specific short- non-invasive diagnostics. The ASD is also involved in an EUPRIM program on term and long-term projects. We also continued our policy to receive groups animal training. The animal trainer followed various courses and a workshop to explain our research and show the institute. for animal training was held at the BPRC. Our success with respect to training was reflected in the invitation to our Head Animal Training to present our approach and its results at an International Animal Training and Husbandry Workshop jointly organised by the European Association of Aquatic Mammals (EAAM) and the International Marine Animal Trainers Association (IMATA). 6 BPRC Annual Report 2011 BPRC Annual Report 2011 7 Alternatives Unit Research within the Alternatives Unit is organized along two main themes. I. We apply in vitro approaches to precede, complement and ultimately replace in vivo experiments performed to answer disease-related questions. To model immune-mediated diseases of the central nervous system we use primary cell culture systems that are started by using ‘left-over’ tissue from deceased rhesus monkeys. The short post mortem time enables us to initiate primary cultures of cells that are sensitive to activation. Such cultures are difficult to reproducibly initiate from human donors where post mortem times and cause of death are considerably less controllable, while still working in a system that is closely comparable to humans. In 2011, we have successfully continued and expanded the use of primary cell cultures as a pre-in vivo test phase. We have submitted a manuscript on the mode of action of statins, drugs that are used to lower cholesterol but that are also known to exhibit immune-modulatory functions, on immune functions of microglia. Our system allowed for a thorough comparison of variables in a well-controlled system using clinically relevant cell types (i.e. primary microglia and primary macrophages). In addition, we have further pioneered the development of a new in vitro technique, the establishment of brain slice cultures, in close collaboration with the Netherlands Institute for Neuroscience in Amsterdam. First attempts demonstrate the potential of this technique to establish long-term cultures of brain cells in their natural micro-context. Finally, we have initiated a new research line on inflammasome-mediated activation of different primary cells. A new PhD student has started and an extensive review on this topic was published. II. We aim to refine the current use of adjuvants in immunization procedures of experimental animals. More specifically, we focus on complete Freund’s adjuvant (CFA), which is notorious for its side-effects such as the formation of granulomas at immunization sites. Our aim is to develop an adjuvant that lacks these unwanted side-effects, but that is equally potent as CFA. CFA is a mixture of paraffin oil, surfactant and heat-killed mycobacteria. The bacteria are responsible for both the desired adjuvating effects of CFA as well as 8 BPRC Annual Report 2011 for most of the side-effects, but these effects are not coupled. We have set up in vitro systems to model the adjuvating effects, i.e. Toll-like receptor (TLR) bioassays, as well as to model the side-effects, i.e. an in vitro granuloma model. Both assays are also used to screen other, new adjuvants for potential adjuvating and side-effects without having to use animals. In collaboration with research groups in Amsterdam and in Baltimore (USA), we use specific mycobacterial strains that lack expression of sets of proteins. These strains reportedly induce less granulomas in zebrafish or in rabbit models. In 2011 we have in vivo tested the different strains that were identified in 2009 and 2010 as potential replacement candidates. The first results indicate that all strains tested cause less side-effects (cutaneous granulomas) than the strain currently used in CFA. We are now carefully validating these results and will further test these strains for their adjuvating potential in vivo shortly. Since CFA is also widely used in experimental animal models where other species than non-human primates are being used, we believe that results from this line of research will have broader applicability than replacing CFA as adjuvant in non-human primates only. In addition to these scientific goals, we serve as a communication hub regarding alternatives to animal experimentation within the BPRC, and we contribute to the public discussion on the use of animals for experimental purposes. We do so by publishing on these topics both in specialized scientific journals and in journals aimed at a broader audience as well as by participating in national and international initiatives for the replacement, refinement and reduction of animals in research e.g. those organized by the Dutch Center for Alternatives (NKCA) and the British National Center for 3Rs (NC3Rs). In addition, we have authored the publically accessible “Proefdierkundig Jaarverslag 2010” (in Dutch: http://www. bprc.nl/BPRCNL/L4/newsdownloads/PDKJ2010.pdf) providing transparency on experimental animal