登革病毒感染的致病機轉---動物模式(II)

行政院國家科學委員會專題研究計畫 期中進度報告

登革病毒感染的致病機轉：動物模式（2/3）

計畫類別： 個別型計畫 計畫編號： NSC92-3112-B-006-017- 執行期間： 92 年 05 月 01 日至 93 年 04 月 30 日 執行單位： 國立成功大學微生物免疫學研究所 計畫主持人： 黎煥耀 共同主持人： 林以行，劉校生 報告類型： 完整報告 報告附件： 出席國際會議研究心得報告及發表論文 處理方式： 本計畫可公開查詢 中 華 民 國 93 年 3 月 24 日

基因體醫學國家型科技計畫

National Research Program for Genomic Medicine

National Science Council, the Executive Yuan, ROC.

計畫名稱-中文

登革病毒感染的致病機轉：動物模式

計畫名稱-英文

Pathogenesis of dengue virus infection: Animal model

報告類別：□ 新進研究計畫 □ 修正後計畫書

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年度成果報告 (New Proposal) (Revised Proposal) (Progress Report) 計畫類別：

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個別型計畫 □ 整合型計畫

(Individual Project) (Program Project) 計畫編號：NSC92-3112-B006-017

計畫主持人 (Principle Investigator)：黎煥耀 共同主持人 (Co-Principle Investigator)： 執行單位 (Institution)：國立成功大學

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National Research Program for Genomic Medicine

National Science Council, the Executive Yuan, ROC.

Progress Report—Research Project

基因體醫學國家型科技計畫

國科會延續性計畫進度報告

個別型計畫 (Individual Project)

Program Classification:

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Genomic Medicine … Bioinformatics

… Proteomics & Structural Genomics … ELSI

Project Number: NSC91-3112-B006-003 (計畫編號) NSC Funding Number: NSC93-3112-B006-001 (93年度國科會預核編號) (in Chinese) 中文 登革病毒感染的致病機轉：動物模式 Title of Project 計畫名稱 (in English) 英文

Pathogenesis of dengue virus infection: Animal model

(in Chinese) 中文

國立成功大學微生物及免疫所

Institution

研究（執行）單位

(in English) 英文

Department of Microbiology & Immunology, College of Medicine, National Cheng Kung University

(in Chinese) 中文 黎 煥 耀 Principle Investigator 計畫主持人 (in English) 英文 Huan-Yao Lei FY 2002 2003 2004 Total Budget 2,057,000 2,084,600 2,067,700 6,209,300

(in NT dollars: 1USD = 34 NTD)

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TABLE OF CONTENTS

Progress Report... ３

1. Response to previous reviewers’ critiques... ３ 2. Specific Aims ... ４ 3. Progress Summary... ５ 4. Projected Timeline & Brief Summary of Plans for Next Year... ９ 5. Personnel ... １０ 6. Publications and/or Patents ... １１ 6a. Publications ... １１ 6b. Patents ... １２

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Progress Report

1. Response to previous reviewers’ critiques

Please describe the previous reviewers’ critiques and how based on the critiques, you made modifications to specific aims, experimental design, or resource allocation etc. Critic of Reviewer 1.

The progress has been somewhat disappointing. Although the funding period has been short so far, more progress should have been expected because this is a continuing project in Dr. Lei’s laboratory. He needs to be more focused in his approach. Overlap between this funding and funding from other sources should also be looked into.

Critic of Reviewer 2.

This grant application was well received by the Study Section during the last review. The four original specific aims of the research proposal were original and hypothesis-driven. There was no change of aims in this progress report and no new budget provided. The research progress was minimal. However, since the grant was not funded until September of last year, the grantee cannot be overlay criticized for this lack of research progress.

We have tried to improve the efficacy of grant execution. The specific goal of this project is to establish a mouse model for dengue virus-induced dengue

hemorrhagic fever/dengue shock syndrome. Based on our previous study on clinical dengue patients, our theory for the immunopathogenesis become more evident. The association between dengue virus infection and anti-platelets and anti-endothelial cells auto-antibody production is confirmed. But its cause-effect needs to be verified in mouse infectious model. We are in the process of the experiments as stated in the following progress report. On the clinical part, we obtained a NHRI intramural contract grant NHRI-CN-CL8901P (The pathogenic studies on dengue virus infection-induced DHF/DSS, 2000/1/1 – 2003/6/30). This is a clinical study on dengue patients, which will complement to our animal study, and provides a clinical relevance to this project.

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2. Specific Aims

Please state the overall goals of the project, and specific aims, as reviewed and approved by the Study Section and actually awarded. If these specific aims as actually funded did not differ in scope from those actually pursued during the grant period, and if the aims have not been modified, state this. If they have been modified, give the revised aims.

