Virological and Immunological Studies of Dengue Virus Infection in Pigtail Macaques (Macaca nemestrina)

VIROLOGICAL AND IMMUNOLOGICAL STUDIES
OF DENGUE VIRUS INFECTION
IN PIGTAIL MACAQUES (MACACA NEMESTRINA)

SUSANA WIDJAJA

SEKOLAH PASCASARJANA
INSTITUT PERTANIAN BOGOR
BOGOR
2010

STATEMENT
Hereby I, Susana Widjaja, do declare that this dissertation entitled
“Virological and Immunological Studies of Dengue Virus Infection in Pigtail
Macaques (Macaca nemestrina)” is my own work and has not been submitted in
any form for another degree or diploma programs (course) to any university or
other institution. The content of the dissertation has been examined by the
advising committee and the external examiner.

Bogor, August 2010

Susana Widjaja
P067050051

ABSTRACT
SUSANA WIDJAJA. Virological and Immunological Studies of Dengue Virus
Infection in Pigtail Macaques (Macaca nemestrina). Supervised by DONDIN
SAJUTHI, JOKO PAMUNGKAS, DIAH ISKANDRIATI, and PATRICK J
BLAIR.
A non-human primate (NHP) model is essential for the study of dengue
hemorrhagic fever (DHF) pathogenesis and the evaluation of dengue (DEN)
vaccine and antiviral drug. Until now, it has been difficult to find an NHP DHF
pathogenesis model. Therefore, an evaluation of a DEN vaccine candidate is
performed in NHPs that show viremia after infected by DEN virus and the
vaccine efficacy is its capability to develop immunity that reduces viremia when
vaccinated NHPs are challenged by DEN virus. In this study, the potential of
pigtail macaque to serve as an animal model for DEN vaccine testing was
evaluated. Homologous sequential DEN challenges were conducted using primary
viral isolates from DEN patients in Indonesia. Two parameters, the ability to
support dengue viremia and to produce sufficient antibody responses were
measured. This study shows that primary infections of all four DEN serotypes

cause consistent, measurable viremia in pigtail macaques. The responses of IgM,
IgG and avidity antibody following primary and secondary DEN infections are
similar with antibody responses in human. The immunity produced by primary
infection is sufficient to protect against homologous virus. This species of
macaque therefore appears to be a suitable alternative model for testing DEN
vaccine candidates. Besides antibody, T lymphocyte also has an important role in
the protection and pathogenesis of DEN diseases. DEN specific T lymphocyte
measurements, ELISPOT and intracellular cytokine staining-flow cytometry (ICFC), were developed to support DEN studies in pigtail macaque. Peripheral blood
mononuclear cells (PBMC) collected before and after DEN infections were tested.
ELISPOT results show increase of DEN specific interferon-γ (IFN-γ) producing
cells as an individual response of pigtail to primary DEN-1, DEN-3 or DEN-4
infections. Using pools of PBMC taken from several animals, ELISPOT and
intracellular cytokine staining-flow cytometry (IC-FC) was run side by side to
quantify DEN specific lymphocytes following primary and secondary DEN-2
infections. ELISPOT revealed an increase of DEN specific IFN-γ producing cells
following primary infection and a significant increase after secondary infection.
Similarly, IC-FC also measured an increase of DEN specific producing IFN-γ
CD3+CD4+ and CD3+CD4- T lymphocytes. As such, ELISPOT and IC-FC can
be applied to measure DEN specific T lymphocytes in pigtail macaques.
Therefore, the application of these assays would be useful in elaborating adaptive

immunity induced by vaccine and the level of protection. Furthermore, the
development of pigtail as DHF model can be evaluated when further research on
the cross-reactive T lymphocyte and antibody responses during secondary
heterologous is conducted.
Keywords: M. nemestrina, dengue infections, viremia, antibody, T lymphocytes.

ABSTRAK
SUSANA WIDJAJA. Studi Virologi dan Imunologi Infeksi Virus Dengue Pada
Satwa Primata Beruk (Macaca nemestrina). Dibimbing oleh DONDIN SAJUTHI,
JOKO PAMUNGKAS, DIAH ISKANDRIATI, dan PATRICK J BLAIR.
Satwa primata sangat dibutuhkan untuk meneliti patogenesis demam
berdarah dengue (DBD) dan mengevaluasi vaksin dengue (DEN), juga obat
antivirus. Sampai saat ini sangat sulit mendapatkan model DBD pada satwa
primata. Jadi evaluasi kandidat vaksin DEN dilakukan pada satwa primata yang
memperlihatkan viremia setelah infeksi virus DEN dan vaksin yang efisien adalah
vaksin mampu menimbulkan kekebalan yang dapat mereduksi viremia pada
primata yang setelah divaksinasi kemudian diinfeksikan virus DEN. Untuk dapat
mengetahui potensi satwa primata beruk sebagai hewan model pada penelitian
vaksin DEN, beruk diinfeksikan berturutan dengan serotipe DEN yang sama.
Virus DEN yang digunakan berasal dari virus yang diisolasi dari pasien-pasien