use and numbers within BPRC. Finally, we coordinate the European research proposal (7th Framework Programme) on the “Development of in vitro technologies to replace, reduce and refine non-human primate studies” under the umbrella of the EU-PRIMNETII network. BPRC Annual Report 2011 9 Department of Immunobiology The research program of the Immunobiology department is focused on three groups of clinical disorders, namely autoimmune neuroinflammation, neurodegeneration and autoimmune joint-inflammation. We use in-house developed disease models in monkey species from the Old World, rhesus macaques, and New World, common marmosets, for our research into pathogenic mechanisms and the preclinical efficacy evaluation of new treatments. The models are continuously refined to reduce the discomfort that is inevitably associated with the modeling of serious diseases in the human population. One example is the banning of the strong bacterial complete Freund’s adjuvant from the EAE model. Another example is the validation of the cutaneous delayed-type hypersensitivity (DTH) reaction for prescreening of new immunomodulatory reagents. Autoimmune neuro-inflammation: Our research in a unique and relevant preclinical model of multiple sclerosis (MS), the rhMOG/CFA-induced experimental autoimmune encephalomyelitis (EAE) model in common marmosets, has resulted in the identification of a new immunopathogenic mechanism that is engaged in late-stage development of MS-like pathology and neurological symptoms (1). Natural killer-type T cells have a central pathogenic role in this non-canonical pathway as main culprits of the induction of pathology in the central nervous system (CNS) and neurological deficit.The characterization of these cells showed functional and phenotypical similarity with effector memory cells present in the normal immune repertoire (2). Upon in vivo activation by immunization of marmosets with peptide 34-56 of myelin/oligodendrocyte glycoprotein (MOG34-56) in incomplete Freund’s adjuvant, these cells display their high reactivity and their capacity to induce demyelination of CNS white and grey matter, which occurs independent of autoantibodies (3). Prominent immunological characteristics are high IL-17A production and specific cytotoxicity towards target cells presenting the specific 10 BPRC Annual Report 2011 epitope MOG40-48 in the context of Caja-E molecules (2). Studies with a monoclonal antibody neutralizing IL-17A support a pathogenic role of IL-17A in the rhMOG/CFA EAE model, albeit not in the disease initiation but rather in the late stage expression of pathology and symptoms (4). By contrast, the lack of activity of human IFNg in the MOG34-56 model argues against a prominent pathogenic role of T helper 1 cells (5). Intriguingly, the demyelinating activity of the T cells is independent of autoantibodies, but B cells do have an important pathogenic role (6). Accumulating evidence from B cell depletion studies with a clinically relevant anti-CD20 monoclonal antibody, shows that B cells are intimately involved in the activation of the pathogenic T cells, most likely as antigen presenting cells (APC). Autoimmune joint-inflammation: The collagen-induced arthritis (CIA) model in rhesus monkeys has been used for the preclinical evaluation of new treatments of rheumatoid arthritis. After successful validation of the model with the clinically relevant drug RoActemra® we have extended the analysis of historical control animals with lipid metabolites Cholesterol, Triglycerides, high-density lipids (HDL) and low-density lipids (LDL)(7). The atherogenic index (AI), which is defined as Cholesterol/HDL ratio, expresses the risk to develop atherosclerosis. Atherosclerosis is responsible for more than 50% of the excess death observed in RA patients. The AI is increased during the period of active inflammation in all rhesus monkeys that had CIA. In addition a second comorbidity, anemia, has been analyzed that is responsible for poor quality of life in over 35% of the RA patients. All rhesus monkeys were anemic during active inflammation as shown by seriously reduced iron together with reduced hemoglobin and hematocrit. These extended analyses can provide insight into the ameliorating effects of new therapeutics on dyslipidemia and anemia that underlie important comorbidities in RA and are currently seriously undertreated. The standardized analysis of rhesus monkeys with CIA BPRC Annual Report 2011 11 Department of Immunobiology has generated a database of historical control data on multiple parameters describing the clinical status, inflammation, bone remodeling and histopathology. This enables a more robust evaluation of new promising therapies. For the CIA models in rhesus monkey and common marmoset it was further established that the antibody response against collagen type II (CII) in the joints is remarkably conserved in both CCP-negative and CCP- positive RA patients. The fine specificity of the anti-CII antibody response is similar between CIA in monkeys and rodents (Lindh et al., 2012, submitted). The model still awaits validation with a clinically