VIROLOGICAL AND IMMUNOLOGICAL STUDIES
OF DENGUE VIRUS INFECTION
IN PIGTAIL MACAQUES (MACACA NEMESTRINA)
SUSANA WIDJAJA
SEKOLAH PASCASARJANA
INSTITUT PERTANIAN BOGOR
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
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 (IC-FC), 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.
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.
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.
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 anti-cytokine antibody on polyvinylidene difluoridemembrane, 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 1, DEN-3 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 helper-1) 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.
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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
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
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.
To Indonesian scientists in health research.
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
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.
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.
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.
TABLE OF CONTENTS
The roles of B and T memory lymphocytes in the pathogenesis of dengue hemorrhagic fever ... Dengue vaccine and antiviral drug ... Animal model for dengue infections ...
Dengue specific cytokine producing T lymphocyte measurements ...
GENERAL METHODOLOGY ...
LIST OF TABLES
page
1 Grading severity of dengue infection ...
2 Dengue vaccine candidates in clinical and pre-clinical trials ...
3 Human T cell subsets ...
4 Homologous anti-DEN neutralizing antibody responses after primary and secondary infections ...
5 DEN specific IFN-γ producing cells in pigtail macaques before and after DEN infection ...
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8
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LIST OF FIGURES
page
1 A schematic presentation of dengue polyprotein ...
2 The life cycle of dengue virus in the cell ...
3 Outline of virus injection and blood collection for pigtail
susceptibility study ...
4 Outline of of virus injection and blood collection for the study of cellular immunity specific to DEN measurements ...
5 The length of viremia in pigtail challenged with DEN viruses ...
6 IgM , IgG and avidity responses after primary and secondary
infections ...
7 Anti-DEN IgG subclasses after primary infection with DEN-4 ...
8 Dengue-1, Dengue-3 and Degue-4 antigen optimization by ELISPOT ...
9 Dengue-2 antigen optimization by ELISPOT and IC-FC ...
LIST OF APPENDICES
page
1 List of reagents for laboratory assays ...
2 PCR cycle condition ...
3 List of reagents for ELISPOT and intracellular staining-flow cytometry ...
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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, ADEI hypothesis) (Halstead 2003). 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 pro-inflamatory cytokines. Thus, altered T lymphocyte functions lead to DHF or DSS (Rothman 2004).
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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 H-thymidine 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.
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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, 40-60 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).
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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.
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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 Leukopenia occasionally.
of the following sings: Trombocytopenia may be present.
headache, retro-orbital pain, No evidence of plasma loss myalgia, arthralgia
* DHF grade III and IV are also called as dengue shock syndrome (DSS)
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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 non-specific 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).
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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/ Vaccine* Monovalent (M)/ Status
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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 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) .
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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).
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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 CD4+ Production of IL-2, IFN- γ and TNF α Th2 CD4+ Production of IL-4, IL-5, IL-6, IL-10 and IL-13
T cytotoxic CD4+ or CD8+ Lyse target cells, production of IFN-γ
and TNF α * IL: interleukin. TNF: tumor necroting factor (adapted from Hickling 1998).
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 International-accredited animal facility.
13
Figure 3 Outline of virus injections and blood collections for pigtail susceptibility study.
Laboratory assays were done in the Viral Diseases Program, NAMRU-2. To detect the presence of DEN virus, serum samples were analyzed by standard tissue culture in C6/36 cells, mosquito inoculation and reverse transcriptase-polymerase chain reaction (RT-PCR). The profiles of IgM, IgG, avidity IgG, subclasses IgG and neutralizing antibody were analyzed. Reagents used for laboratory assays are listed in Appendix 1. The cycles of RT-PCR and semi-nested PCR are in Appendix 2. Detail procedures are described on page 20 to 22.
A development of DEN specific cellular immunity measurements.
14
Figure 4 Outline of DEN injections and blood collections for the study of cellular immunity specific to DEN measurements.