DEN di Indonesia. Dua parameter yang diukur adalah viremia yang terjadi setelah
penyuntikan virus DEN dan antibodi sebagai respon beruk terhadap infeksi DEN
tersebut. Beruk memperlihatkan viremia yang konsisten setelah diinfeksikan
dengan virus DEN-1, DEN-2, DEN-3 dan DEN-4. Respon antibodi IgM, IgG dan
aviditas setelah infeksi primer dan sekunder menyerupai respon pada manusia.
Kekebalan yang terjadi setelah infeksi primer dapat melindungi beruk dari infeksi
sekunder homologus. Hasil ini menunjukkan bahwa beruk dapat digunakan untuk
evaluasi vaksin DEN dan menjadi hewan model alternatif untuk penelitian infeksi
DEN. Tidak hanya antibodi, limfosit T juga memiliki peran penting terhadap
proteksi dan patogenesa infeksi DEN. Pengukuran limfosit T spesifik DEN yang
memproduksi interferon-γ (IFN- γ) yaitu ELISPOT dan intracellular cytokine
staining-flow cytometry (IC-FC) dikembangkan untuk mendukung penelitian
DEN pada beruk. Pengujian dilakukan menggunakan peripheral blood
mononuclear cells (PBMC) yang diambil sebelum dan sesudah infeksi DEN.
Hasil ELISPOT memperlihatkan kenaikan jumlah limfosit T spesifik DEN
sebagai respon individu beruk terhadap infeksi DEN-1, DEN-3 dan DEN-4.
Dengan menyatukan PBMC dari beberapa beruk, ELISPOT dan IC-C dilakukan
secara bersamaan untuk mengukur jumlah limfosit T spesifik DEN setelah infeksi
DEN-2. ELISPOT memperlihatkan kenaikan limfosit T spesifik DEN yang
memproduksi IFN-γ setelah infeksi primer dan kenaikan yang lebih nyata sebagai

respon terhadap infeksi sekunder. Hasil IC-FC, pola kenaikan dari limfosit T
CD3+CD4+ dan CD3+CD4- spesifik DEN yang memproduksi IFN-γ sebagai
respon terhadap infeksi primer dan sekunder serupa dengan respon yang diukur
dengan ELISPOT. Jadi ELISPOT dan IC-FC dapat digunakan untuk mengukur
limfosit T spesifik DEN. Aplikasi kedua uji ini dapat digunakan untuk
mempelajari lebih rinci kekebalan adaptif yang didapat dari vaksinasi dan
kemampuan proteksinya. Demikian pula, pengembangan beruk sebagai model
DBD akan dapat dilakukan melalui penelitian respon reaksi silang dari limfosit T
dan antibodi pada infeksi sekunder heterologus.
Kata kunci: M. nemestrina, infeksi dengue, viremia, antibodi, limfosit T.

SUMMARY
SUSANA WIDJAJA. Virological and Immunological Studies of Dengue Virus
Infection in Pigtail Macaques (Macaca nemestrina). Supervised by DONDIN
SAJUTHI, JOKO PAMUNGKAS, DIAH ISKANDRIATI, and PATRICK J
BLAIR.
Dengue virus infections have caused a major public health problem in
tropical and sub-tropical countries. The geographical distribution, the frequency
of epidemic cycle and the number of cases have been increasing at an alarming
rate and highlighted the urgency of DEN vaccine (WHO 2005; Raviprakash et al.

2009). The clinical manifestations of DEN infections range from mild dengue
fever (DF) with high fever, headache, rash, and bone and muscle pain up to severe
dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with
evidence of thrombocytopenia, bleeding, plasma leakage and shock. The severe
manifestation cause a high mortality rate particularly in children (WHO 2005).
Since the 1980s, epidemiological data revealed that 85% of DHF and DSS were
heterologous secondary infections and immunopathological response to
heterologous secondary infection has been hyphothesized leading to DHF
pathogenesis (Halstead 1983). Antibody produced following primay DEN
infection confers the protection to homologous infection, however, heterologous
secondary infection may still occur. The pre-existing, non-neutralizing antibodies
binds DEN viruses and these complexes, then bind the target cells via the FcγRI
and FcγII, resulting in increased viral load, shortened incubation period and
increased disease severity (Fink et al. 2006). Meanwhile, DEN specific CD4+ and
CD8+ T lymphocytes are suggested to have a low binding affinity for the current
serotype, and consequently, inefficient to clear the infection (Fink et al. 2006).
The limitations of DEN study in human have hampered the understanding of these
two components of adaptive immunity, antibody and T lymphocyte, in the
pathogenesis of DHF.
Dengue vaccine evaluation in non-human primate (NHP) model is

required before the vaccine can be applied in human. Here, we explored the
posibility of pigtail macaque (Macaca nemestrina) as an animal model to evaluate
DEN vaccine. A total of seventeen Flavivirus-free pigtail macaques were
separated into four groups by DEN serotypes. Dengue-1 to dengue-4 viruses were
isolated from DEN patients in Indonesia. Aproximately 105 plaque forming unit
(PFU) DEN virus was injected subcutaneously into each individual in the lateral
chest area. Blood samples were obtained prior to virus injection as baseline
sample and daily for 10 days post-infection for virus detection and on 14, 28 and
87 days post-injection for anti-dengue antibody profile analysis. Consistent
viremia was detected by virus isolation (mosquito inoculation and C6/36 cell
culture) and RT PCR methods. Viremia was detected one or two days postinfection in most of the animals. By RT-PCR and mosquito inoculation methods,
the least number of viremia days occurred with DEN-4 (5±1.4 and 3.3±1 days).
By isolation in C6/36, DEN-3 produced the least (4±0.8). DEN-2 resulted in the
longest average number of days viremia (7.8±0.5, 6.8±1 and 5.8±1 days as
measured by RT PCR, isolation in C6/36 and mosquito inoculation, respectively).
A challenge with homologous serotype six month after the first infection did not

result in any detectable viremia by virus isolation and only one to two days viral
RNA was detected in DEN-4 group. Prior in primary infection, IgM antibody was
detected, then followed by IgG antibody. During secondary infection, IgM was

not detected, whereas IgG increased rapidly. The avidity of IgG increased
overtime following primary infection and secondary infection. Similarly with IgG
and its avidity, high neutralizing antibody was generated following primary
infection and augmented in secondary infection. These antibody responses to
primary and secondary DEN infections were similar with antibody responses in
human. The predominat IgG subclass following primary and seconday infections
was IgG1. These data reveal that pigtail macaque is suitable for the study of DEN
infection. This animal can serve as an alternative model for evaluating DEN
vaccine, since the efficacy of a DEN vaccine is measured by its capability to
reduce viremia after vaccinated animals are challenged with live DEN virus.
To support DEN study in pigtail macaque, ELISPOT and intracellular
cytokine staining-flow cytometry (IC-FC) were established to enumerate DEN
specific T lymphocytes. The ELISPOT assay employs ELISA technique to trap
antigen- induced cytokine secretion around the cells by an immobilized anticytokine antibody on polyvinylidene difluoride membrane, and then visualizes the
complexes by anti-cytokine conjugate and substrate. IC-FC uses brefeldin A to
trap cytokine intracellularly following antigen stimulation. Then, the cells are
permeabilized and specific anti-cytokine fluorescent antibodies can pass into the
cells and react with cytokines. Both assays measure functional T cells after
stimulation by DEN antigen, however, ELISPOT measures secreted cytokine
while IC-FC measures intracellular cytokine (Lecth and Scheibenbogen 2003).