The goal of this project is to establish a mouse model to study the pathogenesis of dengue virus infection. The specific aims as originally proposed are:

1. Set up a dengue infectious mouse model with DHF/DSS manifestation.

2. Understanding the mechanism of dengue virus-induced autoimmunity: the role of molecular mimicry.

3. Modify the antibody-dependent enhancement hypothesis to explain the pathogenesis of DHF/DSS.

4. Propose and verify the new hypothesis of immunopathogenesis for dengue virus

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3. Progress Summary

Summarize concisely the results obtained for each specific aim during the past year (or reporting period). Negative results, if any, should also be included and approaches taken to improve the prospects of the project discussed. (Do not exceed 5 pages, not including figures and references.)

Based on the clinical study of dengue patients, we have proposed an

immunopathogenesis for DHF/DSS. We have proposed a new immunopathogenetic hypothesis for dengue virus infection. A strong over-activation of the immune response not only impair the immune response to clear the virus, but also result in overproduction of cytokines that affect monocytes, endothelial cells, and

hepatocytes. The infection of B cells and monocytes shall play a crucial role in the enhanced production of anti-platelet or anti-endothelial cell autoantibodies. The aberrant generation of anti-NS1 antibodies that cross-react with platelet or

endothelial cells has been associated with dengue disease. Platelets are destroyed by anti-NS1 cross-reactive anti-platelet autoantibodies. Anti-NS1 antibodies also induce endothelial cell apoptosis. Therefore, anti-platelet or anti-endothelial cell autoantibodies might be involved in the clinical manifestation of thrombocytopenia and endothelial cell dysfunction. Dengue-virus-induced vasculopathy and

coagulopathy are also involved in the pathogenesis of hemorrhage, and the

unbalance between coagulation and fibrinolysis activation increases the likelihood of severe hemorrhage in DHF/DSS. Hemostasis is maintained unless the

dysregulation of coagulation and fibrinolysis persists. This immunopathogenesis of DHF/DSS can account for specific characteristics of clinical, pathologic, and epidemiological observations in dengue virus infection. In this theory, several important issues need to be emphasized. First, the association of acute dengue virus infection with the production of auto anti-platelet and anti-endothelial cell

antibodies, this is the first example for acute viral infection to induce autoimmunity. Second, a molecular mimicry exists between dengue virus protein and platelet or endothelial cell antigen. Third, the cytokine profile such as IFN-γ and IL-6 or the auto-antibody responses are similar among primary infected infants, secondary infected children or dengue infected adults or elders. Fourth, in addition to

monocytes, B cells can be infected by dengue virus. All these findings are novel or first-reported in dengue research filed.

In past year, we primarily focused on the specific aim 2 (understanding the mechanism of dengue virus-induced autoimmunity: the role of molecular mimicry) and the specific aim 3 (study the antibody-dependent enhancement hypothesis to explain the pathogenesis of DHF/DSS), and have obtained the following results.

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1. Generate a panel of monoclonal anti-NS1, anti-E, and anti-preM antibody from dengue-infected mice. These reagents provide a very useful tool for our study on dengue virus infection. Interestingly, most of the clones against NS1 will cross-react to platelet while the anti-preM antibody clones will cross-react with endothelial cells.

2. Epitope recognized by anti-preM was mapped to M3 peptide, amino acid 53-67 of preM (CPFLKQNEPEDIDCW). We are in the process to identify the

molecular mimicry on platelet or endothelial cells. The blast search found there are homology between M3 peptide and Chain B of Traf6-Cd40 Complex, Chain A, Structure Of A Dystrophin Ww Domain Fragment In Complex With A Beta-Dystroglycan Peptide, Chain A, Crystal Structure Of The Glycosylated Five-Domain Human Beta2-Glycoprotein I Purified From Blood Plasma, or Chain B, Human Procarboxypeptidase B. The proteomic analysis with two dimension and MALDI-TOF is undergoing.

3. Develop a simple flow cytometric analysis to detect the dengue-infected cells. Using the cytoplasmic staining with anti-E and anti-NS monoclonal antibodies, the percentage of dengue-infected cells can be determined and the viral antigen during virus replication in the cells can be quantitated on a cell basis. This methodology will be very useful to study the mechanism of antibody dependent enhancement (ADE) phenomenon. It can allow us to differentiate the effect of ADE: whether it is to increase the percentage of dengue-infected cells or to enhance the virus replication in the cell.

4. Monocytes are thought to be the major cell target for dengue virus. However, B cells can also be infected by dengue virus. Virus infection also induced B cells to produce IL-6 and TNF-α. In addition, heterologous antibody was able to enhance both virus and cytokine production in B cells. Using B cell lines or B cell hybridomas as models, it was demonstrated that B cells can support dengue virus replication and B cells also undergo activation. But surprisingly, only B cell hybridomas that secret the anti-preM antibody can be infected. The

anti-preM antibody seems to play some role to enhance the dengue virus infection.

