Letter from the Chairman
of the Supervisory Board

Health and public health issues remain highly relevant for

Basic studies on the immunological system remain necessary to make

society. Consequently, containment and/or prevention of

optimal use of the animal models. Furthermore, animal behaviour studies

diseases are of utmost importance. Research at BPRC

are essential to improve animal care and welfare at BPRC, a very important

continues to focus on those health-related problems which

part of the quality of the research.

are difficult to address without the use of non-human

BPRC remains open to the public at large. Being a knowledge institute

primates. On the other hand studies to explore suitable alternatives

it is a prerequisite that the way research is being performed is shared

are also part of the research programme. This annual report offers a

with society.

thorough in depth view on the efforts made by BPRC to unravel suitable

The Supervisory Board is well aware of the professional attitude of

solutions for a number of diseases. Apart from the major infectious

everybody at BPRC which makes it possible to continue the urgently

diseases, including malaria, tuberculosis and HIV/AIDS, emerging virus

needed studies in the interest of society.The annual report is recommended

diseases with a threat to human and veterinary public health, like Dengue

to the general public, but also to professionals in health sciences and to

virus and West Nile virus are studied as well. In the light of the ongoing

policy makers who are offered a well balanced look into a research area

risk of seasonal epidemics by influenza viruses interesting research on the

with a great potential.

development of a new vaccine concept has been performed. Chronic
diseases like multiple sclerosis and rheumatoid arthritis continue to be a
challenge to researchers. Non-human primates offer very valid models to

Professor Joost Ruitenberg

understand the dynamics of the disease processes and to suggest options

Chairman of the Supervisory Board

for treatment.

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3

Message from the Director

Dutch law defines that biomedical research with animals

The relevance of BPRC’s research efforts is highlighted by the fact that our

is not allowed if alternative methods are available. Non-

staff members published 51 manuscripts in peer-reviewed journals, with an

human primates are essential in biomedical research for an

average impact factor of 6.1. Many of these papers were published in high

array of infectious and chronic diseases that are a burden

impact journals. For an overview, please consult the news section of our

to mankind, due to the fact that alternative methods

website (www.bprc.nl).

are not available. For instance, in the case of diseases like Malaria, AIDS,
Tuberculosis, Multiple sclerosis and Alzheimer there is a desperate need
for the development of safe and effective vaccines, therapies or medicines.
The immune system of humans and non-human primates share many
similarities. As a consequence, many new medicines or vaccine candidates
can only be tested for safety and efficacy in a proper non-human primate
model, as those components are not reactive, for instance, in rodent species.
Next to that, BPRC has a reputed research line based on the so called 3Rs
principle (Reduction, Replacement and Refinement), which is in line with
our societal Responsibility (fourth R). BPRC also puts a lot of effort in

As can be seen later in the report, the year 2013 was closed with a small

behavioural research in collaboration with the University of Utrecht.

positive financial result. Finally, I would like to thank all the BPRC employees,

In concert with our high research standards, the BPRC continues to

but also all the other dedicated persons, who have helped us to maintain

improve its animal welfare issues, and much effort has been put in training

and improve BPRC’s research and animal welfare standards.

animals. All animals are housed in pairs or larger social configurations in

4

large indoor and/or outdoor facilities matching the best standards. The

Prof. dr. Ronald Bontrop

BPRC breeds its own animals and has become independent of importation.

Director BPRC

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5

Ethology

Aim of the ethological research

Like humans, primates are highly social animals.

Why study non-human primates

Since primates are our closest living relatives, insight in their social behavior and

They spend their whole life in a group and their

intelligence provides crucial knowledge for understanding the evolution of human

social behavior is varied and complex. Studies

sociality and intelligence. In addition, such knowledge is crucial to the improvement

comparing different species indicate that social

of primate husbandry and housing in all captive settings.

complexity has selected for relatively large brains.
Moreover, a relatively large brain size has been connected to animal intelligence.
However, how smart primates actually are and how they employ these capacities

Results in 2013

The output concerns scientific papers and reports in the popular press on primate

in their behavior is still an open question. The ethology research at the BPRC aims

social strategies, primate smartness and primate communication.

to address these questions with observational studies and non-invasive behavioral

The social complexity of primate groups is evident in the differentiated

experiments.

relationships that members of primate groups maintain. Relationships with kin and
friends involve by high rates of proximity and grooming. However, whether these

Contribution to knowledge

6

relationships are actively maintained by the animals or are only side-effects of

The smartness of primates and their relationship to social behavior is explored in

dominance interactions is unclear. We found that macaques maintain relationships

a research program that studies monkey social behavior and primate intelligence.

over a long period. The longest stable relationship lasted at least 3.5 years. This

Humans are part of the so-called ape lineage, that also includes apes like

is consistent with the idea that macaques maintain their social relationships by

chimpanzees and gorillas. Less closely related to humans are monkeys, including

seeking particular social partners. Moreover, while dominant males would like to

baboons and macaques. The least related are prosimians, such as lemurs from

prevent subordinate males from mating, these subordinate males and also the

Madagascar and nocturnal bush babies from Africa. It has been proposed that apes

females in the group seek mating opportunities. We showed that they enhance

(e.g. chimpanzees) are smarter than monkeys (e.g. macaques) and that these are

their options to mate, by showing sneaky mating behavior when dominant males

(again) smarter than prosimians. However, while apes are studied at numerous

are absent.

locations, research on the intelligence of monkeys is relatively scarce. Therefore,

Humans can not only predict how others will behave, but also what others

the lack of evidence on monkey-intelligence may also be due to the relative

know. While at other institutes behavioral experiments with chimpanzees showed

small research effort. Moreover, the evolution of human capacities can only be

that they understand that another chimp can see them, monkeys failed at such tasks,

understood when also monkeys form part of the picture.

indicating that apes are indeed smarter than monkeys. Our research counters this

At the BPRC, this research focuses on monkeys, in particular on macaques. We

idea, by showing that macaques, a monkey species, do know whether a dominant

study their social interactions and their social communication.

can or cannot see food and adjust their behaviour accordingly.