relevant therapeutic. The combination of this new model with the EAE model in the same species creates a powerful platform for research into the pathogenic mechanisms that drive chronic inflammation in brain and joint. Parkinsonism: Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases in the human population. Current medications only suppress disease symptoms, but do not affect the progressive degeneration of dopaminergic neurons. Hence, there is a pressing need for novel treatments for protecting the brain against neurodegeneration. This requires the identification of targets for neuroprotection during the very early phase of the pathogenic process when brain damage (cell loss) is not yet (clinically) apparent. Identification of changes in the expression of gene transcripts and proteins during this phase can be the key to finding these new targets for neuroprotection. Our previous studies in mice have shown that in the very early phase of PD a change in protein expression occurs, probably due to the initial insult and not resulting from degeneration per se (Thesis Nelleke Verhave, Free University, Amsterdam, 2011). In order to repeat this in a more translational approach with an open eye towards human validity, we have implemented a new progressive chronic PD model in marmosets. This chronic progressive model is based on a long-term chronic injection of low weekly 12 BPRC Annual Report 2011 doses of the neurotoxin MPTP. The ensuing disease best reflects idiopathic PD and is comparable with the accidental disease induction in drug users exposed to MPTP. Motor as well as non-motor aspects, such as REM sleep behaviour disorder (8, 9) were used to identify the symptomatic dynamics during disease development. In this induction protocol the behavioral effects were less acute and appeared gradually over time. In order to find genetic susceptibility factors for developing neurodegenerative disease via a proteomic approach we selected offspring of five different marmoset breeding families. Interestingly, we observed that low and high responsiveness segregates with family relations. This may suggest an inherited influence on the susceptibility of neurodegeneration, which has to be confirmed by transcriptomic analysis. Delayed-type hypersensitivity: The development of new biologicals for the treatment of immune-mediated inflammatory conditions, such as autoimmune diseases and graft rejection, requires efficacy and safety testing in appropriate species. Non-human primates (NHP) are often the species of choice for such preclinical evaluation. The ultimate proof that a new drug is effective for a particular indication is testing the drug in a relevant animal model: arthritis model for RA, transplantation model for graft rejection. However in NHP such models cause serious discomfort to the animals. Complete disease development is not always required to evaluate a drug in the equivalent of the human clinical situation. Immunomodulatory effects can also be evaluated using models with less discomfort. We examined whether the cutaneous delayed type hypersensitivity (DTH) reaction can be used to predict immunomodulating effects of new drugs. We have shown that the local response to recall antigens (OVA and TT) shares several histological characteristics with immune events in synovia of collagen induced arthritic rhesus monkeys and rejected kidney allografts (Jonker et al, in prep 2012). BPRC Annual Report 2011 13 Department of Immunobiology References 1. Kap, Y. S., P. Smith, S. A. Jagessar, E. Remarque, E. Blezer, G. J. Strijkers, J. D. Laman, R.Q. Hintzen, J. Bauer, H. P. Brok, and B. A. ‘t Hart. 2008. Fast Progression of Recombinant Human Myelin/Oligodendrocyte Glycoprotein (MOG)-Induced Experimental Autoimmune Encephalomyelitis in Marmosets Is Associated with the Activation of MOG34-56-Specific Cytotoxic T Cells. J Immunol 180: 1326-1337. 2. Jagessar, S. A., N. Heijmans, E. L. Blezer, J. Bauer, J. H. Blokhuis, J. A. Wubben, J. W. Drijfhout, P. J. van den Elsen, J. D. Laman, and B. A. Hart. 2012. Unravelling the T-cell-mediated autoimmune attack on CNS myelin in a new primate EAE model induced with MOG(34-56) peptide in incomplete adjuvant. European journal of immunology 42: 217-227. 3. Jagessar, S. A.,Y. S. Kap, N. Heijmans, N. van Driel, H. P. M. Brok, E. L. A. Blezer, J. D. Laman, J. Bauer, and B. A. ‘t Hart. 2010. Induction of progressive demyelinating autoimmune encephalomyeitis in common marmoset monkeys using MOG34-56 peptide in incomplete Freund’s adjuvant. J Neuropathol Exp Neurol 69: 372-385. 4. Kap,Y. S., S. A. Jagessar, N. van Driel, E. Blezer, J. Bauer, M. van Meurs, P. Smith, J. D. Laman, and B. A. t Hart. 2011. Effects of early IL-17A neutralization on disease induction in a primate model of experimental autoimmune encephalomyelitis. J Neuroimmune Pharmacol 6: 341-353. 5. Jagessar, S. A., B. Gran, N. Heijmans, J. Bauer, J. D. Laman, B. A. t Hart, and C. S. Constantinescu. 2011. Discrepant Effects of Human Interferon-gamma on Clinical and Immunological Disease Parameters in a Novel Marmoset Model for Multiple Sclerosis. J Neuroimmune Pharmacol. 