To determine the response of pigtail to DEN infection, blood samples collected before and one month after animal injected with DEN were used. Antigen optimation was done using samples from uninfected animals that were taken for the selection of Flavivirus-free animals and samples from infected animals that were taken at the second month after challenged.
Dengue antigen was prepared from Vero cultures infected by either DEN-1 strain 16007, DEN-2 strain 16881, DEN-3 strain 16562 or DEN-4 strain 1036. Both intra and extracellular DEN proteins were collected. Control antigen was prepared similarly using uninfected Vero cells. The protein concentration in each antigen was determined using bicinchoninic acid kit (Pierce, Rockford, IL). ELISA and western blot were done to confirm the presence of dengue proteins.
Pigtail macaque
(Macaca nemestrina) and Dengue Virus Infectivity:
a Potential Model for Evaluating Dengue Vaccine Candidates
*Susana Widjaja 1, Imelda Winoto1, Jonathan Sturgis1, Chairin N Maroef1, Erlin Listiyaningsih1, Ratna Tan1, Joko Pamungkas2,3, Diah Iskandriati3,
Patrick J Blair1, Dondin Sajuthi3,4** and Kevin Randall Porter 5
1
Naval Medical Research Unit #2, Jalan Percetakan Negara 23, Jakarta 10560, Indonesia;
2
Department of Animal Diseases and Veterinary Public Health, School of Veterinary Medicine, Institut Pertanian Bogor, Jalan Agatis, Bogor 16680, Indonesia;
3
Primate Research Center, Institut Pertanian Bogor, Jalan Lodaya II/5, Bogor 16151, Indonesia;
4
Department of Clinic, Reproductive and Pathology, School of Veterinary Medicine, Institut Pertanian Bogor, Jalan Agatis, Bogor 16680, Indonesia;
5
Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, Maryland 20910, USA
**Corresponding author: Phone/Fax: +62-251-8314371, E-mail: [email protected]
17
ABSTRACT
Pigtail macaque (Macaca nemestrina) has been shown to respond to infectious disease agents, such as HIV, and is more sensitive compared to other species of macaques such as rhesus (M. mulatta) and cynomolgus monkeys (M. fascicularis). To evaluate pigtail macaque for the ability to support dengue (DEN) viremia and serve potentially as an improved model for testing DEN vaccines, a series of experiments were conducted using primary viral isolates from individuals with DEN virus infections. This study shows that pigtail macaques develop consistent, measurable viremia with all four DEN serotypes and produce immune responses sufficient to protect against homologous virus. Anti-dengue antibodies generated after infection are predominately IgG1. This species of macaque therefore appears to be a suitable model for testing DEN virus vaccine candidates. Keywords: dengue infection, Macaca nemestrina, viremia, antibody.
INTRODUCTION
Dengue fever (DF) and dengue hemorrhagic fever (DHF) are the most important arthropod-borne viral diseases worldwide. An estimated 100 million DF cases occur every year in dengue (DEN) endemic regions of the world (Halstead 1988). DHF, the more severe form of DEN infection, is associated with a mortality of 1% to 5% and may be as high as 30% to 40% in untreated patients. The tremendous public health impact of this disease emphasizes the need for an effective preventive DEN vaccine.
There are many DEN vaccine candidates in clinical and pre-clinical trials. Pre-clinical trials usually involve the evaluation of promising vaccine candidates in non-human primates (NHP). Since DHF manifests only in humans, the model for
testing the efficacy of a DEN vaccine centers on the vaccine’s ability to prevent, or
18 2006). However, the susceptibility of the pigtail macaque (M. nemestrina) to infection with DEN has not been tested.