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7

Ethology

Animals communicate through facial expressions and vocalizations. We established

for these parasites. However, the administration of metronidazole in drinking

in common marmosets the meaning of several facial expressions. These facial

water was not successful in rhesus monkeys due to low fluid intake attributable

signals can invoke friendly behavior and animals can signal stress and fear resulting

to unaccepted taste. Therefore another anti-amoebic medicine, clioquinol, was

in a higher rate of affiliative behaviour. We could demonstrate that despite the

studied. Together with the Department of Pharmacy of the Veterinary Faculty in

fact that they live in small groups in trees, common marmosets use several visual

Utrecht a method was developed to dissolve this drug, which is not soluble in

expressions to communicate among each other.

water.This medicated solution was well taken by the animals. Currently, the efficacy

Furthermore, research on primate vocalizations in collaboration with the Utrecht

of this treatment in naturally infected animals is being evaluated. Other research

University showed that wild and zoo-housed orang-utans have flexibility in their

parts included improved methods to allow acquisition of data without the need to

call production. These apes can learn a new sound, namely to whistle, and can

handle the animals, such as by using biotelemetry.

modify their sounds by using their hand or leaves. This indicates that some of the

To monitor inflammation in macaques more efficiently, we analysed together

flexibility that is typical for human language is also present in apes.

with the Department of Veterinary Clinical and Animal Science, University of

The research has received attention in the popular press. In particular the sneaky

Copenhagen, various methods to determine serum proteins and to correlate

mating behaviour in macaques and the signals in common marmosets were high-

them with ongoing inflammatory events. Early detection of these compounds can

lighted.

be used to monitor the animals and provide early treatment when necessary.

Animal Science

Animal Training and Enrichment

optimal care for the animals and support for the research groups. In addition, the

BPRC investigators have implemented various animal training programs. In these

ASD plays an important role in the continuous further improvement of animal

programs the animals are trained to voluntarily cooperate within the specific

care and welfare at the BPRC.

studies. As partner within the EU-subsidised EUPRIM-Net program, various

The main task of the animal science group is the provision of husbandry and

To improve sampling using methods that disturb the animals as little as possible,

lectures have been prepared for training animal caretakers and active training

8

Veterinary research

programs are in place. The BPRC considers training and continuous improvement

The veterinary group is involved in research to improve veterinary methods and

of the training methods essential.

increase animal welfare. Housing of animals in open settings, such as done at the

In 2013 we studied the impact of various forms of enrichment in the experimental

BPRC, also increases the risk of infections, e.g. with parasites such as Entamoeba spp.

facilities on the animals. The aim of this study was to provide evidence-based

and Giardia. These parasites can cause severe diarrhoea and treatment is then

selection of the most optimal enrichment for these animals. The data are currently

mandatory. In veterinary medicine, metronidazole is the most common treatment

analysed.

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Alternatives for research in animals

Next to research for the benefit of human health, the second focus of research
at the BPRC is the development of alternatives for animal experiments. These
alternatives are developed along the guidelines of the three R’s of Refinement,
Reduction and Replacement. Efforts within the BPRC are organized at multiple
levels. The three R’s are deeply embedded in the Animal Science Department
and all research departments educate a minimum of one PhD student who
will work on Alternatives. In addition, there is a separate Unit that is dedicated
to the development of Alternatives for animal experiments.
Organotypic and cell culture methods
A straightforward alternative for performing animal experiments is to try to
step-by-step replace them by methods that can be performed in laboratory
dishes (in vitro). Such in vitro methods can try to model an entire organ or can
try to model specific cell types. At the BPRC we are setting up methods to
model the liver, skin and brain. These organotypic culture methods are started
from tissue material from deceased animals that would otherwise have been
discarded. The material is carefully prepared, sliced and put into plastic culture
dishes where it can be used for experiments.
In addition, we use the tissue to start primary cell cultures. Cultures of primary
cells are much closer to normal cells than the more often used cell lines or
tumour cells that can be kept in culture indefinitely. Primary cell cultures have
a limited life span and should be started over and over again. Not all cell types
can be easily cultivated in lab dishes, and in particular cells of organs like the
brain are difficult to cultivate. Within the Unit Alternatives a large variety of
primary cell culture methods has been developed and characterized. These
methods enable researchers to test their research questions on relevant cell
types before performing animal experiments. Such a pre in vivo test phase
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leads to a considerable reduction in the number of animal experiments. It
is important to note that organotypic and primary cell cultures are always
initiated from ‘rest material’ derived from donor animals that were euthanized
as a result of e.g. other experiments. Thereby the end of one experiment is
the beginning of another.

Last year new data with particular relevance for neuroinflammation were
obtained demonstrating that cells from the brain respond differently than their
counterparts in the blood. We have not only demonstrated that they respond
differently, but also describe why they respond differently. Such insights are
important when clinical therapies are developed that should work in the brain.
In addition, a series of pilot experiments were performed to set up protocols
for the cultivation of stem cells. Stem cells can be kept alive indefinitely and

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11

Alternatives for research in animals

can, in theory, be transformed into any cell type from the body by providing
them with the right stimuli at the right time. This would open additional in vitro
possibilities, especially for cell types like neurons that we are currently unable
to grow as primary cells.
Refinement of experimental methods
Much Refinement is achieved by the Animal Science Department in the
housing, training and daily lives of the animals. Refinement can also be achieved
by optimisation of experimental procedures to reduce animal discomfort. One
line of research with the Unit Alternatives aims to refine the use of adjuvants.
Adjuvants are additives that are used in vaccines to enhance immune responses
directed against pathogens or in experimental animal models for e.g. human
autoimmune diseases. However, many potent adjuvants cause adverse effects.
Most notable is the development of granulomatous skin lesions, causing various
degrees of discomfort to non-human primates in biomedical experiments.
There is therefore an urgent need for new and better adjuvants.
Recently it has become clear that the immune-enhancing effects of adjuvants
can be attributed to activation of receptors of the innate immune system.
Based on this knowledge we have engineered a series of luminescent cell lines
and used these bioassays to screen new adjuvant candidates for their immuneenhancing effects. In addition, we have developed a 3D in vitro granuloma assay
to screen adjuvant candidates for their potential to cause adverse effects.
Combining these assays as a robust integrated test system has generated
detailed information on a number of new candidate adjuvants. Last year we
have finalised these in vitro screenings and we have selected several candidate
adjuvants that will be tested in experimental models next year.