14 BPRC Annual Report 2011 6. 7. 8. 9. Jagessar, S. A., N. Heijmans, J. Bauer, E. L. A. Blezer, J. D. Laman, N. Hellings, and B. A. ‘t Hart. 2012. B-cell depletion abrogates antibody nondependent CNS demyelination in the marmoset EAE model. J. Immunol. resubmitted. Vierboom, M., E. Breedveld, and B. A. t Hart. 2012. New drug discovery strategies for rheumatoid arthritis: a niche for nonhuman primate models to address systemic complications in inflammatory arthritis. Expert opinion on drug discovery 7: 315-325. Verhave, P. S., M. J. Jongsma, R. M. Van Den Berg, R. A. Vanwersch, A. B. Smit, and I. H. Philippens. 2012. Neuroprotective effects of riluzole in early phase Parkinson’s disease on clinically relevant parameters in the marmoset MPTP model. Neuropharmacology 62: 1700-1707. Verhave, P. S., M. J. Jongsma, R. M. Van den Berg, J. C. Vis, R. A. Vanwersch, A. B. Smit, E. J. Van Someren, and I. H. Philippens. 2011. REM sleep behavior disorder in the marmoset MPTP model of early Parkinson disease. Sleep 34: 1119-1125. BPRC Annual Report 2011 15 Department of Parasitology With a portfolio continued in 2011 to focus on diseases of poverty, i.e. malaria and tuberculosis, the department continued its efforts to make an impact on vaccine and drug development for these important human health problems. Apart from preclinical and even early clinical evaluation, being the red thread through our research , we aim to understand the infectious agent biology and its interaction with the host, as a basis for the rational design of better vaccines and understanding disease mechanisms. The initial focus of the malaria programme on Plasmodium falciparum vaccine development has in the last few years expanded to include P. vivax malaria drug development as well as studies with an important zoonosis, the primate malaria P. knowlesi.The focus for P. falciparum remained on the development of vaccines, especially the in-house developed blood stage vaccine candidate PfAMA1. The focus for P. vivax has been on the development of new drugs against the liver stages and for P. knowlesi on the studies of parasite-host interactions exploiting systems biology approaches. The development of PfAMA1 as a malaria blood stage candidate vaccine antigen has entered a critical phase. Pilot studies for clinical grade (GMP) production of three artificial variant AMA1 proteins, developed and patented by BPRC (the DiCo proteins), were completed in 2010 and the proteins were produced in 2011. We have further developed assays that will be needed for the Phase Ia/b clinical testing, expected to begin in 2012. These assays comprise a quantitative DiCo-specific ELISA, that allows the measurement of specific immune responses to each of the three proteins and a potency assay for evaluating the vaccine quality over time. Furthermore, adjuvant selection studies in mice and rabbits were completed. Adjuvants, two of which were very potent in our studies, are currently being selected for the clinical trial. Finally, one of our African PhD students completed his PhD thesis on PfAMA1 polymorphism and the impact on vaccine development. 16 BPRC Annual Report 2011 For P. vivax drug development we currently focus on the liver stages of the parasite. Liver stages are the first developmental stages of malaria parasites in the human body, following a bite of an infected mosquito. The liver stages develop without any disease symptoms in 6-9 days to very large numbers of parasites within the infected liver cell. These parasites are released into the blood stream and then cyclically infect red blood cells, giving rise to the classical malaria disease symptoms. P. vivax has a unique characteristic, the formation of dormant stages (hypnozoites) in the liver that can re-activate through unknown mechanisms. Hypnozoites thus give rise to a new blood stage infection without being bitten again by infected mosquitoes. This complicates treatment and future eradication of malaria tremendously and new drugs to target hypnozoites are urgently needed. Only in two of the human and some primate malarias hypnozoites are formed. We have developed a platform for in vitro culture of liver stages, including hypnozoites, of the primate malaria P. cynomolgi. We have continued to use this in vitro platform in 2011 to screen the in vitro activity of new compounds against hypnozoites. One of the identified active compounds was further optimized in 2011 and a lead candidate was selected. In vivo activity of this lead will be investigated in 2012 using the gold standard P. cynomolgi sporozoite-infected rhesus monkey model. This will be the first in vivo evaluation of a lead compound that has been identified in an in vitro hypnozoite assay without using an in vivo screen. This has meant a substantial reduction of primate use for this type of drug discovery programs. Novel research lines focussed in 2011 on parasite-host interactions, exploiting our knowledge of the P. knowlesi-rhesus monkey model. As we had previously adapted P. knowlesi blood stage parasites to long-term in vitro growth, we are in an excellent position to compare in