5. Anti-preM antibody can mediate the ADE. The anti-dengue virion antibody such as anti-E antibody can enhance the dengue infection on Fc receptor bearing cells, such as monocytes, macrophages, dendritic cells, and K562 cells. This enhancement is observed at the sub-neutralization antibody level.

However, we found that anti-preM antibody can also mediated the

enhancement of dengue virus infection on Fc receptor bearing cells. This enhancement was not sub-neutralization related. It can even enhance the dengue virus infection on non-Fc receptor bearing cells such as BHK cells.

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6. With the cooperation of Dr. Hsiao-Sheng Liu, we have generated dengue-2 viruses produced from the infections full-length cDNA clones. The full-length viral RNA in vitro transcribed from above cDNA was injected intracerebrally and propagated in the brain of the suckling mice. The mice will paralyze at two weeks post injection, the dengue virus is demonstrated to replicate in brain and can be isolated from brain. Dengue virus were then isolated and cultured in C6/36 cells. Using this protocol, we can generate mutant form as well as chimera dengue virus with substituted genes (from other three serotypes) on the same serotype 2 backbone. The E, preM or core genes from four different serotypes will be switched one another to generate chimera virus, and will be used in the sequential infection experiments. The virus virulence can also be studied using this approach of chimera cDNA from different serotypes of dengue virus

7. Dengue virus antigens can stimulate a unique early-type hypersensitivity (ETH) as well as classical delayed-type hypersensitivity. The 1 h-early type

hypersensitivity has the characteristics of increased vasopermeability. When dengue antigen is injected intravenously into dengue antigen-primed mouse, the mouse will develop hemoconcentration as well as thrombocytopenia. Some of the mice will die within half an hour due to the loss of effective blood volume. If the mice were infected with type 2 dengue virus, two months later, they were challenged with the dengue antigen intravenously. The

thrombocytopenia was manifested at 10 min after challenge. We proposed that dengue virus once infect the mouse would stimulate the dengue

antigen-specific ETH. When the mouse is re-stimulated with the dengue antigens, the anaphylactic response such as hemoconcentration and thrombocytopenia is manifested. This is a two-stage reaction, it needs

re-stimulation with dengue virus antigen. We are in the process to manipulate the response to develop the hemoconcentration and thrombocytopenia in one step of dengue virus infection.

8. Our previous study showed that dengue virus can infect mouse, induce immune deviation, and generate transient thrombocytopenia that resembles only DF. However, the clinical manifestation cannot progress to DHF/DSS. The dengue genome can only be detected in blood at day 2 post dengue virus injection. No other organs such as spleen, lymph node, liver, lung, heart, thymus, brain, kidney reside dengue virus after 3 days post injection. We suspect that the inoculated dengue virus is cleared rapidly, there is no chance to replicate further. Therefore no high viral load was observed and no subsequent responses

followed. We found that the anti-preM hybridoma can be infected easily. We are in the process to transplant the hybridoma into the syngenic BALB/c mice, then infect the hybridoma with dengue virus. It is expected that dengue virus

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can continue to replicate in the hybridoma an spread into other tissue to produce more damage.

In summary, DHF/DSS can develop in either primary dengue virus infection or secondary infection although the possibility to develop DHF/DSS is higher in secondary than primary infection. Both the clinical symptom, the cytokine profile and autoantibody response to platelet and endothelial cells are similar. But, high dengue viremia titer was associated with increased disease severity. Peak virus titers were 100- to 1000-fold higher for patients who developed DSS than those with DF in Thailand’s dengue-infected children. Patients with a secondary antibody response were twice as likely to have DHF, compared with those with a primary antibody response. Apparently, virus load is a major contributing factor for development of DHF/DSS. Whether virus load is reflected on its virulence or high growth rate in vivo or antibody enhancement requires further investigation. We think that the antibody-dependent enhancement is the key element to solve the puzzle of

DHF/DSS pathogenesis. Therefore, we focus on this specific aim in the past year. Together with the exploration of cellular targets for dengue virus infection and its interaction with the enhancing antibody, the mechanism of enhancing antibodies (increasing the infected cell mass, enhancing the virus replication, or preventing apoptosis) can be delineated. The animal model of DHF/DSS can be developed eventually.

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4. Projected Timeline & Brief Summary of Plans for Next Year

Provide a short paragraph to describe the plans for next year including a realistic timetable and appropriate milestones for the project, based on the progress reached so far. (Do not exceed 1 page.)