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BPRC Annual Report 2013

Communication, transparency and discussion
To enhance transparency the BPRC annually publishes a report on the use of
experimental animals in the facility (www.bprc.nl/download/BPRC-PDKJ2012.
pdf). Next to activities within the BPRC that are aimed at animal welfare, BPRC
employees are also active in national (www.nkca.nl) and international initiatives
(Expert working group ‘Non-human primates in monoclonal antibody studies’
organized by the UK national centre for 3Rs (www.nc3rs.org.uk/) to stimulate
the use of alternatives.

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13

Immunogenetics

The MHC in humans
It is well known that recipients of an organ transplant may reject the organ. To
reduce the risk of this negative response, recipients and the candidate donors
are tissue-typed, and the patient who matches best receives the available organ.
In fact,tissue typing involves testing for the proteins of the Major Histocompatibility
Complex (MHC), which in humans is named Human Leukocyte Antigen (HLA).
MHC proteins are present on the cell-surface of particular body cells. The
genes that encode these proteins are highly polymorphic, which means that
they vary widely within the population. The MHC has not evolved to hamper
the artificial process of organ transplantation. In the immune response, MHC
proteins play a pivotal role in the recognition of foreign structures such as
bacteria and viruses. The MHC proteins bind fragmented particles of microbial
antigens, and present them to T lymphocytes, leading to specific immunological
defence reactions. Particular variants of MHC genes (alleles) have been shown
to be associated with susceptibility or resistance to infectious or autoimmune
diseases.
MHC-typing in non-human primates
Since non-human primates are used as animal models to study immune-related
diseases, their MHC genes are thoroughly investigated within the Department
of Comparative Genetics and Refinement, with the aim of selecting the most
suitable animals for certain types of investigations. Typing is based on DNA
sequencing of the MHC genes. DNA, the chemical substance of which genes
are composed, is a long string-like molecule formed by series of four different
bases. The sequence of these bases in a particular MHC gene determines the
structure of the encoded protein on the cell-surface, and hence the capacity to
bind antigens and generate immunological defence reactions.
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Recent investigations
Considerable research has been carried out in genetically characterizing the
breeding colony of rhesus macaques at the BPRC. Almost 1400 animals have
been tested for their most significant MHC genes, and 140 different haplotypes
have been distinguished. A haplotype is defined as a combination of MHC
genes on one chromosome, which are usually inherited “en bloc”. An individual
has two haplotypes, each inherited from one of the parents. All the data have
been stored in a database, which minimises the need to obtain samples of
blood or tissue from the animals.
MHC genes vary within the population, and polymorphisms are observed
mostly in the part of the gene that encodes the antigen-binding region of the
protein. The antigen-binding motifs for a few rhesus monkey genes have been
explored, with the aim of contributing to a better understanding of T-cellmediated immune responses.
The Department has recently started to investigate the MHC of the common
marmoset (Callithrix jacchus). Some functional aspects of the MHC in these
animals appear to differ substantially from those in humans. In humans the
HLA-A, -B, and C proteins perform the classical antigen presentation function
(activation of the immune defence), whereas HLA-G is expressed only in the
placenta.This gene is thought to play an important role in protecting the foetus
from potential hostile immune responses from the mother, directed against
MHC polymorphisms inherited from the father. In the common marmoset, the
most profound antigen presenting MHC protein is encoded by a gene, which
is structurally related to HLA-G.These results demonstrate the plasticity of the
MHC in primates.

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Multiple sclerosis

What is MS?
Multiple sclerosis (MS) is a progressive neurological disease that affects
exclusively the central nervous system, comprising brain and spinal cord
(CNS). The cause of the disease is not known, but it is widely believed that in
individuals with genetic susceptibility to MS, infection with certain viruses or
bacteria triggers a complex pattern of immunological reactions against the CNS.
The primary target of this attack is the myelin sheath, being a multi-lamellar
structure that winds around nerve tracks (axons), produced by specialized
cells of the CNS, called oligodendrocytes, (Figure 1). An intact myelin sheath
protects axons against damage and ensures a rapid conduction of electrical
pulses from nerve cells to the organ or tissue on which they project. The
sheath contains multiple proteins, each with a different function, each of which
can be a target of the immune attack. The focal infiltration of immune cells,
such as B and T cells or macrophages, and immune factors, such as antibodies,
from the blood into the CNS causes inflammation and destruction of myelin
(demyelination); such demyelinated areas are called lesions. In the early phase
of the disease inflammation can be suppressed by counter-regulatory immune
mechanisms. Demyelination can be stopped and reversed by the formation
of new myelin (re-myelination). The episodic flare and waning of immune
activity underlies the typical relapsing-remitting (RR) course of early MS, where
episodes of neurological function loss (relapse) alternate with recovery. Later
in the disease recovery becomes incomplete and ultimately disappears, i.e.
secondary progressive MS (SPMS). In a minority of patients (±15%) the disease
is progressive from the onset, i.e. primary progressive MS. The reason of the
transition from RR to SP MS is unknown and thus subject of intensive research.