vitro developing parasites versus parasites grown under increasing immune pressure in the host. BPRC Annual Report 2011 17 Department of Parasitology We have sampled in vitro grown parasites and in vivo-derived parasites at different time points and extracted RNA from all parasite samples. This RNA is currently being sequenced. In addition, we harvested white blood cells from two monkeys over two rounds of infection with P. knowlesi and extracted RNA for sequencing. Analysis and modelling of the sequence data of host and parasite will begin in 2012. Another line focussed on methods to develop in vitro culture of P. vivax blood stage parasites. This parasite needs very young red blood cells, reticulocytes, for growth and in collaboration we are working to establish in vitro production of reticulocytes from precursor cells. Currently we have shown that when we express the Duffy blood group antigen (the reticulocyte receptor for P. vivax and also for P. knowlesi) on the surface of precursor cells, P. knowlesi is able to bind to the precursor cells, whereas no binding is seen with non-transfected parent precursor cells. This promising first step in red cell invasion and parasite growth will be followed up in 2012. Finally, to create marker parasites for our in vitro hypnozoite studies we have generated transgenic P. cynomolgi that express two fluorescent marker proteins in all stages of their life cycle. We have also shown that we can purify hypnozoites from in vitro cultures by flow cytometry, which we plan to follow up in 2012 for further biological studies on hypnozoites. In recent years, tuberculosis (TB) research within the department of Parasitology was focussed on the preclinical evaluation of new TB vaccine candidates. In 2011 we have extended our efforts mostly to retrospective analyses in particular of the protective effects of standard BCG control vaccination groups. Via a comparative analysis of (partially) protected phenotypes induced by BCG versus the non-vaccinated controls we could identify clinical surrogates that can be used for accellerated and refined, longitudinal and quantitative 18 BPRC Annual Report 2011 assessment of TB disease and vaccine effects in our NHP-TB models. In the retrospective analysis we have also investigated immune correlates of the variable BCG efficacy in several studies at BPRC over the last few years. The results hinted towards distinctive kinetics in the induction of T cell immunity, assayed by the antigen specific induction of IFNg and IL2, determining the efficacy or failure of BCG vaccination in the rhesus monkey model.The variable BCG efficacy in this model provides great research potential and is setting the stage for our NHP-TB activities in the nearby future, aiming at the identification of mechanisms of (vaccine induced) protection against TB. Proned by the definition of clinical surrogates in rhesus monkey TB we have pioneered the common marmoset (Callithrix jacchus) TB model. The experiment was designed to establish the dose and time relationship of M. tuberculosis infection in these animals; the evaluation is still ongoing. A marmoset model would hold promise for TB drug and/or combination therapy studies with the perspective to fill a critical gap in the translation from in vitro via in vivo drug testing in lower vertebrates (mostly rodents) towards clinical evaluation. In our continuous effort to improve vaccination regimes for clinical application we have been able to demonstrate the effectivity of recombinant adenovirusvectored immunisation in combined heterologous prime-boosting regimes using different adenoviral vectors, which were either or not combined with adjuvant formulated protein immunisation. We used a lead candidate for pre-erythrocytic malaria vaccination for immunological read out. In particular, protein in adjuvant priming followed by heterologous adenoviral vector boosting provided enhanced the kinetics of the humoral antigen specific immune responses. BPRC Annual Report 2011 19 Department of Virology Infections caused by a variety of emerging, and re-emerging pathogens, such as Mycobacterium tuberculosis, Vibrio cholera, helminths, influenza viruses, severe acute respiratory syndrome (SARS), coronavirus, human immunodeficiency virus (HIV) and hepatitis C virus (HCV), pose severe health problems to the world’s population. These health threaths are particularly serious for people living in developing countries where hygiene can be poor and in people with conditions compromising immunity. HIV and HCV viruses, for instance, have infected approximately 40 and 170 million people, respectively, worldwide and their prevalence is expected to rise globally. Most infected individuals have subclinical symptoms, are unaware that they are infected, and will therefore continue to transmit the virus to other people. Although some (relatively expensive) anti-viral drugs are available which can slow diseases caused by HIV or HCV infection, they have not been able to stop the epidemic.Vaccination, which aim at enhancing our immune system’s capacity to combat infections, appears a promising