Similar to rhesus, pigtail macaques possess dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) that has similar characteristics and functions as human DC-SIGN (Baribaud et al. 2001). This type II membrane protein with a C-type lectin functions as a receptor binding domain for dengue virus and several viral pathogens such as HIV-1 and influenza A/H5N1. This fact supports the possibility of pigtail macaques as a model for DEN infection. However, unlike other macaques, pigtails have been shown to be susceptible to HIV infection. Pigtail macaques possess the cytoplasmic body protein TRIM5ø, which is incapable of restricting HIV replication after viral entry to host cells. Other
macaques posses TRIM5α, which inhibits reverse transcription of retrovirus
(Stremiau et al. 2004, Brennan et al. 2007). The pigtail is the most potential macaque model in which HIV can cause as AIDS-like syndrome in non-human species (Agy et al. 1992, Baroncelli et al. 2008, Hatziioannou et al. 2009).
In this study, we evaluated the ability of dengue to replicate in pigtail macaques. A series of experiments where pigtail macaques were inoculated with all four serotypes of dengue virus were conducted. Viremia and anti-dengue antibody responses were studied and revealed that this monkey species may serve as a suitable model for evaluating experimental dengue vaccines.
MATERIALS AND METHODS
19 The study was conducted under a protocol approved by the Institutional Animal Care and Use Committee of the Naval Medical Research Unit #2 number 98AUC02.
Virus Inoculation and Blood Samples. The animals were separated into four groups of four animals each. One monkey was used as an alternate to replace any animal that needed to be excluded during the study for any reason. Each group was assigned to receive either DEN-1, DEN-2, DEN-3 or DEN-4 virus. Each group received two inoculations of virus. For the first inoculation, two animals in each group received virus and two received phosphate buffered saline (PBS). For the second inoculation, all animals in the group received live virus. Each group (DEN virus serotype) therefore consisted of four examples of primary infection and two of secondary infection. The extra monkey was later included in the DEN-3 group.
The DEN-1 virus used was isolated from a DEN fever patient hospitalized in Jakarta, Indonesia and passaged five times in C6/36 cell culture. DEN-2, DEN-3 and DEN-4 isolates were derived from patients hospitalized in Palembang, Bandung and Yogyakarta, Indonesia, respectively. The DEN-2 isolate was passaged five times and the DEN-3 and DEN-4 isolates were passaged 4 times in C6/36 cell culture. All isolates were obtained in 1998 with the exception of the DEN-4 isolate that was obtained in 1996. Virus stocks were prepared from clarified cell culture supernatant and stored at –70oC until used. The cells were used to confirm DEN serotype and to rule out a possibility of other related viruses contamination by indirect fluorescence assay using monoclonal antibodies to DEN-1 through DEN-4, polyclonal antibodies to Flavivirus and Alphavirus. For each inoculation, approximately 105 plaque-forming units (PFU) were administered subcutaneously in the lateral chest area. Prior to inoculation, the site was shaved and cleaned with 70% alcohol.
20 days post-inoculation, additional blood samples were obtained for anti-dengue antibody profile and antibody avidity analysis.
Dengue Virus Detection by RT-PCR. Qiamp Viral RNA mini kit (QIAGEN Gmbh, Hilden, Germany) was used to extract viral RNA from 140 μL of serum sample following manufacturer’s instruction. A total of 60 μL RNA was obtained and 5 µL used in the RT-PCR reaction. The methods of Lanciotti were used for the RT-PCR and semi-nested PCR (Lanciotti et al. 1997). PCR products were resolved by electrophoresis using a 2% agarose gel and ethidium bromide staining. Dengue viremic serum and negative serum were used as control positive and negative.
Dengue Virus Isolation. Serum samples obtained post-infection for 10 days were analyzed for the presence of virus by standard tissue culture in C6/36 cells and by mosquito inoculation. Virus isolation in cell culture was performed as described by Graham et al. (1999). For mosquito inoculation, Toxorhynchites
mosquitoes were used following the method ofYamamoto et al. (1987).