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Figure 1: The lower part of the figure depicts a neuron (the stellate cell on the right) with its
extension (axon), which is enwrapped by bands of myelin interspaced with non-myelinated
nodes of Ranvier. The nodes are essential for fast pulse conduction, which jumps from one node
to the next. The upper part depicts an enlarged segment, showing the layers of myelin in which
a selection of essential proteins is indicated. Note that only myelin oligodendrocyte glycoprotein
(MOG) is exposed on the myelin surface. (figure: Mayer and Meinl, Ther. Adv. Neurol. Dis. 2012)

Our research approach
Intensive research over the past decades has delivered a number of therapeutic
agents with reasonable efficacy in RRMS. However, for progressive MS there
is no effective treatment available. The main reason for this situation, which is
rather disappointing taken the substantial research efforts, is our still very limited
understanding of the factors that underlie the induction and perpetuation of
the disease. In our preclinical research we integrate exploratory research into

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17

Multiple sclerosis

18

disease mechanisms and the causal relation between infection and MS with
applied research into how new therapies work to maximize relevance for the
patient (Figure 2).

The non-human primate EAE model
The impressive biotechnological development in the past decades has created
the possibility to generate biological molecules of high quality and sufficient
quantity for the treatment of patients. With these “biological bullets” cells and
molecules that induce and/or perpetuate the disease can be functionally or
physically eliminated in a highly specific manner that spares tissues and organs
not involved in the disease.
Over the years we have developed well-validated EAE models in the smallbodied common marmoset (Callithrix jacchus), which recapitulate essential
clinical and pathological aspects of relapsing-remitting and of progressive MS.

Figure 2: integration of exploratory and applied research in our preclinical research

Major achievements

The role of animal models in MS research
Despite the many uncertainties, relative consensus exists on the concept that
MS is caused by the interaction of genetic and environmental factors, which
together induce the pathogenic immune reactions. This concept is the basis
of most MS research. For genetic and immunological studies in patients blood
and, albeit more difficult to collect, the cerebrospinal fluid that surrounds
the CNS, are used. However, the CNS itself is such a vulnerable organ that
tissue samples for research cannot be collected without the risk of irreversible
damage.These limitations have created a need for valid animal models in which
clinical and pathological aspects of MS can be investigated and new therapies
can be developed.
The elected animal model in MS research is experimental autoimmune
encephalomyelitis (EAE), which has been established in mice, rats, guinea pigs
and monkeys, which resembles the situation in humans at best.

• Implementation of magnetic resonance imaging (MRI) techniques with
which development of MS-like lesions in the brain could be longitudinally
monitored. The possibility to perform brain MRI has been an essential tool
for assessing effects of a new treatment on existing disease.
• The discovery that the initiation and progression of EAE in marmosets are
driven by distinct pathogenic mechanisms. The specificity, MHC restriction
and mode of action of the T cells that operate in EAE initiation and
progression has been resolved.
• The discovery that virus-infected B cells have a key pathogenic role.
Development of a highly refined EAE model induced with a synthetic
peptide in oil.

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Rheumatoid Arthritis

With a prevalence of 1%, rheumatoid arthritis (RA) is one of the most common
immune-mediated inflammatory diseases in the human population. RA primarily
affects the joints of all extremities and is pathologically characterized by severe
inflammation and progressive destruction of cartilage and subchondral bone.

Severe inflammation of a healthy joint (A); thickening of the synovial membrane and the
involvement of immune cells (B) and subsequent destruction of cartilage and subchondral bone.

In the preclinical research of RA, aiming at a better understanding of disease
mechanisms and development of safe and effective treatments, animal models
have an important role. It is of critical importance that the animal model used
is sensitive to the pharmacological action of the tested drug and that the
tissue distribution and pharmacological properties of the molecules targeted
by the treatment are comparable to those observed in patients. The BPRC
has developed 2 NHP models of inflammatory arthritis: A collagen induced
arthritis (CIA) model in the rhesus monkey and in the common marmoset.

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CIA in the rhesus monkey
With the use of genetically susceptible rhesus monkeys we have developed a
NHP model of inflammatory arthritis. A one-time injection with collagen type
II (CII) in a strong immune-stimulating formulation results in a mild to severe
swelling of the peripheral joints. Onset of clinical arthritis occurs 3 weeks after
the immunization. Frequent physical examination and blood analysis provides
a detailed analysis of the inflammatory response developing in these animals.
The available biomarkers provide quantitative information on: 1) the clinical
status (e.g. disease score, soft tissue swelling of the joints, bodyweight and body
temperature), 2) severity of inflammation (e.g. serum C-reactive protein, IL6, platelets, neutrophils, haematocrit), 3) immune status (serum autoantibody
levels, T-cell reactivities and immune phenotyping) 4) joint pathology (serum
alkaline phosphatase, urinary excretion rate of collagen crosslinks and end-stage
histopathology). The size of the animal allows for frequent blood-sampling for
in depth analysis of immune parameters. The rhesus monkey CIA model has
been validated with clinically relevant therapeutic agents, namely a monoclonal
antibody against the human IL-6 receptor (Tocilizumab) and a fusion protein
CTLA-4Ig (Abatacept).
CIA in the common marmoset
Recently we developed a new CIA model in a New World primate, the
common marmoset. Induction of visible arthritis required immunization with CII
in a strong adjuvant followed by several immunizations with CII in oil emulsion.
This model displays 2 major disease patterns. In about 50% of the animals
clinical signs develop approximately 4 weeks after immunization.The other half
of the animals develops a more chronic disease. Both helper T and cytotoxic T
cells as well as antibody responses (IgM & IgG) against CII were implicated in