and much-needed approach for the treatment and prevention of these diseases. To develop vaccines, detailed knowledge of immunological and pathological mechanisms is of the utmost importance. For several years the Department of Virology has taken a multidisciplinary approach by developing expertise in cellular, humoral and mucosal immunology, immunopathology, and molecular virology. These skills have been applied to rational immunogen design and pre-clinical evaluation of a number of leading HIV and HCV vaccine candidates. In the area of HIV vaccine development we are currently concentrating on exploring the beneficial effect of different vaccine modalities in a prime-boost strategy using combinations of DNA as the priming immunization followed by poxviruses or smaller peptide particles (synthetic long peptides: SLP) as a booster regimen. The poxviruses (different NYVAC vectors) were developed within a consortium funded by the EU (EuroVac) and a consortium funded by the Bill and Melinda Gates Foundation (BMGF). The BPRC was a partner in both consortia. The SLP as vaccine principle has been developed by the Leiden University Medical Center (Prof. C. Melief) and was successfully applied in a clinical trial in patients with cervical cancers. We tested this vaccine modality 20 BPRC Annual Report 2011 (against HIV) for its effectivity in a SIV infection model (Koopman et al, in preparation). Furthermore as part of a consortium (a fruitful collaboration that has been on-going for the last 10 years) funded by the United States National Institutes of Health (NIH)) we have been trying to establish vaccine strategies aimed at inducing protective humoral immunity against HIV. In 2011 we immunized macaques with recombinant envelope glycoproteins, which had been triggered/changed into the configuration that they adopt after first binding to the cell surface. This conformation exposes parts of the molecule that are engaged in the next stage of infection.These structures are shared by most HIV1 isolates. We showed that the immunizations did induce antibodies to these novel structures. However, the macaques were not protected against repeated challenge with very low doses of a SHIV, although the used virus was relatively sensitive to neutralization. Other strategies to combat HIV infection have been explored as well. Recently, we exploited the concept of using autologous apoptotic cells for homologous gene transfer to introduce HIV genes into the host for vaccination purposes. With this method vaccine antigens can be expressed over extended time intervals, presented by professional antigen presenting cells, which should result in the induction of more effective anti-HIV responses (Koopman et al., Vaccine, 2012 Feb 5). Within the scope of the 3 R’s (Reduction, Refinement, Replacement), we recently published a study in which we have characterized our macaque population for specific HIV protective alleles:TRIM5 allelic polymorphism in species/populations of different geographic origins (de Groot et al., 2011).Armed with this knowledge we can now better select suitable animals for vaccine efficacy studies against HIV by avoiding animals with “protective” alleles, thereby contributing to the Refinement/Reduction in animal experimentation. In line with this, we identified, together with collaborators from several European primate centres (as part of the European funded EUROPRISE network: SIVNeutNet activities), some macaque serum samples and mAbs with neutralizing activity that could serve as useful reference reagents. Together with these partners we have performed various neutralization assays and have established and identified some samples BPRC Annual Report 2011 21 Department of Virology that could act as neutralization reference reagents for future SIV studies. All these initiatives will eventually result in a further reduction of the number of experimental animals and in standardization of experiments. HCV research is hampered by the inability of the virus to survive and replicate in cell culture. Besides humans the only animal species that can be infected with HCV is the chimpanzee. Because the use of chimpanzees for biomedical research has been banned in Europe, the Department of Virology is exploring an alternative model of HCV infection, the GBV-B virus infection in marmosets. This virus, originating from tamarins (also a South-American primate species) is similar to HCV, and causes hepatitis in tamarins and marmosets. We are setting up a cell culture technique for this virus, which can be used for research of certain aspects in the life cycle of the virus in vitro. Furthermore, the department is investigating if the GBV-B marmoset infection model is suitable for developing and evaluating different vaccination strategies against HCV. Fundamental research in the last couple of years has been, and still is, aimed at understanding vaccine induced immunity to HIV and HCV, and the elucidation of those specific host immune responses in individuals that are able to control or eliminate virus infection (i.e. HCV). Identification of (a) correlate(s) of protection