IgM and IgG Analysis. Anti-dengue IgM and IgG antibodies were detected using a commercially available antibody capture ELISA kit (Focus Diagnostic, Cypress, CA). Assays were performed following manufacturer’s procedures. A numerical index was calculated by dividing the OD of sample with OD of the cutoff control. A sample with an index greater than or equal to 1 was considered DEN antibody positive. Serum samples drawn on day 0, day 14 and day 28 were tested by IgM ELISA, while samples drawn at day 0, day 14, day 28 and day 87 were tested by IgG ELISA.
21 dilution. For samples giving an OD less than 0.6, samples were re-tested at two-fold dilutions starting at 1:10.
For samples tested at a single 1:100 dilution, the avidity index was calculated by dividing the OD from the urea treated sample by the OD of the untreated control. For samples requiring serial dilutions, fine determinations of the avidity indexes were calculated by dividing the dilution of the urea-treated curve necessary for a defined OD by the respective dilution of the control curve at the same OD. The defined OD was selected in the range 0.25–0.60 fold of the maximal OD.
Antibody Subclass Analysis. The subclasses of anti-DEN IgG produced in response to live virus infection were studied. The method of Shearer et al. (1999) was used for this analysis. The distribution of IgG subclasses was examined with the use of indirect ELISA. Briefly, dengue cell lysate antigen (DEN ag) and Vero-76 cell lysate antigen (mock ag) in carbonate-bicarbonate buffer pH 9.6 were coated onto five U8 Maxisorb microtiter plates (Nunc, Roskilde, Denmark). The plates were incubated overnight at 4oC. The plates were washed with phosphate buffer saline pH 7.4 containing 0.1% Tween 20 (PBS-T) six times and 100 μL of serum (diluted 1/100 in PBS with 0.1% Tween 20 and 5% defatted milk powder) was added into each well. After 1 hr serum incubation in 37oC, plates were washed. Horseradish peroxidase labeled sheep anti human IgG1 to IgG4 (The Binding Site, Birmingham, UK) at 1/50 dilution and horseradish peroxidase labeled goat anti human IgG (Accurate, Westbury, USA) at 1/1000 dilution were added into each plate, so plate 1 was incubated with anti-IgG1, plate 2 with anti-IgG2 and so on. The plates were incubated 1 hour in 37oC. The 2.2’-azino-di[3-ethyl-benzthiazoline sulfonate (6)] (ABTS) substrate (Kirkegaard and Perry, Gaithersburg, USA) was added after the plates were washed. Following another one hour incubation at 37oC, the absorbance was measured at 415 nm. The specific absorbance for each serum sample was calculated as the mean A 415 nm DEN ag - the mean A 415 nm mock ag.
Checkerboard titrations of Den ag and the peroxidase labeled anti-Flavivirus 4G2 (Kikergard and Perry, Gaithersburg, USA) were performed to determine the optimal dilution of DEN-1 to DEN-4 cell lysate ags.
22 neutralization as performed by Morens et al. (1985) and the results expressed as the reciprocal dilution that produces a 50% reduction in plaque count. Plaque assay was performed to determine the virus titer during viremia by inoculating 1:10 diluted serum in PBS/BA into BHK12 Clone-15 suspension.
RESULTS
Clinical Signs and Symptoms. Following infection, the animals were monitored daily for any signs and symptoms of DEN infection. There were no abnormal changes in temperature or respirations following live virus injection. The
animals’ weights remained stable before, during and after viremia. No obvious decreases in food consumption were noted.
DEN Viremia Following Infection. There were 16 episodes of experimental primary infection among the animals. Figure 5 shows the days of viremia for each DEN serotype according to the different virus detection methods. Almost all of the animals became viremic within 1 to 2 days after DEN injections. As expected, RT-PCR was the most sensitive method for detecting viremia. However, consistent viremia was detected in each of the animals by all three methods. DEN-2 resulted in the greatest average number of days viremia at 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. If going by RT-PCR and mosquito inoculation methods, the least number of viremia days occurred with DEN-4 (5±1.4 and 3.3±1). By isolation in C6/36, DEN-3 produced the least (4± 0.8).