Dengue antigens were generated from intra- and extra-cellular proteins of DEN
virus culture in Vero cells. The application of DEN antigen for in vitro stimulation
of T lymphocytes reduce the complexity of DEN specific T lymphocyte assays,
since the generation of antigen presenting cells or prior knowledge of antigenic
peptides is not required (Mangada et al. 2004). Homologous T cell responses were
observed. Peripheral blood mononuclear cells (PBMC) pre- and post DEN
infections had been isolated from heparinized blood collected during several
previous DEN studies and stored in LN2 until assayed. ELISPOT detected 0-40
DEN specific interferon-γ (IFN-γ) producing cells from PBMC before DEN
infection and 28-440 cells after DEN infections. Increase of DEN specific IFN-γ
producing cells was detected as an individual response of pigtail to DEN-1, DEN3 or DEN-4 infections. ELISPOT and IC-FC was run simultaneously to quantify
DEN specific lymphocytes following primary and secondary DEN-2 infections
using pools of PBMC taken from several animals. An increase of DEN-2 specific
IFN-γ cells following primary infection and a significant increase after secondary
infection were detected by ELISPOT. Similarly, increase CD3+CD4+ (T helper1) and CD3+CD4- (T cytotoxic) specific DEN after primary and secondary
infections were detected. These results show that both ELISPOT and IC-FC can
be used to measure DEN specific T lymphocytes.
Based on the susceptibility of pigtail macaque to the infections of all four
DEN serotypes, pigtail macaque is suitable model to study DEN infection and can
be used as an alternate NHP model to evaluate DEN vaccine or anti-viral.

Furthermore, the availability of DEN specific T lymphocyte measurements allow

more detail exploration on immunity induced by vaccination that protects the
pigtail from DEN challenge. As DHF is associated with heterologous secondary
infections, to evaluate whether pigtail macaque is suitable as the model, further
study on serotype cross-reactive antibody and T lymphocytes responses should be
investigated.

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VIROLOGICAL AND IMMUNOLOGICAL STUDIES OF
DENGUE VIRUS INFECTION IN PIGTAIL MACAQUES
(MACACA NEMESTRINA)

SUSANA WIDJAJA

Dissertation
submitted in partial fulfillment of the requirements
for the degree of Doctorate in the Primatology Major,
Graduate Program, Institut Pertanian Bogor.

GRADUATE PROGRAM
INSTITUT PERTANIAN BOGOR
BOGOR
2010

External examiners in private defense:
1 Dr. Irma Suparto, M.D., M.S.
2 Drh. Surachmi Setiyaningsih, Ph.D.
External examiners in public defense:
1 Bachti Alisjahbana, M.D., Ph.D
2 Tjahjani Mirawati Sudiro, M.D., Sp.M.K., Ph.D.

Title

: Virological and Immunological Studies of Dengue Virus
Infection in Pigtail Macaques (Macaca nemestrina)

Name

: Susana Widjaja

Student Number

: P067050051
Approved by
Advisory Commitee

Prof. Drh. Dondin Sajuthi, M.St., Ph.D.

Dr. Drh. Joko Pamungkas, M.Sc.

Major Advisor

Co-Advisor

Dr. Drh. Diah Iskandriati

Patrick J Blair, Ph.D.

Co-Advisor

Co-Advisor

Acknowledged by
Chairman, Major Primatology

Dean of Graduate School

Prof. drh. Dondin Sajuthi, M.St., Ph.D. Prof. Dr. Ir. Khairil A. Notodiputro, M.S.

Date of final examination: 8 October 2010

Date of graduation:

To Indonesian scientists in health research.
Let science be our first priority to achieve welfare for all Indonesians.

PREFACE
“Virological and Immunological Studies of Dengue Infection in Pigtail
Macaques (Macaca nemestrina)” consists of two research publications entitled
“Pigtail Macaque (Macaca nemestrina) and Dengue Virus Infectivity: a Potential
Model for Evaluating Dengue Vaccine Candidates” and “The Measurements of
Dengue Specific Interferon-γ Producing T Lymphocytes in Pigtail Macaques
(Macaca nemestrina)”. These two studies are intended to explore pigtail macaque
as a non-human primates (NHP) model for dengue research, therefore, more
diverse NHP species can be utilized. The urgency of available licensed dengue
vaccine draws attention to NHP requirements in the pre-clinical phase of vaccine
trial. And the lack of dengue hemorrhagic fever NHP model may be solved by
certain susceptible NHP species. Another purpose is to bring more opportunities
of pigtail macaque to be used in biomedical research. As pigtail macaque is
endemic NHP in Kalimantan and Sumatra islands, the use of Indonesian “natural
resource” in biomedical research without threatening the existence of the species
in its natural habitats in Indonesia, hopefully, can open a better chance for the
welfare of the people and NHP in Indonesia.