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Rheumatoid Arthritis

the development of the disease. At the level of histopathology, tissue analysis
showed less destruction of bone and cartilage compared to the rhesus
monkey CIA model. We also observed unique extra-articular manifestations
of inflammation near the bone outside the joint and subcutaneous tissues in
acute responders to disease induction. Because of the small size of an adult
monkey (± 400 grams) the model is ideal for the efficacy evaluation of a
new drug at early stage development, when only limited amounts of drug are
available. A unique feature of the common marmoset is that they are born
as non-identical twins or triplets with chimeric bone marrows due to fusion
of the placental bloodstreams. This creates the unique situation that the twin
sibling of a monkey in an experimental group can be used as the optimal
control. The model still awaits validation with a clinically relevant therapeutic.
Both models provide an important platform for the evaluation of human specific
therapeutics. These models can also identify the selective impact that a drug
might have on processes that show comparable expression and regulation in
humans and NHP, but are lacking in rodent models because of the evolutionary
distance. The models also provide the opportunity to perform exploratory
research into comorbidities of RA, including anemia and atherosclerosis.
Recent comparison of the MHC genomic regions of the common marmoset
and rhesus monkey revealed presence of MHC alleles that contain the shared
epitope. This is a motif in the peptide binding groove of the DRB1 molecule
that is linked to more severe arthritis, the presence of diagnostic antibodies
against citrullinated proteins and smoking. Especially, a large proportion of the
common marmoset express MHC molecules with this particular motif. This
creates a specific niche exploring the impact of this genetic trait in the disease
course of arthritis.
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Alzheimer & Parkinson’s disease

The increasing prevalence of neurodegenerative diseases is a growing concern
for ageing societies. A highly prevalent age-associated neurodegenerative
disorder is Alzheimer’s disease. At this moment there are about 35 million
patients worldwide and every 2 seconds, this disease affects a new person.
Another major neurodegenerative disorder is Parkinson’s disease. Nowadays
about five million patients have been diagnosed with Parkinson’s disease
worldwide.
Parkinson’s disease (PD) is a motor dysfunction
disorder caused by specific progressive degeneration
of dopamine containing nerve cells (neurons) in the
substantia nigra, a small nucleus in the brain that is involved
in control of movements. This results in a wide range of
motor symptoms, such as slowness of movements, tremor,
rigidity and/or postural instability. At the time the patient
is diagnosed at least 50-60% of the dopamine producing
cells in the brain are already lost.
Alzheimer’s disease (AD) is a brain disease that
slowly affects memory, and the ability to carry out simple
tasks of daily living. About 50-80% of all cases of dementia
are caused by AD. As cells die over time, affected brain
regions begin to shrink. This degeneration seems to be
woven by aggregation of abnormally-folded proteins, such
as amyloid plaques, that are causally linked to cellular
stress and inflammation in the brain.

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BPRC Annual Report 2013

Treatment of neurodegenerative diseases
AD as well as PD are both irreversible, progressive brain diseases for which
no cure exists. Current medications suppress disease symptoms, but do not
affect the progressive degeneration of neurons in the brain. Hence, there is
a pressing need to tap into treatments aiming at protecting the brain against
neurodegeneration. Since the actual cause of PD and AD is still unknown, it
is difficult to develop disease-modifying treatments to stop or slow down the
disease progression at an early stage.
• In AD, protein misfolding, after it has originated locally, spreads through
the brain as if it is a “contagious” or prion-like disease. An inflammatory
response may well be responsible for this progressive spread throughout
the brain. These aggregations of proteins hinder communication in the
brain that finally results in disconnections, brain cell death, and memory
loss. Treatment of AD is still focussed on mitigation of cognitive decline for
as long as possible. As the progression continues this battle will be lost and
memories will fade away.
• The PD patient encounters the same problem as treatment relies on
dopamine replacement therapies that are focussed on improving motor
function. Besides the fact that progressive degeneration continues,
these therapies induce severe side effects, such as dyskinesia (abnormal
involuntary movements) caused by levodopa medication, which is one of
the most important concerns of the patient. Strategies to limit these side
effects are of high priority in order to improve the quality of life of the
patient. There is a strong need to develop treatments to restore normal
function after levodopa treatment in PD patients permitting long-term

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25

Alzheimer & Parkinson’s disease

medication. The Michael J. Fox Foundation has awarded a study to evaluate
a candidate compound, HE3286 (Triolex), to prevent these undesired side
effects. HE3286 blocks the activation of the transcription factor pathway in
the neuron and blocks the NMDA receptor on the surface of the neuron,
which is involved in the dyskinesia and in the calcium homeostasis, a feature
in neurodegeneration in general (Figure 1).

The relevance of monkeys in the research
Animal models offer an opportunity to link low-level neurodegeneration to
disease manifestation and a possibility to investigate strategies for intervention
therapy. While physiological and pharmacological questions can often be
studied in rodent models, issues concerning complex behavior and cognition
can be addressed more accurately in non-human primates (NHP), which
optimize the extrapolation of the results to human.
The common marmoset (Callithrix jacchus) is an established model in neuroscience:
• Marmosets develop, like humans, natural protein plaque progression during

aging and cognitive decline, which might reflect their genetic, physiological
and behavioral proximity to humans (Figure 2). Injections of beta-amyloid
protein in the brain of marmosets in combination with an activated
immune system might trigger the spread of plaque formation in the brain
as seen in AD.This model validation is currently being performed at BPRC.

Figure 1: schematic overview of the interaction between protein misfolding or toxins with
supporting cells, named glia, and with neurons in the brain and the mechanism of action in these
neurons. Over-activation and phosphorylation of the NMDA receptor by damaging factors (a.o.
toxins, activated glia cells) causes a disturbed calcium homeostasis leading to cell death as seen in
PD and AD. Inflammatory mediators increase this process.The NMDA receptor and activated
transcription factors are also responsible for levodopa induced dyskinetic side effects in PD.

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BPRC Annual Report 2013

Figure 2: Example of spontaneously
developed diffuse amyloid plaques
(black spots) in brain tissue of a 7-year
old female marmoset monkey with
wasting syndrome that had been
exposed to inflammation during
the last 6-months of her life. Stained
with a Campbell-Switzer staining.

