||Study on the protective effects of antibodies against C-terminal region-modified dengue virus nonstructural protein 1 in mouse model
||Department of Microbiology & Immunology
nonstructural protein 1
登革病毒 (dengue virus; DV) 是一種由蚊子所傳播的病毒，感染病人所造成的臨床症狀包括輕微的登革熱 (dengue fever; DF) 以及嚴重可能會致死的登革出血熱 (dengue hemorrhagic fever; DHF) 和登革休克症 (dengue shock syndrome; DSS)。在病人身上所出現的臨床症狀主要包括血管病變滲漏、血小板低下、出血時間延長以及大量細胞激素的產生。每年都有超過五千萬的登革感染病例，然而至今仍舊未研發出良好的抗病毒藥物以及疫苗可供使用。實驗室先前研究顯示，抗登革病毒非結構性蛋白1 (NS1) 抗體會交叉反應到人類的內皮細胞以及血小板，導致內皮細胞凋亡以及血小板凝集機制異常。依據蛋白質體學序列分析顯示NS1蛋白質之C端序列與自體抗原有高度相似性，考量以NS1為疫苗發展的安全性，我們將具有交互作用的抗原片段 (胺基酸271-352) 去除，產生了名為C NS1之蛋白質，另外也將此去除的片段由日本腦炎病毒 (JEV) NS1蛋白質之C端置換成名為DJ NS1之蛋白質。過去實驗室研究結果顯示，抗C NS1以及抗DJ NS1抗體對人類內皮細胞以及血小板的交叉反應能力較抗DV NS1抗體低。我們進一步於對DV更有感受性之STAT1-/-小鼠建立起DV感染的動物模式，實驗結果顯示，DV2感染STAT1-/-小鼠會造成小鼠出血時間延長、脾臟腫大以及大量細胞激素的產生。進一步於此動物模式觀察抗體治療性效果，結果指出，抗C NS1以及抗DJ NS1抗體能有效抑制由DV2感染所造成之小鼠出血時間延長以及與疾病嚴重程度相關之血清介白素 6 (IL-6) 以及巨噬細胞發炎蛋白質1 (MIP-1) 的產生。此外，抗C NS1以及抗DJ NS1抗體也被證實能有效降低由另一血清型登革病毒DV3感染所造成之小鼠出血時間延長、單核球趨化蛋白質1 (MCP-1)之產量以及巨噬細胞的浸潤程度。綜合以上結果，抗C NS1以及抗DJ NS1抗體不僅能提供同一血清型病毒感染治療性效果，對於不同血清型病毒的感染也提供一定的治療性效果，這也提供了登革疫苗發展所需之重要資訊。
Dengue virus (DV) is a mosquito-transmitted virus which may cause dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Dengue patients show symptoms with plasma leakage, thrombocytopenia, bleeding tendency, and elevated cytokines. Over 50 million cases of DV infection occur each year, but there is still no approved antiviral treatment or vaccine available. Our previous studies showed that antibodies (Abs) against DV nonstructural protein 1 (NS1) cross-reacted with human endothelial cells and platelets and induced endothelial cell apoptosis and platelet dysfunction. Based on sequence analysis, the C-terminal region of DV NS1 protein contains homologous sequences with self-antigens. For safety concerns of vaccine development, we generated a C-terminal region (amino acid 271-352)-truncated DV NS1 protein or replaced the C-terminal region with JEV NS1 protein, designated C NS1 and DJ NS1, respectively. Anti-C NS1 Abs and anti-DJ NS1 Abs showed lower binding activity to human endothelial cells and platelets than that of anti-DV NS1 Abs. We further established a DV infection model in STAT1-/- mice which are more susceptible to DV as compared with the wild-type mice. Results showed that DV infection in STAT1-/- mice caused prolonged bleeding time, splenomegaly, and elevated levels of serum cytokines and chemokines. The therapeutic effects of anti-C NS1 Abs and anti-DJ NS1 Abs were further investigated in this mouse model. DV2-infected mice treated with anti-C NS1 Abs and anti-DJ NS1 Abs showed significant reduction of prolonged bleeding time and serum interleukin-6 and macrophage inflammatory protein-1 production, which were associated with disease severity. In addition, anti-C NS1 Abs and anti-DJ NS1 Abs also reduced DV3-induced prolonged bleeding time and monocyte chemotactic protein-1 production both in serum and local infection sites. Furthermore, macrophage infiltration to the local infection site was also inhibited in DV3-infected mice by treatment with anti-C and anti-DJ NS1 Abs. Taken together, these studies show not only the therapeutic effects of anti-C and anti-DJ NS1 Abs in DV2-infected mice but also cross-protective effects in DV3-infected mice. The results also provide important information for DV vaccine development.