Animals that were infected with each DEN serotype were re-challenged with the homologous virus. None of the animals re-challenged with DEN-1, DEN-2 or DEN-3 developed viremia. Two animals with secondary DEN-4 infections both developed breakthrough viremia as detected by RT-PCR. DEN-4 was detected in one animal on days 6 and 7 and in the other only on day 7.
23
Figure 5 The length of viremia days from each individual by (a) C6/36 isolation, (b) mosquito inoculation and (c) RT-PCR. Each bar represents the number of viremia days from each individual.
24 anti-DEN IgM antibody index was detected on day 14. Some monkeys showed no IgM antibody at day 28. Anti-DEN IgG was detectable on day 28 and showed a steady increase up to day 87. In animals that received secondary infections, the highest anti-DEN IgG ELISA index was detected earlier on day 14, while anti-DEN IgM did not increase at all (Fig 6b). The avidity of anti-DEN IgG antibodies steadily increased after primary infection (Fig 6a). Following secondary infection there was a further increase in antibody avidity indicating additional maturation of anti-DEN IgG (Fig 6b).
25
Figure 7 Anti-DEN IgG subclasses after primary infection with DEN-4.
Figure 7 shows the anti-DEN IgG subclass antibody response to DEN-4 virus. After primary infection, anti-DEN IgG1 was detected primarily and increased through day 87. The other IgG subclasses were either undetectable or detectable at low levels. Following secondary infection, IgG1 continued to predominate at higher levels. Increases in the proportions of the other IgG subclasses were also noted (data not shown). By 87 days post secondary infection, all levels showed a decrease but were still detectable. Similar patterns were observed with DEN-1 and DEN-2 (data not shown). Anti-DEN-3 IgG subclass analysis was not performed.
26 Table 4 Homologous anti-DEN neutralizing antibody responses after primary and
secondary infections.
This study demonstrated that pigtail macaques are susceptible to infection with all four wild type DEN viruses, giving rise to consistent viremia for several days as detected by three different viral detection methods. Since macaques do not manifest obvious symptoms of dengue disease, vaccine developers gauge the
protective efficacy of experimental dengue vaccines by evaluating the vaccine’s
27 detection, making comparisons between different macaque species regarding their ability to produce viremia following live dengue virus infection may be inappropriate. Nevertheless, the days of viremia caused by DEN-1 and DEN-4 primary infections in this study were slightly longer than the days of viremia seen in cynomolgus macaques infected with these serotypes (Koraka et al. 2007). In addition, the overall length of viremia in pigtails was longer than that seen in earlier rhesus monkey studies (Halstead et al. 1973, Freire et al. 2007). One prior study with rhesus monkeys showed that several strains of DEN viruses failed to produce viremia (Freire et al. 2007). The current study utilized only a single wild type dengue viral isolate for each serotype. It remains to be determined if the viremia characteristics seen with this set of viruses is characteristic of the viremia that would occur if other viruses were used. While in theory, the same level of viremia should occur with other viruses, further evaluation of the pigtail macaque with other isolates is warranted.
28 There was no anti-pigtail IgG subclass available commercially, when IgG subclass assay was set. Anti-human IgG subclasses were chosen, since these reagents bound more anti-pigtail IgG compared with anti-macaque IgG subclasses (data not shown). Anti-DEN IgG1 was primarily detected and predominant IgG subclass following primary and secondary infections in pigtails. In humans, IgG1 and IgG3 were the predominant IgG subclasses throughout the course of illness regardless of whether the illness was characterized as DF, DHF or DSS (Thein et al. 1993; Koraka et al. 2001). In this study, it is not further determined whether this different due to different species response or less cross reaction of anti-human IgG3 than anti-human IgG2.