BPRC Annual Report 2013

27

Alzheimer & Parkinson’s disease

• A well-validated approach for the induction of PD is exposure to the
neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTPtreated marmosets show optimal face validity with a wide range of
parkinsonian behaviors, including the levodopa-induced dyskinesia. We
have implemented a progressive-chronic PD model in the marmoset,
which is based on long-term chronic exposure of low doses of MPTP,
which selectively damage dopamine neurons that play a role in motor
function. This approach best reflects idiopathic PD and is comparable with
the accidental disease induction in drug users exposed to MPTP.
Sensitive non-invasive read-out systems for motor function, cognition but
also for sleep disturbances and other behavioral symptoms with an open eye
towards human validity are used to identify the symptomatic dynamics during
disease development and the effects of candidate treatments. Post-mortem
proteomics and transcriptomics of key brain structures are used to identify
changes in protein and gene expression to find new targets for neuroprotection.

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BPRC Annual Report 2013

29

Malaria

Malaria-the disease and research
Malaria is the biggest parasitic killer in the world. Malaria is caused by Plasmodium
parasites which are transmitted from man-to-man by mosquito. In humans, the
parasite first infects the liver, where it multiplies several thousand-fold in a week
or so, without giving any disease symptoms. Then, the parasites escape from
the liver and infect red blood cells.The ten-fold cyclical multiplication in only 48
hours gives rise to malaria symptoms.
There are two major human malaria parasites, P. falciparum and P. vivax.
P. vivax is very difficult to study as, unlike P. falciparum, the parasite cannot be
cultured in vitro. Thus, the only sources of parasites are patients, or certain
experimentally infected South American monkeys that are very scarce.
Malaria research in the Department is resting on three pillars: drug development,
vaccine development, and malaria parasite biology. The biology, including
parasite-host interactions, forms a red thread through all our research as we
believe that if we understand the parasite better, we can make better working
vaccines and drugs for the combat of malaria.
Malaria drug development model
For our drug development programme we focus on dormant liver stage
parasites of P. vivax, a unique stage that is only present in P. vivax and a few
closely related primate malaria species. Due to the difficulties described above,
we are using a relative of P. vivax, the primate malaria P. cynomolgi. Using infected
rhesus monkeys as a source of parasites, we have developed an in vitro drug
assay for the dormant liver stage parasites that we use for the screening of
potential new medicines. This is important, as in humans these stages form a
hidden parasite reservoir that can give rise to new blood stage malaria at any
time.
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BPRC Annual Report 2013

New medicines are necessary as currently only a single medicine exists and
parasites become resistant to this medicine. In 2013, we have optimized an
active compound which will be further tested in vivo in the P. cynomolgi-rhesus
monkey model for activity. Furthermore, to study the biology of the dormant
liver stages, we have been able to purify sufficient dormant parasites from in
vitro culture to allow extraction of genetic material for analysis. The results will
tell us which building blocks are present in the dormant parasites, which may
lead to new targets for medicine development.
(Pre)clinical malaria vaccine development
In our vaccine programme we focus on the blood stages of P. falciparum, for
which we have developed a good vaccine candidate, PfAMA1. In the past we
had developed in vitro assays to be able to measure whether immune responses
against PfAMA1 can inhibit parasite growth, and used rhesus monkeys to
evaluate safety and immune responses of different PfAMA1 formulations,
suitable for usage in humans. The immune responses were measured using
in vitro assays; as rhesus
monkeys cannot be infected
with P. falciparum, protective
efficacy of the vaccination
cannot be measured. Based
on all these studies, we have
selected suitable formulations
in 2013 and made preparations
for clinical evaluation of the
PfAMA1 vaccine candidate in
2014.

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31

Malaria

Biological studies in non-human primates
We focus our biological studies on non-human primate malarias that are close
relatives of P. vivax (P. cynomolgi and P. knowlesi) because 1. the parasites are
closely related to human parasites, and 2. the host, the rhesus monkey, is closely
related to humans.Thus, outcomes of studies can be easier extrapolated to the
human situation. In 2013 we started a study where we vaccinate rhesus monkeys
with P. knowlesi-infected mosquito bites in the presence of a strong anti-blood
stage malaria medicine, which does not interfere with liver stage development
but prevents blood stage malaria. This is a known procedure to experimentally
protect people from infection, but we do not understand exactly how this
works. The purpose of this experiment is to find mechanisms of protection by
detailed analyses of protective responses in general, but uniquely also locally in
the liver, spleen and lymph nodes, important immunological organs, that cannot
be obtained in human studies. Results will follow in 2014.
Female Anopheles mosquito, carrying malaria parasites in the saliva, used for vaccinating rhesus
monkeys (Picture from www.sciencebuzz.org)

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33

Tuberculosis

A Global Health Challenge
Worldwide, tuberculosis (TB) kills one human being every 20 seconds. The
infection is caused by inhalation of Mycobacterium tuberculosis (M.tb) and
relatively difficult to treat with antibiotics.To fight TB at a global scale, vaccination
– that is prior harnessing of immune defense to prevent pathogenic M.tb from
doing harm - will be most effective. The current vaccine, live Mycobacterium
bovis BCG, however, often fails in different populations. Since the mechanisms of
protective immunity are poorly understood, it remains a formidable challenge
to improve TB vaccine therapy beyond the level of BCG.
Why Using Non-Human Primates?
More research is required and clinical studies are informative, yet suffer greatly
from the extended incubation time that can occur upon (latent) infection
with M.tb. Experimental infection of humans to control kinetics, however,
is considered as undesirable. Thus, model systems are indispensible and, so
far, only animal models harbour the immuno-physiological complexity that
underlies disease development (pathogenesis) and protective immunity.
We use non-human primates (NHP) and macaques specifically, which – in
contrast to most if not all other TB models - are closely related to humans,
are naturally susceptible to M.tb, and present the best recapitulation of the
human disease. NHP are used for testing new vaccination strategies and for
investigating mechanisms of protection.
Our previous NHP studies have suggested that different cohorts of macaques
respond differently to infection with M.tb and vaccination with M.bovis BCG,
resembling the situation in clinical TB. To exclude any possible variation due
to disparity in mycobacterial challenge and/or experimental conditions, we
planned a head-to-head comparison of two distinct breeding populations of
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BPRC Annual Report 2013