May 1, 2004 to April 30, 2005:

We will continue studying the mechanism of dengue virus-induced autoimmunity: the role of molecular mimicry. The relationship of molecular mimicry between dengue NS1 or preM and platelet or endothelial cell will be established. The NS1 or preM transgenic mice will be generated, then infected with dengue virus. The cause-effect of dengue virus-induced autoimmunity can be determined. The ADE phenomenon will be verified in vivo. The mouse DHF/DSS model is expected to be set up.

I. Understanding the mechanism of dengue virus-induced autoimmunity: the role of molecular mimicry.

A. The platelet auto-antigen determination recognized by anti-NS1 cross-reactive monoclonal antibody by Proteomic-based analysis.

B. The endothelial cell auto-antigen determination recognized by anti-preM cross-reactive monoclonal antibody by Proteomic-based analysis.

C. The cause-effect of autoimmunity-induced thrombocytopenia and endothelial cell damage.

II. Study the antibody-dependent enhancement mechanism A. The working mechanism of ADE

a. Increasing the infected cell mass b. Enhancing the virus replication c. Preventing apoptosis

B. Confirm the ADE in vivo

a. Secondary infection with the same serotype of dengue virus

b. Sequential infection with different serotypes of chimeria dengue virus c. Set up a mouse dengue virus infection for DHF/DSS

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5. Personnel

Summarize the personnel involved in the project during the grant period. List the personnel in accordance to the following categories: (1) senior investigators, including visitors; (2) postdoctoral fellows; (3) graduate students; (4) technicians or research assistants. Specify for each individual the period of involvement and the percentage commitment of effort.

Name

In Chinese In English Position Title

Education Degree % of personal effort on this project Job Description or Responsibilities 黎煥耀 湯麥可 黃國珍 楊育靜 Huan-Yao Lei Michael Theron Kuo-Jean Huang Yu-Ching Yang Senior investigator Technician Ph.D. student Master student Ph.D. Master Master Bachelor 30% 100% 100% 100%

P.I. of this project

Help executing this project Perform the animal

experiments

Perform the monoclonal antibody analysis

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6. Publications and/or Patents 6a. Publications

List the title and complete references (author(s), journal or book, year, page number) of all publications directly resulting from studies supported by the project (i.e., with citation of this grant in the acknowledgement section). List the publications for the project in accordance to the following categories: (1) manuscripts published and accepted for publications; (2) manuscripts submitted; and (3) conference proceedings. Provide one copy of each publication not previously reported to the National Science Council in the Appendix.

1. Hung, N.T., N.T. Lan, Y-S. Lin, C-F. Lin, L-B. Lien, K-J. Huang, T-M. Yeh, D.Q. Ha, V. T. Q. Huong, L-C. Chen, L. T. My, J.-H. Huang, C-C. Liu, and H-Y. Lei. Anti-platelet and

anti-endothelial cell autoantibodies in Vietnamese infant and children with dengue hemorrhagic fever. (submission).

2. Hung, N.T., N.T. Lan, H-Y. Lei, Y-S. Lin, L. B. Lien, K-J. Huang, C-F. Lin, D.Q. Ha, V. T. Q. Huong, L. T. My, T-M. Yeh, J.-H. Huang, C-C. Liu, and S. B. Halstead. The association between sex, nutritional status and the severity of dengue hemorrhagic fever, and immune status in infants with dengue hemorrhagic fever. (in preparation).

3. Lee, Y.R., K-J. Huang, H-Y. Lei, S-H. Chen, T-M. Yeh, Y-S. Lin, and H-S. Liu. Establishment of a suckling mice system to rapidly screen and produce infections dengue-2 virus from full-length cDNA. (submission).

4. Huang, K-J., Y-S. Lin, T-M. Yeh, H-S. Liu, H-S. Chen, C-C. Liu, and H-Y. Lei. Generation of anti-platelet autoantibody during dengue virus infection (submission).

5. Huang, K-J., Y-S. Lin, T-M. Yeh, H-S. Liu, S-H. Chen, C-C. Liu, and H-Y. Lei. Dengue virus infects endothelial cells ex vivo and induce inflammation. (in preparation).

6. Lei, H-Y., Y-S. Lin, K-J. Huang, and C-C. Liu. Dengue virus infection-induced pathogenesis: cellular targets, enhancing antibody, autoantibody and molecular mimicry (in preparation). 7. Lei, H-Y., Y-S. Lin, T-M. Yeh, K-J Huang, H-S. Liu, S-H. Chen, C-C Liu, N.T. Hung, and N. T.

Lan. 2004. Immunopathogenesis of Dengue Hemorrhagic Fever. (In Vietnamese) 8. Huang K-J., Y-S. Lin, H-S. Liu, T-M. Yeh, C-C. Liu, and H-Y. Lei. Anti-preM

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6b. Patents

List all inventions disclosed, patents filed, and patents granted. Please note the inventors, assignee, title of patent, country or area where patent applied for, filing or issued number and date.

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