High-titer anti-DEN neutralizing antibodies were produced in response to dengue virus infection in each animal and demonstrated cross-reactivity against the DEN serotypes used in the plaque reduction neutralization assay. These antibodies resulted in solid protection against challenge with the homologous dengue virus serotype, although minimal break-through viremia occurred in two DEN 4 animals as detected by RT-PCR. Because no live virus was detected in these animals, this could have just represented residual RNA from non-viable virus.
The ability of pigtail monkeys to support the replication of all four serotypes of dengue raises the question of whether this species is suitable for evaluating the efficacy of experimental dengue vaccines and anti-DEN therapeutic drugs. Further studies are planned to evaluate the efficacy of DNA-based vaccines using the pigtail model.
ACKNOWLEDGMENT
This work was supported by the Naval Medical Research Center work unit 61102A.S13.S.S1415. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the US Government. Authors (SW, IW, JS, CNM, EL, RT, PJB, KRP) as employees of the U.S. Government or military service members, conducted the work as part of their official duties. Title 17 U.S.C. §105
provides that ‘Copyright protection under this title is not available for any work of
the United States Government.’ Title 17 U.S.C. §101 defines a US Government work as a work prepared by a military service member or employee of the US
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The Measurements of Dengue Specific Interferon-
γ P
roducing T
Lymphocytes in Pigtail Macaques (Macaca nemestrina)
Susana Widjaja 1,2,*, Dasep Purwaganda1, Victor A Sugiharto1, Imelda L Winoto1, Gary T Brice1, Timothy H Burgess1, Kevin R Porter 3, Charmagne G Beckett3, Maya Williams1, Joko Pamungkas2,4,5, Diah Iskandriati2,5, Dondin Sajuthi2,6 and
Patrick J Blair1
1
Naval Medical Research Unit #2, Jalan Percetakan Negara 23, Jakarta 10560, Indonesia;
2
Primatology Major, Graduate Program, Institut Pertanian Bogor, Jalan Lodaya II/5, Bogor 16151, Indonesia;
3
Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, Maryland;
4
Department of Animal Diseases and Veterinary Public Health, School of
Veterinary Medicine, Institut Pertanian Bogor, Jalan Agatis, Bogor 16680, Indonesia;
5
Primate Research Center, Institut Pertanian Bogor, Jalan Lodaya II/5, Bogor 16151, Indonesia;
6
Department of Clinic, Reproductive and Pathology, School of Veterinary Medicine, Institut Pertanian Bogor, Jalan Agatis, Bogor 16680, Indonesia;
34
ABSTRACT
Pigtail macaques (Macaca nemestrina) have been reported susceptible to the infections of all four dengue (DEN) serotypes and suitable for DEN vaccine evaluation. To enhance DEN study in pigtail macaques, ELISPOT and intracellular cytokine staining-flow cytometry (IC-FC) were developed to measure DEN specific interferon-γ (IFN-γ) producing T lymphocytes. Peripheral blood mononuclear cells (PBMC) collected before and after DEN infections were tested. ELISPOT results show increase of DEN specific IFN-γ producing cells as an individual response of pigtail to DEN-1, DEN-3 or DEN-4 infection. ELISPOT and intracellular cytokine staining-flow cytometry (IC-FC) were run side by side to quantitate DEN specific lymphocytes following primary and secondary DEN-2 infections using pools of PBMC. ELISPOT revealed an increase of DEN specific IFN-γ producing cells following primary infection and a significant increase after secondary infection. The patterns of DEN specific IFN-γ producing CD3+CD4+ and CD3+CD4- T lymphocytes by IC-FC were similar with the pattern of DEN specific IFN-γ producing cells. Therefore, ELISPOT and IC-FC can be used to study DEN specific IFN-γ producing T lymphocytes in pigtail.
Keywords: Dengue, T lymphocytes, pigtail macaque, IFN-γ
INTRODUCTION