ACKNOWLEDGMENTS
I praise and thank God for His good hand is upon me in each step so I am
able to complete this dissertation. And this dissertation holds far more than the
culmination of research. It is also a result of great correlation with many brilliant,
generous, inspiring and lovely people.
My deepest gratitude goes to Prof. Kevin Porter, M.D., who had the
original idea and initial study of the pigtail macaque as an animal model for
dengue infection. Also, this dissertation would not be completed without
subsequent research and kind-hearted continual support from all the former Viral
Diseases program Directors: Charmagne G Beckett, M.D., Patrick J Blair, Ph.D.,
Timothy H Burgess, M.D., M.P.H., and Maya Williams, Ph.D.
My heartfelt gratitude also goes out to my supervisors, Prof. Drh. Dondin
Sajuthi, M.St., Dr. Drh. Joko Pamungkas, M.Sc., Dr. Drh. Diah Iskandriati, and
Patrick J Blair, Ph.D whose untiring effort, commitment, encouragement,
guidance and support helped me greatly in exploring the studies and writing the
dissertation.
My special thank

to Gary T Brice, Ph.D.,

for tutoring the cellular

measurements, and for the long discussions that helped me sort out the technical
details of the work.
I am grateful to Prof. Dr. Ir. Sri Supraptini Mansjoer, Drh. Ikin Mansjoer,
M.Sc., Dr. Irma H. Suprapto, M.D., Dr. Erni Sulistiyawati, D.V.M, Dr. Ir. Dyah
Perwitasari and other lecturers in Primatology Major for teaching good research,
also giving continuous guidance and encouragement.
I acknowledge valuable direction and advice to finalize this dissertation
from the external examiners: Dr. Irma H. Suparto, M.D., M.S., Drh. Surachmi
Setiyaningsih, Ph.D., Bachti Alisjahbana, M.D., Ph.D., Tjahjani Mirawati Sudiro,
M.D., Sp.M.K., Ph.D.
My thank to the Primatology staff for assisting me with the administrative
tasks necessary for completing my doctoral program: Yanti and Yana.
I am in debt to my invaluable, supportive, forgiving, generous and loving
colleagues: Ratna Tan, Chairin Maroef, Imelda Winoto, Sri Hadiwidjaya, Ungke
Antonjaya, Sherly, Dasep, Deni, Haditya, Anton, Gustiani, Yuanita, Nurhidayah,

Nurhayati, Ester, Melinda, Santo, Mara, Anti, Ovi, Saraswati and other US
NAMRU-2 staff. Their incredible hard work and dedication to the US Navy and
scientific society inspire me for always doing high quality work. I am most
indebted to Herman Kosasih M.D. and Victor Sugiharto for abiding friendship,
careful review and discussion that graciously provided throughout all stages of
this dissertation fruition.
I am grateful to Sylvia, Tuah, Harri, and Suyanti for the friendship and
encouragement during and after the master degree program.
I greatly value the care and confidence from my best friends, Linda
Martini and Bimo Wicaksana, whose friendship have helped me keep moving on
and stay focus through the years.
Most importantly, none of this would have been possible without my
family; my sister and brothers: Susanti, Susanto and Sugiharto, my husband:
Herjadi, my children: Calista and Aldwin whose patience and love sustain me
through all my endeavours to complete this dissertation.
Jakarta, August 2010
Susana Widjaja

CURRICULUM VITAE
The author was born on the 3rd of May in 1964 in Jakarta. She is the
second daughter of the four children from the late Bakri Widjaja and Betty
Gomulya. She was married with Laurentius Herjadi and has blessed with talented
daughter, Saphire Calista, and thoughtful son, Lotharius Aldwin.
She received Doctor of Veterinary Medicine from the Faculty of
Veterinary Medicine , Institut Pertanian Bogor in 1987. She entered the Graduate
Program at the Institut Pertanian Bogor for a master degree in Primatology Major
in 2003, then approved to continue directly to doctorate degree in 2005.
The author started to serve at the United States Naval Medical Research
Unit-2, Jakarta in December 1988. In this prestigious infectious diseases research
laboratory, she got the amazing opportunity to develop her skill and knowledge
from technical ability as a bench laboratory staff up to managerial flair as the
head of the Tissue Culture and Immunology Division of the Viral Diseases
Program. She was a member of Institutional Animal Care and Use Committee
since 2003. She received a visiting scientist scholarship in 2002-2003 and trained
for the measurements of dengue humoral and cellular immunity in Naval Medical
Research Center, Maryland. After completion of this training, she received
outstanding visiting scientist award. Over 20 years of faithful and exceptional
service, she was granted numerous awards and letters of recognition. She also
produced many scientific publications as the author or co-author together with
briliant US and Indonesian scientists. She proudly continued to serve at the US
NAMRU-2 until its unfortunate and sudden closure in 2010.

TABLE OF CONTENTS
page
LIST OF TABLES

xiv

LIST OF FIGURES

xv

INTRODUCTION ........................................................................................

1

LITERATURE REVIEW ............................................................................. 4
Dengue virus ................................................................................................ 4
Dengue infections ........................................................................................ 6
The roles of B and T memory lymphocytes in the pathogenesis of dengue
hemorrhagic fever ......................................................................................... 7
Dengue vaccine and antiviral drug ............................................................... 7
Animal model for dengue infections ........................................................... 9
Dengue specific cytokine producing T lymphocyte measurements ............ 10
GENERAL METHODOLOGY ...................................................................

12

PIGTAIL MACAQUE (MACACA NEMESTRINA) AND DENGUE
VIRUS INFECTIFITY: A POTENTIAL MODEL FOR EVALUATING 16
DENGUE VACCINE CANDIDATES ........................................................
THE MEASUREMENT OF DENGUE SPECIFIC INTERFERON-γ
PRODUCING T
LYMPHOCYTES IN PIGTAIL MACAQUES
(MACACA NEMESTRINA) .......................................................................... 33
GENERAL DISCUSSION ........................................................................... 48
CONCLUDING REMARKS ....................................................................... 51
REFERENCES ............................................................................................. 52
APPENDIX .................................................................................................. 55

LIST OF TABLES
page
1

Grading severity of dengue infection ..............................................

6

2

Dengue vaccine candidates in clinical and pre-clinical trials .........

8

3

Human T cell subsets .......................................................................

11

4

Homologous anti-DEN neutralizing antibody responses
after primary and secondary infections ............................................

26

DEN specific IFN-γ producing cells in pigtail macaques before
and after DEN infection .................................................................

