Download Annual Report 2011 - Biomedical Primate Research Centre

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.
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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
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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
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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
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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
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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).
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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
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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.
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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
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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
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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
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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).
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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.
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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.
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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.
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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.
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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
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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.
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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
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(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
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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
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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
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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
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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
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BPRC Annual Report 2011
2007
37
2008
2009
60
2010
-76
2011
BPRC Annual Report 2011
29