would be a major benefit to the development of an HIV vaccine. However, the best way to identify such correlates is from the results of a successful human efficacy trial. The BPRC can now offer an in vitro assay, which correlates with protection following simian-human immunodeficiency virus challenge of rhesus macaques. Antibodies, which come closest to the traditional definition of neutralization, are protective at doses that can be induced in the monkeys by vaccination. The antibodies are able to protect against a relatively high dose of virus as long as it is sensitive to neutralization. Against virus, which is more resistant, protection can also be seen but this requires doses of antibody, which can only be provided by passive transfer. However, at physiological concentrations the antibody may be able to protect against low doses of the resistant virus (Davis et al. 2011). Furthermore, new assays have been developed to characterize and quantify other immune interactions correlating with protection. A dendritic cell-T cell co-culture system has been 22 BPRC Annual Report 2011 developed to improve the screening of new vaccine delivery systems as well as new antiviral therapies (Tel et al., 2012 in press). In this way we will contribute to Reduction of the need for experimentation in life animals. Besides the HIV work, much effort has been invested on materials collected in the past from previous HCV infection studies in chimpanzees. We have looked for correlates of protection using cells, which have already been stored for many years. Here, we have also found that some correlates are of importance and might be useful for design of future human trials (Verstrepen et al, submitted). Over recent years much work has been dedicated to the development of diagnostic assays for primate viral infections and the characterization of novel primate viruses. Primate polyomaviruses are a relatively new research topic. In humans, several polyomaviruses have been described that can cause serious disease in immunocompromised hosts, JCV and BKV for example. Three new viruses have been discovered in the last few years (WU, KI, and MePyV). Because of their likely impact on colony health, and their potential threat to humans, we have initiated the development of several new diagnostic tools. These tools have enabled us to characterize new viruses from chimpanzees (Deuzing et al., 2010), orangutans (Groenewoud et al., 2010), macaques (Dijkman et al., 2009; Fagrouch et al., 2011), and squirrel monkeys. Immediately linked to diagnostics is fundamental virology research regarding the spread and the evolution of specific viruses, such as retroviruses, herpesviruses and polyomaviruses. The available diagnostic tools have provided the BPRC with the opportunity to set up breeding colonies of specific-pathogen free (SPF) animals. In general, this means that our animals are free of several retroviruses, like SIV, STLV and SRV, and of herpes B virus. Via our Primate Viral Diagnostic unit, diagnostic services are provided to third parties all over Europe, including zoos, research centres etc. and to animal rehabilitation centres in Africa, Asia and South-America. The number of requests as well as the numbers of third parties is increasing every year. PhD theses: Erik D.I. Rutjens: Differences in cellular immunity between humans and chimpanzees in relation to their resistance to AIDS. University Leiden. February 3, 2011 BPRC Annual Report 2011 23 Department of Comparative Genetics and Refinement Next-generation sequencing of receptors of the immune system The immune system is essential for fighting pathogens, controlling the growth of malignant cells, and by necessity is tightly regulated to prevent autoimmunity. Cellular receptors that mediate these diverse immune functions are encoded by genes. The differences in the immune response between individuals can in part be attributed to variation (polymorphism) within the genetic code (DNA) of these genes. Relevant non-human primate immune receptor genes that are being studied include those of the major histocompatibility complex (MHC) class I and II (1, 2), killer cell immunoglobulin-like receptor (KIR) (3), and tripartite motif-containing protein 5 (TRIM5) genes (4). For all these polymorphic genes it is known that particular variants influence the outcome of infection and disease in non-human primates. The inherited variation of these genes – genotype – is determined by examining the genetic code of individuals. This information is analyzed to see if it can be linked to the functional characteristics – phenotype – of the immune system. The genetic information of these genes is read by a technique known as DNA sequencing. In recent years, this technology has rapidly evolved, to the point where so called next-generation sequencing (NGS) is now becoming the gold-standard for any in-depth genetic study. NGS yields vast amounts of genetic data in a cost-effective manner, resulting in an emphasis-shift from datagathering to data-analysis. This NGS technology has been adapted in house to perform comparative immunogenetic studies on