41

5

LIST OF FIGURES
page
1

A schematic presentation of dengue polyprotein ...............................

5

2

The life cycle of dengue virus in the cell ...........................................

5

3 Outline of virus injection and blood collection for pigtail
susceptibility study .............................................................................

13

4 Outline of of virus injection and blood collection for the study of
cellular immunity specific to DEN measurements ............................

14

5 The length of viremia in pigtail challenged with DEN viruses ..........

23

6 IgM , IgG and avidity responses after primary and secondary
infections .............................................................................................

24

7 Anti-DEN IgG subclasses after primary infection with DEN-4 .........

25

8 Dengue-1, Dengue-3 and Degue-4 antigen optimization by
ELISPOT ...........................................................................................

40

9 Dengue-2 antigen optimization by ELISPOT and IC-FC ...................

42

10 ELISPOT and IC-FC results of DEN-2 primary and secondary
infections ............................................................................................

43

LIST OF APPENDICES
page
1

List of reagents for laboratory assays ...............................................

55

2

PCR cycle condition .........................................................................

58

3 List of reagents for ELISPOT and intracellular staining-flow
cytometry ...........................................................................................

59

INTRODUCTION
Dengue (DEN) virus infections have threatened more than one third of the
world population (WHO 2005). It has been estimated that there are 50-100 million
dengue fever (DF) cases annualy of which 2-4% result in severe forms of the
disease, dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), a life
threatening disease particularly in children (WHO 2005). In Indonesia, more than
150 000 DHF and DSS cases with 1-2% mortality rate were reported in 2007 (Dit
Jen P2MPL 2008). Thus, dengue vaccine has become a priority of world health
research for effective prevention (Raviprakash et al. 2009).
Dengue virus consists of four distinct serotypes (DEN-1 to DEN-4) with up
to 30% dissimilarity among serotypes (Irie et al. 1989). While primary infection
confers protective immunity to the same serotype, heterologous secondary infection
has been hypothesized to be responsible for the immunopathogenesis of DHF or
DSS. Original antigenic sin theory has enlightened the role of B and T lymphocytes
(B and T cells) during heterologous secondary infections (Halstead et al. 1983).
Although the similarity between serotypes of primary and secondary infection result
in rapid expansion of pre-existing memory B and T cells, it generates low-avidity
antibodies and T cells to the infecting serotype. The antibodies bind, but do not
neutralize the virus. Instead, they augment virus entry to target cells through Fc
receptor (antibody dependent enhancement of infection,
(Halstead 2003).

ADEI hypothesis)

As consequent, increase of viral replication and increase of

infected cells result in more antigen presenting cells to stimulate T cells. Low
avidity T cells have less ability for viral clearance and produce predominantly proinflamatory cytokines. Thus, altered T lymphocyte functions lead to DHF or DSS
(Rothman 2004).
An animal model of DEN infections will be invaluable to study the
pathogenesis of DHF or DSS, since study in humans has had many limitations
(Beckett et al. 2005; Raviprakash et al. 2009). Non-human primates (NHP)
commonly used in DEN research are rhesus (Macaca mullata) and cynomolgus (M.
fascicularis) macaques as they develop detectable viremia and antibodies following
DEN infections (Bente and Rico-Hesse 2006; Raviprakash et al. 2009). They are
used to test the efficacy of a DEN vaccine and antiviral drug which is evaluated by

2
their abilities to prevent, or to significantly reduce, viremia when animals are
challenged with live DEN virus. Until now, NHP as DHF animal model is still
difficult to find. As pigtail macaque (M. nemestrina) has been shown exceptional
suceptibility

to

human

immunodeficiency

virus

(HIV)

and

simian

immunodeficiency virus (SIV) (Baroncelli et al. 2008), it may also be studied to see
whether it is better, compared to other non human primates, as the animal model
for DEN infections. This animal has never been reported as a model for DEN
infection (Raviprakash et al. 2009).
Compared with B cells and antibodies, T cells and their functions have been
limited to study. Conventional measurements of antigen specific T cells, such as Hthymidine proliferation assay, Cr-release cytotoxic assay and secretion of cytokines
in bulk lymphocyte cultures are laborious and time consuming. Also, they produce
insensitive and inconsistent results (Hickling 1998, Gauduin et al. 2004). The
enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining-flow
cytometry (IC-FC) assays measure T functional cells and employ the antigen
specific secretion of cytokines to detect specific T cells on a single cell level (Lecth
and Scheibenbogen 2003). These assays have become preferential, since they are
more straightforward and faster than conventional assays (Pahar et al. 2003). To
quantify DEN-specific T cells in cynomolgus macaques, Koraka et al. (2007 a,b)
employed ELISPOT and applied APC derived from autologous B cells to stimulate
interferon-γ (IFN-γ) producing T cells. An alternative technique for in vitro
stimulation of DEN specific T cells was an application DEN lysate antigen in bulk
human peripheral blood mononuclear cells (PBMC). Mangada et al. (2004) applied
these antigens and IC-FC assay to detect DEN specific IFN-γ producing T cells in
human.
In order to explore the possibility of pigtail macaque as a model for DEN
infection, DEN study in pigtail macaques was conducted. Virological and
immunological examinations were done thoroughly including virus isolation and
RT PCR for virerima detection, IgM, IgG, avidity IgG, subclass IgG and
neutralizing antibodies for the evaluation of humoral responses. Another study was
conducted for the development of ELISPOT and IC-FC assays, in order to enhance
DEN study in pigtail macaque. These assays used DEN antigens to stimulate T
cells. Our study shows that pigtail macaques support DEN replication resulting in

3
viremia and antibody responses that are similar with viremia and antibody responses
in human. Therefore, pigtail macaques are appropriate as animal model for vaccine
and antiviral evaluations. The ELISPOT and IC-FC revealed increase of DEN
specific IFN-γ Tcells after DEN infections.