Chinese Abstract I
Table and Figure List VIII
Characteristics of dengue virus 1
Life cycle and target cells of dengue virus 3
Epidemiology of dengue virus 4
Clinical symptoms, classification, and diagnosis of dengue disease 5
The pathogenesis of dengue virus infection 7
Animal models of dengue virus infection 12
Vaccine development for dengue virus 13
Objective and Specific Aims 15
1. Preparation of Abs against C-terminal region-modified DV NS1 proteins and study of their protective effects. 16
2. Evaluation of the therapeutic effects of anti-C NS1 and anti-DJ NS1 Abs in DV2-infected STAT1-/- mice. 16
3. Investigation of the cross-therapeutic effects of anti-C NS1 and anti-DJ NS1 Abs in DV3-infected STAT1-/- mice. 16
Materials and Methods 18
A. Materials 18
A-1 Mice 18
A-2 Cell lines 18
A-3 Virus 18
A-4 Preparation of recombinant proteins and Abs 19
A-5 Drugs and reagents 19
A-6 Antibodies 22
A-7 Kits 22
A-8 Consumables 23
A-9 Instruments 24
B. Methods 25
B-1 Antibody titer determination 25
B-2 Mouse tail bleeding time determination 25
B-3 Cell cultures 25
B-4 Plaque assay 26
B-5 Infection of endothelial cells with DV2 26
B-6 Flow cytometry 26
B-7 Western blotting 27
B-8 Histopathology 27
B-9 Animal protection model 27
B-10 Detection of serum cytokine levels 28
B-11 Detection of serum chemokine levels 28
B-12 Immunohistochemistry staining 28
B-13 Statistics 29
1. Preparation of Abs against C-terminal region-modified DV NS1 proteins and study of their protective effects. 30
1.1 Preparation of the DV NS1, JEV NS1, C NS1, and DJ NS1 proteins. 30
1.2 Preparation of Abs against DV NS1, JEV NS1, C NS1, and DJ NS1 proteins. 30
1.3 Anti-C NS1 Abs can recognize NS1 protein expressed on DV-infected endothelial cells. 31
2. Evaluation of the therapeutic effects of anti-C NS1 and anti-DJ NS1 Abs in DV2-infected STAT1-/- mice. 31
2.1 A DV-infection model in STAT1-/- mice. 31
2.2 Anti-C and anti-DJ NS1 Abs reduce DV2-induced prolonged bleeding time. 32
2.3 Anti-C and anti-DJ NS1 Abs reduce DV2-induced cytokine and chemokine production in STAT1-/- mice. 33
3. Investigation of the cross-therapeutic effects of anti-C NS1 and anti-DJ NS1 Abs in DV3-infected STAT1-/- mice. 34
3.1 DV3-infected STAT1-/- mice show similar pattern as DV2-infected STAT1-/- mice of prolonged bleeding time and cytokine and chemokine production. 34
3.2 Anti-C and anti-DJ NS1 Abs reduce DV3-induced prolonged bleeding time. 34
3.3 Anti-C and anti-DJ NS1 Abs may reduce serum cytokine and chemokine production in DV3-infected STAT1-/- mice. 35
3.4 Anti-C and anti-DJ NS1 Abs reduce MCP-1 production, both in serum and local infection sites, and macrophage infiltration. 35
Table and Figures 53
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