登革熱 (Dengue fever) 是一種好發於熱帶、亞熱帶夏季，主要藉由埃及斑蚊及白線斑蚊來傳播的傳染病，是由登革熱病毒 (Dengue virus) 所引起，登革熱病毒是單股正鏈RNA病毒，依照抗原性分類可分為Ⅰ、Ⅱ、Ⅲ、Ⅳ四型，每一型都具有感染的能力。感染其中一型後身體會產生抗體並對該型終生免疫，但只要重複感染不同型的登革熱病毒，原本的抗體會因為對其它血清型病毒的中和能力不足而誘發宿主產生抗體依賴性增強反應 (Antibody-dependent enhancement, ADE)。也因為ADE的特性使得在疫苗及藥物研發上增加不少困難。至今，尚未有安全及有效的登革熱疫苗及抗病毒藥物。
我們先前研究[1]說明成熟類登革病毒顆粒不具有感染性且能誘發較高的免疫反應，它的結構具有二十面體對稱性，位於二對稱軸的E蛋白二聚體內有凹槽 (groove) 特殊結構，此特殊結構，讓結構鬆散，暴露了病毒所會隱蔽的中和表位。此外，我們從老鼠篩選出廣泛型中和抗體 DM25-3，此抗體也在研究中被證實能夠識別並中和四型登革熱。在本研究，我們使用由中興大學微衛所趙黛瑜老師實驗室所提供的pVD2i-C18S質體以及DM25-3細胞株，成功製備成熟類登革病毒顆粒 (mature dengue virus-like particle) 與DM25-3抗體，並使用人體溫度37℃做為成熟類登革病毒顆粒與抗體結合形成免疫複合物的環境溫度。接著我們使用低溫電子顯微鏡 (cryo-EM) 及立體結構重組技術解析複合物結構，我們發現DM25-3抗體接在E蛋白二聚體 (dimer) 兩端，其中抗體的重鏈 (heavy chain) 接在包含融合環 (fusion loop) 的 Domain II上，輕鏈 (light chain) 則是接在另一個E蛋白的Domain III上，說明抗體接合在E蛋白二聚體內 (Inter-dimer interface) 及E蛋白二聚體間 (Intra-dimer interface)。更重要的是，成熟類登革病毒顆粒有穩定結構，並沒有因為培養在溫度37度而膨脹或是接上抗體而改變結構，說明類病毒顆粒是個可以控制操作、攜載抗原的穩定平台，可以做為未來登革熱疫苗及抗病毒藥物研發提供參考。

Dengue fever which caused by dengue virus is a mosquito-borne infectious disease in the summer of tropics and subtropics. The dengue virus (DENV) is a single positive-stranded RNA virus of the family Flaviviridae with four serotypes, DENV-1 to DENV-4, and all four serotypes have infective capacity. After being infected with one serotype, we produced antibodies and were immune to that serotype, but the antibodies could not protect us from another serotypes, instead, the antibodies would cause antibody-dependent enhancement (ADE). ADE plays an important role in dengue pathogenesis. So far, there is no effective vaccine or antiviral drug against the DENV.
In our pervious study[1], we found mature dengue virus like particles (mD2VLP) was non-infectious and had highly immunogenic response. It had icosahedral symmetry structure with a groove located within the E-protein dimers near the 2-fold vertices that therefore exposed highly overlapping, cryptic neutralizing epitopes. DM25-3 is thus the cross-reactive neutralization antibodies that could recognize and neutralize four serotypes DENV produced from mice. In this study, DM25-3 cell line and pVD2i-C18S plasmid were kindly supported by Prof. Day-Yu Chao in NCHU. I have successfully produced and purified mD2VLP particles and antibody DM25-3 and formed the immune-complex at 37˚C, I would like to study the complex structure of mD2VLP and DM25-3 by cryo-EM and 3D reconstruction approach. Then we analyzed the cryo-EM structure of this immune-complex. We found that the three complementarity-determining regions (CDRs) in heavy chain were bound to DII including the fusion loop of one E protein, whereas the three CDRs in light chain bound to DIII of another, meaning that DM25-3 bound to quaternary epitopes. Most importantly, mD2VLP was not expanded or change the structure when induced by higher temperature or antibody binding, showing that VLP could be served as tailorable platform for loading antigenic carriers and has potential for being an effective dengue vaccine candidate.