LITERATURE REVIEW
Dengue virus. According to International Committee on Taxonomy of
Viruses (ICTV), a subgroup of Virology Division of the International Union of
Microbiology Societies, dengue virus belongs to the Flavivirus genus of the
Flaviviridae family (Calisher and Gould 2003). The virus particle is spherical, 4060 nm in diameter. Its icosahedral core consists of a capsid protein (C)
encapsulating a positive-sense, single-stranded RNA genome about 11 kilobases
(kb) in length. This RNA contains a 5’ cap (m7G5’ppp5’A) and functions as a
messenger RNA. The core is surronded by a lipid bilayer envelope with two viral
proteins, membrane (M) and envelope (E) protein (Lindenbach and Rice 2001,
2003).
The Flavivirus structure and replication is reviewed in detail by Lindenbach
and Rice (2001, 2003). The genome directs the synthesis of polyproteins.
Translation of one single open reading frame produces a large polyprotein that is
cleaved co- and post translationonally into three structural proteins: capsid (C),
precursor M (prM) and envelope (E) proteins, and seven non-structural (NS)
proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Figure 1 shows
schematic of DEN polyprotein. The C protein presumably mediates RNA
interaction, membrane association and also serves as signal peptide for ER
translocation of prM. The prM protein has chaperone-like activity protecting E
protein from undergoing rearrangement in the reduced pH environment of the early
secretory pathway. Then, the conversion of immature virus particles to mature
virions occurs in the secretory pathway with cleavage of prM into pr and M by the
Golgi resident furin or furin-like enzyme. The E glycoprotein is the major virion
surface proteins, which mediates binding and membrane fusion. The E protein is a
major target of humoral immunity. While stuctural proteins construct the viral
particle, non-strctural proteins support viral replication. An interaction of NS1 and
NS4A is required at a very early stage in RNA replication. Non-structural2A is
involved in coordinating the shift between RNA packaging and replication. Nonstructural2B is a co-factor for the serine protease of NS3. The carboxy terminal of
NS3 carries three enzymatic activities: a helicase to unwind double-stranded nucleic
acid during RNA replication, a NTPase to hydrolyse ATP to generate energy

5
neccesary during replication, and a RTPase to remove the terminal phosphate group
from the newly synthesized RNA for the formation of the viral cap structure at the
5’ end of genome. The NS5 methyl-tranferase (MTase) adds the cap (two methyl
groups) to the nucleotide. The RNA-dependent RNA polymerase (RdRp) produces
“copy-back” RNA.

Figure 1 A schematic presentation of dengue polyprotein. Dots represent enzyme
activity domains. Prot: protease, Hel: Helicasee/NTPase/RTPase, Mtase:
methyl-transferase, RdRp: RNA-dependent RNA polymerase.
(Lindenbach and Rice 2003).
Dengue virus enters into a host through the skin during mosquito feeding.
The replication of DEN virus begins when the virions infect a permissive host cells.
The primary target cells are mononuclear phagocytes and the entry is facilitated by
receptor mediated endocytosis. The best-characterized receptor that can mediated all
four serotypes of DEN virus is DC-SIGN. The virus is internalised into the
endosomal compartment where the acidic pH triggers a fusion of its envelope to the
endosomal membrane and deliver the viral genome into the cytoplasm. The viral
polyprotein is synthesized in association with the endoplasmic reticulum and is
processed into structural and non-structural protein by viral and cellular proteases.

Figure 2 The life cycle of dengue virus in the cell (Fink et at. 2006).

6

A viral replication complex is formed on the membrane of the endoplasmic
reticulum which facilitates replication of DEN genome. Newly synthesized viral
genomes are packed by core, envelope and membrane proteins along the secretory
pathway. Immature virus particles are transported by the secretory pathway to the
cell wall where furin cleaves the prM protein into M protein and the mature virion is
released. In a secondary infection, DEN virus binds to antibody from a previous
infection (antibody dependent enhancement of infection, ADEI) and is then
endocytosed by Fc receptor bearing cells, such as monocytes. The life cycle of
DEN virus was reviewed by Clyde et al. (2006) and Fink et al. (2006) (Fig 2).
Dengue infections. Incubation period usually varies from 3 to 14 days with
average 4 to 7 days (Gubler 1998). All four DEN infections in human may be
asymptomatic or may lead to undifferentiated fever, dengue fever (DF), dengue
hemorrhagic fever (DHF) or dengue shock syndrome (DSS) (WHO 2005).

Table 1 Grading the severity of dengue infection
Grade

Symptoms

Laboratory

DF

Fever with two or more
of the following sings:
headache, retro-orbital pain,
myalgia, arthralgia

Leukopenia occasionally.
Trombocytopenia may be present.
No evidence of plasma loss

DHF I

Above signs plus
positive tourniquet test.

Thrombocytopenia ≤ 100 000
Hematocrit rise ≥ 20%

DHF II

Above signs plus
spontaneous bleeding

Thrombocytopenia ≤ 100 000
Hematocrit rise ≥ 20%

DHF III*

Above signs plus
circulatory failure
(weak pulse, hypotension,
restlessness)

Thrombocytopenia ≤ 100 000
Hematocrit rise ≥ 20%

DHF IV*

Profound shock with
undetectable blood
pressure and pulse

Thrombocytopenia ≤ 100 000
Hematocrit rise ≥ 20%

* DHF grade III and IV are also called as dengue shock syndrome (DSS)
(WHO 2005)