rhesus macaques in a BCG vaccination and M.tb infection study. By investigating
the differential macaque responses we hope to learn of mechanisms that
determine TB susceptibility and protective efficacy or failure of BCG vaccination
as it occurs in man.
Our Latest Results
Our head-to-head comparative study confirmed that animals from different
breeding origin differ in susceptibility to M.tb infection and TB disease. Rhesus
macaques of Indian breeding origin presented with significantly more pathology
as compared to the Chinese-type rhesus macaques. Accordingly, bacterial
burden and various clinical measures were differentially affected between the
two cohorts.
We also found that only one of two groups showed reduction of disease as
a result of prior BCG vaccination (although the potential of BCG to protect,
does not strictly correlate with breeding origin). Remarkably, upon vaccination,
T cell immune responses that are typically addressed by vaccination, were
not found to be different between the two rhesus monkey groups. On the
other hand, changes in myeloid phagocytic cells, which play an important
role in orchestrating T cell immunity, were distinctive already one week after
vaccination. Similarly, phagocytic myeloid cells showed differential responses
between cohorts early after M.tb infection. Recent clinical studies suggest that
the response status of such myeloid cells, rather than T cells, predicts the risk of
developing severe TB disease. Our rhesus macaque study results would extend
upon those observations.
Additionally, we investigated local vaccination by unconventional pulmonary
(lung) administration of BCG. Most notably, in the rhesus cohort where
standard intradermal (skin) BCG treatment had no effect, vaccination via the

BPRC Annual Report 2013

35

Tuberculosis

lung still did reduce lung pathology significantly.This underpins the relevance of
addressing the lung compartment by local vaccination to reduce pulmonary
TB, and provides for a treatment strategy that should be pursued in our future
research.
This study has provided a large set of data and biosamples that need to
be explored further and from which we may expect to learn about the
mechanisms that determine TB disease suceptibility and the success (or failure)
of BCG vaccination. Our data immediately impact on refined use of NHP
macaques, and on the design of future studies to unravel the mechanisms of
local immunity. Ultimately, these findings and the ongoing analyses contribute
to our knowledge-base and should be instructive towards developing better
therapies for human TB in the near future.

Recent results from our NHP studies and reports in literature do underpin the notion that the
lymphatic system is critically involved and also at early stages of TB infection. Early changes in the
systemic myeloid immune compartment (or the inflammatory status) seem to correlate with
intradermal (parenteral) TB vaccine effects. At the same time we have shown that targeting local
immunity by vaccination can significantly improve towards TB lung disease. These interactions of
local and systemic immune modulation will shape our future research strategies.

36

BPRC Annual Report 2013

BPRC Annual Report 2013

37

Viruses & Vaccines

Viruses are very small particles made of proteins and lipids encapsulating their
genetic material (DNA or RNA). Viruses cause familiar infectious diseases
such as cold, flu, diarrhea, and warts, and can also cause severe illnesses such
as HIV/AIDS, smallpox and hemorrhagic fevers. Viruses behave like hijackers
as they invade cells and use them for multiplication and production of new
virus particles. This can eventually kill infected cells and cause disease. Viral
infections are hard to treat. Antibiotics work on bacteria, but do not act on
viral infections. Viruses that persist inside the body’s cells are “protected” from
most medicines. Vaccines can protect against getting viral infections and/or
diseases. Therefore vaccination is the best approach for the treatment and
prevention of viral infectious diseases. At the department of Virology we focus
on developing and testing potential vaccines against several viral infections.
In our research animal infection models are used for studying the efficacy of
potential vaccine candidates. We have established infection models for HIV/
AIDS, Influenza virus, and West Nile virus in non-human primates (NHP). The
latter two models were established in 2013. Besides testing the efficacy of
various potential vaccine candidates, we also investigate how vaccines work in
the combat of viral infections in order to improve vaccine candidates.
Use of non-human primates in virus research
Animals (including humans) have dedicated cells and tissues to deal with
the threat of infection. Infection usually leads to the induction of an immune
response. Some responses occur immediately so that an infecting agent can be
quickly contained. Other responses are slower but are more focused to the
infecting agent. Infection leads to disease. The human immune system has two
levels of immunity: specific and non-specific immunity. Through non-specific
immunity, also called innate immunity, the human body protects itself against
foreign material that is perceived to be harmful. These first lines of defense
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BPRC Annual Report 2013

include outer barriers like the skin and mucous membranes. When pathogens
(like bacteria and viruses) breach the outer barriers, for example through a
cut in the skin or when inhaled into the lungs, they can cause serious harm. In
contrast to innate immunity, specific immunity, also called adaptive, allows for a
targeted response against a specific pathogen that can last for years because
memory immune cells are induced.
Vaccination stimulates a specific immune response that will generate the
expansion of immune cells (memory B and T cells) specific to a certain
pathogen. The memory cells persist in the body and can lead to a quick and
effective response should the body encounter the pathogen again. Part of
these processes can be studied in laboratory dishes (in vitro), but the complex
interactions between many of these immune cells can only be studied in a living
animal (in vivo). Because we develop vaccines against human viral diseases, it is
important to use animal models that 1) display susceptibility to infection with
these human viruses, 2) mimic disease progression in humans, and 3) have
immune systems that are comparable to humans to study the mechanism(s)
of protection. Only with adequate animal models we can study the efficacy of
potential vaccine candidates for human purpose. For instance human influenza
virus and West Nile virus can both infect mice. However, when infected with
these viruses the animals die within a couple of days. This does not mirror the
human infection where the virus is normally cleared from the body within
2-3 weeks after an initial disease. Another example is the HIV/AIDS model.
This virus cannot infect other mammals than non-human primates. Although
humanized mice have recently been developed as a model to study HIV
infection, the immune system and responses in non-human primates most
closely mimics that of humans and the non-human primate model is therefore
essential for evaluation of human vaccine candidates.