the macaque MHC class I, II, and KIR genes. A major challenge in the field is to use NGS to accurately determine the variation of particular genes in large groups of individuals and subsequently trace this information back to the correct sample. At the department of Comparative Genetics and Refinement an innovative methodology has been developed and 24 BPRC Annual Report 2011 implemented that allows for exactly this feat.This offers the advantage that many animals from a colony can be rapidly screened for variation of target genes. In addition to expanding the knowledge of complex gene systems that have previously been studied by first-generation sequencing, the opportunity now arises to explore the diversity of new families of relevant immune receptors. References 1. Otting, N., A. Morner, and R. E. Bontrop. 2011. Novel major histocompatibility complex class I alleles extracted from two rhesus macaque populations. Tissue Antigens 77: 79-80. 2. Doxiadis, G. G., N. de Groot, N. Otting, J. H. Blokhuis, and R. E. Bontrop. 2011. Genomic plasticity of the MHC class I A region in rhesus macaques: extensive haplotype diversity at the population level as revealed by microsatellites. Immunogenetics 63: 73-83. 3. Blokhuis, J. H., M. K. van der Wiel, G. G. Doxiadis, and R. E. Bontrop. 2011. The extreme plasticity of killer cell Ig-like receptor (KIR) haplotypes differentiates rhesus macaques from humans. Eur J Immunol 41: 2719-2728. 4. de Groot, N. G., C. M. Heijmans, G. Koopman, E. J. Verschoor, W. M. Bogers, and R. E. Bontrop. 2011. TRIM5 allelic polymorphism in macaque species/ populations of different geographic origins: its impact on SIV vaccine studies. Tissue Antigens 78: 256-262. PhD theses: Jeroen H. Blokhuis: Complexity and evolution of KIR genes in rhesus macaques. University Utrecht. June 22, 2011 BPRC Annual Report 2011 25 Department of Facilities and Support Mission almost completed In the contribution of the 2010 Annual Report our ambition was expressed enter the final term of eleven years construction and renovation activities. This to have BPRC’s Mission to achieve its home-make-over completed at the will concern the furnishing and redecoration of the “landscape” around and end of 2011. However, reality forces now a rephrasing of this ambition as between the buildings SG, KL and PG. A parking place will be realized at the “Mission almost completed”. In the spring of 2011, the whole Animal Science entrance of the BPRC premises. Only cars of suppliers, service and immobilized department staff was moved into their new housing in building PG (former 157) people will be allowed to enter the area between the building KL, SG and PG. and the animal housing on the first floor of the PG building was completed. The inside area, as mentioned before, will be the area for pedestrians. Bikers The renovated animal housing on the second floor is now ready for use. As is will get their facility under building PG. In conclusion, we expect that in 2012 all always the case with construction activities, delays can rarely be prevented.The building activities of the BPRC will be finished. housing of the animals in building PG will be realized by the end of October 2011. Despite this slight delay, the new BPRC will be realized soon. During all these activities the outside wall cover of the whole PG building has been renewed. The air control units inside the technical facility on the third floor have been changed and adapted for their new purpose. At the time this message was written, December 2011, the technicians are finishing the renovation works of the old computerized systems for building control (20 years old). The old radiological facilities in building 139 were cleaned and the responsibility for these facilities was returned to TNO. The old gamma source that was used for irradiation of cells has been handed over to the COVRA for waste storage and the new C-lab facility for radiological work came into use. An X-ray generator type RS2000 was purchased for the irradiation of cells used in in vitro experiments. In November we started the demolishing of the buildings 7 to 9a. This will take several months and in the end March 2012 the buildings, we expect, will be completely vanished. At that moment we will 26 BPRC Annual Report 2011 BPRC Annual Report 2011 27 Department of Finance The income realized in 2011 approximately equals to the revenues in 2010. A slight increase in costs causes that the break-even result is not achieved in 2011. A reduction in the human capacity, inspired by the portfolio, caused a reduction of salable hours. In addition, a limited indexation occurred for the basic funding. Staff The number of employees decreased by 2.7% compared to 2010. The turnover per employee increased by 2.5% compared to 2010. The BPRC Financial Annual Report for 2011 has been audited and approved by our external accountant. Result development BPRC 2002 - 2011 450 389 All figures x Euro 1.000 400 350 300 284 250 313 210 200 150 100 50 0 81 21 2002 10 2003 2004 2005 2006 Year 28 BPRC Annual Report 2011 2007 37 2008 2009 60 2010 -76 2011 BPRC Annual Report 2011 29