7
The grades of DEN diseases are described in Table 1. Dengue fever is
characterized by the sudden onset of high fever (38-40oC) and a variety of nonspecific symptoms, including headache, retro-orbital pain, myalgia and arthralgia.
Dengue infection has an unpredictable course where most patients have a febrile
phase lasting 2 to 7 days and this is followed by a critical phase which is of
about 2 to 3 days duration. Usually during this defevercence phase, patient are at
risk of developing DHF/DSS. Symptoms and laboratory findings in DHF grade I
and II include trombocytopenia (less than 100 000) and a rise in hematocrit level
more than 20%. Spontaneous bleeding such as rash, bleeding from nose and gum or
melena distinguish DHF grade I and grade II. Weak pulse, hypotension or
undetectable blood pressure pulse indicate DHF grade III or IV.
The role of B and T memory lymphocytes in the pathogenesis of dengue
hemorrhagic fever. At the early phase of heterologous secondary infection, the
complexes of DEN virus and non-neutralizing antibody allow viral uptake via the
Fc portion of the antibody to FcγRI and FcγRII bearing cells (Littaua et al. 1990).
Consequently, a greater number of cells are infected resulting in increased viral
load, shortened incubation period and increased disease severity (Fink et al. 2006).
The preferential expansion of memory T cells with lower avidity for the infecting
serotype causes altered T cell functional responses (Mathew and Rothman 2008).
Cross-reactive CD8 (clusters of differentiation8) T cells with low binding affinity
for the current infection have less cytolitic activity. This may exacerbate the
infection and lead to significant immune-mediated tissue damage as more T cells
die and release cytokines (Mathew and Rothman 2008). In addition, low affinity
cross-reactive CD4 T cells also produce predominantly proinflamatory cytokines
and lyse bystander uninfected cells (Mathew and Rothman 2008, Rothman 2004).
Dengue vaccine and antiviral drug. Dengue vaccine has been expected as
an effective control for DEN infections. In spite of great efforts over the last seven
decades, a licenced vaccine has not been produced. All these efforts
summarized in Table

2.

are

Live attenuated vaccines (LAV) have led in the

development and clinical trials (Reviewed in Chaturvedi et al. 2005, Raviprakash et
al. 2009). However, these vaccines have
complications

due

to

their

been

associated

with

clinical

reactogenicity. As an attenuation to produce

adequate immunogenicity with minimal reactogenicity is the biggest handicap. To

8
overcome, recombinant LAV has been developed by mutation or deletion in the
viral genome (Reviewed in Raviprakash et al. 2009).

However, formulating

monovalent combinations to attain tetravalent long-lasting protective immunity has
been a big problem because of serotype dominance and competition. Also, concern
regarding an application of replicating vaccine has been a long debate due to the
possibility of mutation or recombination that can initiate virulence. Therefore,
recombinant adenovirus vector and DNA shuffling technology offer an advantage of
expressing multiple antigens from a single vector and make multivalent vaccine
easy to produce (Raviprakash et al. 2009). Nevertheless, non-replicating vaccines
are not as effective as replicating vaccines, since they can not replicate in host cells
and mimick natural infection that induces adequate long lasting immunity
(Reviewed in Chaturvedi et al. 2005, Raviprakash et al. 2009).

In addition,

genetically engineered vaccines based on particular components of DEN virus have
limitations for the immunity against other structural and non-structural components.
The utilization of more than one vaccine platform in a prime-boost strategy have
also been tried to discover an ideal DEN vaccine, which is tetravalent effective, safe
and globally affordable. The development of DEN vaccine still requires long-term
intensive studies.

Table 2 Dengue vaccine candidates in clinical and pre-clinical (NHP) trials
Replicating/
Non-replicating
Replicating
Replicating
Replicating
Non-replicating
Non-replicating
Non-replicating
Non-replicating
Non-replicating

Vaccine*

LAV
rLAV
YFV-DV
Ad-vectored
PIV
Subunit
DNA
VRP

Monovalent (M)/
Tetravalent (T)
T
T
T
T
M
M
T
M

Status

Clinical trial
Pre-clinical trial
Clinical trial
Pre-clinical trial
Pre-clinical trial
Clinical trial
Pre-clinical trial
Pre-clinical trial

*LAV: live attenuated vaccine, rLAV: recombinant LAV, YFV-DV: Yellow fever
virus-dengue virus chimera. Ad-vectored; Adeno vectored vaccine, PIV: purified
inactivated vaccine, Subunit: recombinant subunit protein vaccine, DNA: DNA
vaccine, VRP: venezuelan equine encephalitis replicon particle (Adapted from a
review by Raviprakash et al. 2009).

9

Compared with DEN vaccine, the development of antiviral to DEN virus
infection is still near the begining. There has been only few reports of DEN antiviral
drugs in NHP pre-clinical phase and their inhibition effects were not satisfactory.
Prophylactic ribavirin given one day before DEN infection was inefficient to inhibit
viremia in rhesus macaques (Malinoski et al. 1990). A recombinant human IFN-α
that was injected one day after onset of viremia could reduced viral burden and
improved viral clearance, however, further studies for more suppression is still
required (Ajariyakhajorn et al. 2005). Most of DEN antiviral drugs are still in
design or in vitro evaluation (Noble et al. 2010).
Either DEN vaccine or DEN antiviral should be evaluated in NHP, before
clinical evaluation. However, the lack of DHF animal model has hampered the
evaluation of DEN vaccine and antiviral capability to prevent DHF. Since the level
of viremia is associated with severity of disease, both vaccine and antiviral are
evaluated based on their capability to protect the animals from viremia
(Raviprakash et al. 2009, Nobel at al. 2010) .
Animal model for dengue infection. A total of 18 species from six families
of NHPs were experimentally infected by DEN virus (Reviewed by Bente and RicoHesse 2006). The Old World monkeys, the Cercopithecidae family: Japanese
macaque (M. fuscata), rhesus macaques (Macaca mulatta), cynomolgus macaques
(M. fascicularis), green monkeys (Cercopithecus aethiops), patas monkeys
(Erythrocebus patas), yellow baboons (Papio cynocephalus) and mangabeys
(Cercocebus sp.); the New World monkeys: night monkeys (Aotus sp.), squirrel
monkeys (Saimiri scureus), saimiri monkeys (Saimiri orstedii), white face monkeys
(Cebus capucinus), cotton-top marmosets (Sanguinus oedipus) and marmosets
(Marikini geoffroyi), black spider monkeys (Ateles fusciceps), red spider monkeys
(A. geoffroyi), howler monkeys (Aluoatta palliata); and some Apes: chimpanzees
(Pan troglotdytes) and white handded gibbons (Hylobates lar) were used to study
DEN infection. Some of them were susceptible to DEN infections in terms of
detectable viremia and/or antibody response. However, none of these animals shows
clinical sign. Rhesus and cynomolgus macaques are the most common animal
model for the evaluation of DEN vaccine and antiviral.