BPRC Annual Report 2013

39

Viruses & Vaccines

Results obtained in 2013
In 2013 we have established different influenza virus and West Nile virus
infection models.These models are now being used to study vaccine candidates
for their immunogenicity (does the vaccine stimulate high immune responses?)
and efficacy (are the induced responses potent enough to protect?) against
viral infections and/or disease progression. Additionally, these models can also
be used for the study of other intervention strategies for instance anti-viral
monoclonal antibodies and anti-microbial compounds.
Emerging viruses
Emerging virus diseases are a major threat to human and veterinary public
health. Viruses like Dengue virus, Chikungunya virus, and West Nile virus
have in recent years caused epidemics in Europe. In nature, West Nile virus
(WNV) cycles between mosquitos and birds, but is incidentally transmitted to
humans and other mammals, like
horses, by infected mosquitoes
(see figure). In most humans,
WNV infection goes unnoticed
or leads to a flu-like illness, but
in 1% of human WNV infections
it leads to a serious, sometimes
fatal disease. In the recent years
we have set up a WNV infection
model in rhesus macaques and
common marmosets that can
be used for vaccine testing or evaluation of antiviral therapies. In the past
year we successfully evaluated two WNV vaccines in rhesus macaques. Both
vaccines, both based on the WNV envelope protein, fully protected macaques
against virus in the blood, and drastically reduced the number of infected cells
40

BPRC Annual Report 2013

in various tissues in the vaccinated animals. Additionally, we have seized this
opportunity to develop serological assays for the detection of WNV and
Dengue virus antibodies in sera of non-human primates. These assays are now
part of the Primate Viral Diagnostics service that we offer to primate-keeping
institutes around the world.
Influenza viruses
During seasonal epidemics,
influenza viruses cause
approximately
250,000500,000 deaths annually.
Young, elderly and immune-compromised people are those mainly at risk
for developing severe disease. Recently, influenza A viruses of swine origin
have caused the first pandemic of the 21st century. Animal models play an
important role in studying the pathogenesis of influenza virus infections.
Non-human primates are closely related to humans and show immunological
and physiological resemblances/similarities that make them a highly relevant
model in pre-clinical safety, immunogenicity and efficacy evaluation of vaccines
and therapies. In 2013 we have set up an influenza virus infection model
with a swine origin pandemic influenza A virus and compared susceptibility
to infection in three different non-human primate species. The information
obtained allows us to select the most optimal model for evaluation of novel
“universal” vaccine candidates, which are currently being developed by the EUfunded EDUFLUVAC consortium (www.euvaccine.eu/portfolio/project-index/
edufluvac). This consortium is working on the development of a vaccine that
will protect against many different influenza virus variants, including seasonal
variants that emerge each year as well as newly emerging viruses. With such a
vaccine the yearly vaccination would no longer be necessary and the threat of
future influenza epidemics would be reduced.
BPRC Annual Report 2013

41

Finance

The BPRC financial annual report has been audited and approved by our
financial accountant.

Result development BPRC 2003 - 2013

450

389

All figures x Euro 1.000

400
350
300
250
200

284

313

210

150
100

81

50
0
-50

10
2003

2004 2005

-100

2006

2007
Year

37

101
60
-76

2008

2009

2011

2013
(K€)

2012
(K€)

Turnover projects (extern)
Turnover projects (subsidy)

5.781
6.418

5.711
6.312

Total turnover projects

12.199

12.023

Other excluding interest

392

1.261

392

1.261

CURRENT ASSETS

Total turnover

12.591

13.284

STOCKS

External direct project costs
Staff costs
Depreciation
Other operating charges

770
7.537
508
3.910

867
7.775
625
4.087

Total operating costs

12.725

13.354

Profit/loss on ordinary activities

134-

70-

Interest

235

103

Profit for the financial year

101

33

Tax

-

-

Profit for the financial year after tax

101

33

ASSETS

2013
(K€)

2012
(K€)

FIXED ASSETS
Buildings and structures
Tangible fixed assets

41.224
1.617

42.841

42.886
1.761

44.647

52

38

DEBTORS DUE WITHIN ONE YEAR
Work in progress
Receivables from contracts
Receivables tax
Other receivables

1.614
386
255
60

2.315

1.384
1.544
463
258

3.649

Cash at bank and in hand

18.979

15.902

Total assets

64.187

64.236

LIABILITIES

2013
(K€)

2012
(K€)

EQUITY

33
2012

2010

BALANCE SHEET 31 DECEMBER

PROFIT AND LOSS ACCOUNT

EFFECTIVE PERSONNEL

2013

Equity
Revaluation reserve buildings
Result current year

2013

2012

Service Departments
Animal Sience Department
Research

15.1 14%
43.9 40%
49.9 46%

15.3
46.3
54.2

13%
40%
47%

Total

108.9 100%

115.8

100%

2.317
7.642
101

10.060

2.284
8.252
33

10.569

PROVISIONS
Primates
Deferred tax liabilities
(Flexibel) retirement
Repairs buidings

104
2.547
6.009

8.660

205
2.750
5.189

8.144

LONG TERM DEBTS
Bank
Received in advance on asstes

26.803
11.567

38.370

27.569
11.688

39.257

SHORT TERM DEBTS
Received in advance on projects
Received in advance on assets
Received in advance subsidy
Accounts Payable (TAX)
(Flexibel) retirement
Accounts Payable
Commitment Bank
Other liabilities
Total liabilities

42

BPRC Annual Report 2013

3.153
130
160
476
473
766
1.939

7.097
64.187

2.193
210
480
524
428
729
1.702

6.266
64.236

BPRC Annual Report 2013

43