10
In spite of some interesting findings and increasing demand of pigtail
macaque (M. nemestrina) in the studies of human immunodeficiency virus (HIV),
there has been no report of pigtail macaque as a model for DEN study. Similar with
human and rhesus, pigtail possesses dendritic cell-specific intercellular adhesion
molecule-3-grabbing non-integrin (DC-SIGN), a type II membrane protein with a
C-type lectin functions as a receptor binding domain and transmission factor for
several viral pathogens (Baribaud et al. 2001). Unlike rhesus and cynomolgus
macaques that have tripartite motif 5α (TRIM5α), pigtail has TRIM5

or TRIM5

factor which is incapable to inhibit the reverse transcription of viral replication. This
fact has been associated with the exceptional susceptibility of pigtail macaque to
HIV and simian immunodeficiency virus infections (Brennan et al. 2007).
Dengue specific cytokine producing T lymphocyte measurements. The T
helper (Th) and T cytotoxic (Tc) cells are the central of cellular adaptive immunity
(Janeway et al. 2001). The main function of Th is to initiate the responses of other
cells. They are divided into two functional classes: Th1 and Th2 cells. The function
of Th1 is to activate the microbicidal properties of macrophages and to induce
memory B cells to produce IgG antibodies that are effective at opsonizing
extracellular pathogens for uptake by phagocytic cells. T helper2 cells secrete
cytokines which activate naïve antigen specific B cells to produce IgM antibodies.
The Tc cells have ability to lyse target cells.
A naïve T cell must recognize a foreign peptide bound to a self major
histocompatibility molecule (MHC) which is expressed by professional antigen
presenting cell (APC) such as macrophage, dendritic cell and B cell in order to be
activated. Peptides from intracellular pathogens that multiply in the cytoplasm are
carried into the cell surface by MHC class I molecules and activated Tc cells to kill
the cells and produce cytokines. Pathogens that replicate in intracellular vesicles or
extracellular pathogens and proteins that are internalized into the intracellular
vesicles are degraded by proteases within the vesicles. These peptide fragments bind
to MHC class II molecules and they are delivered to the surface membrane of APC
to activate Th cells. The details of degradation, transportation and presentation of
antigens by MHC class I and II molecules were reviewed by Hickling (1998).
T cell subsets and the cytokine produced are shown in Table 3. The CD stands
for cluster of differentiation, a term for a cell surface molecule that is associated

11
with one or more functions on the cells. The CD4 is usually used as a marker for Th
cells, while CD8 is mostly a marker for Tc. Interferon-γ is the most frequent
cytokine used to determine specific Th1 or Tc responses, since it is produced by
much higher percentage of T cells.

Table 3 Human T cells
T cell subset

Phenotype

Functions

Th1
Th2

CD4+
CD4+

T cytotoxic

CD4+ or CD8+

Production of IL-2, IFN- γ and TNF α
Production of IL-4, IL-5, IL-6, IL-10
and IL-13
Lyse target cells, production of IFN-γ
and TNF α

* IL: interleukin. TNF: tumor necroting factor (adapted from Hickling 1998).

Antigen specific T cells can be detected and enumerated after a short term in
vitro antigen stimulation followed by ELISPOT or IC-FC to detect T cells on a
single cell level (Lecth and Scheibenbogen 2003). The ELISPOT uses 96-well
membrane plate and coats the surface of the membrane with anti-cytokine antibody
to traps antigen induced cytokine secretion around the cells. Then, additional of
enzyme coupled second anti-cytokine antibody and substrate visualizes bound
cytokine. This is a sensitive assay that can count 10 cytokine secreting cells per one
million PBMC (Lecth and Scheibenbogen 2003). Whereas IC-FC uses brefeldin A
to trap cytokine intracellularly following antigen stimulation. Subsequently, the
cells are permeabilized, thus, specific anti-cytokine antibody conjugated with
fluorocrome can pass into the cells and react with cytokines (Lecth and
Scheibenbogen 2003). The advantage of IC-FC is its ability to phenotype the cells.
By applying anti CD-3 (CD-3 is a T cell marker), anti-CD4 and anti-CD8
monoclonal antibodies conjugated with different fluorocromes, the flow cytometer
quantify characterized T cells by the fluorocromes that are bound on and inside the
cells (Lecth and Scheibenbogen 2003).

GENERAL METHODOLOGY
Two studies were conducted for the development of pigtail macaque as
animal model in DEN research. The first study explored the possibility of pigtail
macaque as an animal model for DEN infection. It was conducted under approved
protocols by the Institutional Animal Care and Use Committee (IACUC), Naval
Medical Research Unit-2 (NAMRU-2) number 98AUC02. The second study was a
development of DEN specific cellular immunity measurements. This study utilized
samples collected during other DEN studies under protocols approved by the
IACUC of the NAMRU-2 or Animal Care and Use Committee of Primate Research
Center, Institut Pertanian Bogor. The approval numbers were 02AUC05 for DEN-1
and DEN-4, 99AUC01 for DEN-2, and P.09-08-IR for DEN-3.
Study of pigtail macaque susceptibility to DEN infection. Specific
pathogen free (tuberculosis, simian retrovirus, simian immunodeficiency virus,
simian T-lymphotropic virus, and Flavivirus) adult pigtail macaques were selected
and housed in mosquito-proof rooms at the NAMRU-2 AAALAC Internationalaccredited animal facility.
Figure 3 shows an outline of DEN injections and blood collections in this
study. The animals were separated into four groups assigned to receive either DEN1, DEN-2, DEN-3 or DEN-4 virus. Each group received two inoculations of virus.
The first inoculation, two animals in each group received live virus and another two
received phosphate buffered saline (PBS). The second inoculation, all animals in
the

group received live virus.

Each