1. Shen, W.-F., et al., Epitope resurfacing on dengue virus-like particle vaccine preparation to induce broad neutralizing antibody. eLife, 2018. 7: p. e38970.
2. Organization, W.H., Global strategy for dengue prevention and control 2012-2020. 2012.
3. Simmons, C.P., et al., Dengue. New England Journal of Medicine, 2012. 366(15): p. 1423-1432.
4. Bhatt, S., et al., The global distribution and burden of dengue. Nature, 2013. 496(7446): p. 504-507.
5. Organization, W.H., Dengue and severe dengue. 2014, World Health Organization. Regional Office for the Eastern Mediterranean.
6. Lin, R.J., T.H. Lee, and Y.S. Leo, Dengue in the elderly: a review. Expert Review of Anti-infective Therapy, 2017. 15(8): p. 729-735.
7. S, A.H., et al., Dengue structural proteins as antiviral drug targets: Current status in the drug discovery & development. European Journal of Medicinal Chemistry, 2021. 221: p. 113527.
8. Guzman, M.G. and E. Harris, Dengue. The Lancet, 2015. 385(9966): p. 453-465.
9. Centers for Disease Control, M.o.H.a.W., R.O.C.(Taiwan), Statistics of Communicable Diseases and Surveillance Report 2015. 2016.
10. Centers for Disease Control, M.o.H.a.W., R.O.C.(Taiwan), Statistics of Communicable Diseases and Surveillance Report 2019. 2020.
11. Thisyakorn, U. and T. Tantawichien, Dengue vaccine: a key for prevention. Expert Rev Vaccines, 2020. 19(6): p. 499-506.
12. Idrees, S. and U.A. Ashfaq, RNAi: antiviral therapy against dengue virus. Asian Pacific journal of tropical biomedicine, 2013. 3(3): p. 232-236.
13. Rouvinski, A., et al., Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope. Nature Communications, 2017. 8(1): p. 15411.
14. Rodenhuis-Zybert, I.A., et al., Immature Dengue Virus: A Veiled Pathogen? PLOS Pathogens, 2010. 6(1): p. e1000718.
15. Midgley, C.M., et al., An In-Depth Analysis of Original Antigenic Sin in Dengue Virus Infection. Journal of Virology, 2011. 85(1): p. 410-421.
16. Guzman, M.G., et al., Dengue: a continuing global threat. Nature Reviews Microbiology, 2010. 8(12): p. S7-S16.
17. Tully, D. and C.L. Griffiths, Dengvaxia: the world’s first vaccine for prevention of secondary dengue. Therapeutic Advances in Vaccines and Immunotherapy, 2021. 9: p. 25151355211015839.
18. Biswal, S., et al., Efficacy of a Tetravalent Dengue Vaccine in Healthy Children and Adolescents. New England Journal of Medicine, 2019. 381(21): p. 2009-2019.
19. Mukhopadhyay, S., R.J. Kuhn, and M.G. Rossmann, A structural perspective of the flavivirus life cycle. Nature Reviews Microbiology, 2005. 3(1): p. 13-22.
20. Zhang, X., et al., Cryo-EM structure of the mature dengue virus at 3.5-Å resolution. Nature Structural & Molecular Biology, 2013. 20(1): p. 105-110.
21. Lok, S.-M., The Interplay of Dengue Virus Morphological Diversity and Human Antibodies. Trends in Microbiology, 2016. 24(4): p. 284-293.
22. Kuhn, R.J., et al., Structure of Dengue Virus: Implications for Flavivirus Organization, Maturation, and Fusion. Cell, 2002. 108(5): p. 717-725.
23. Wahala, W.M.P.B. and A.M.d. Silva, The human antibody response to dengue virus infection. Viruses, 2011. 3(12): p. 2374-2395.
24. Screaton, G., et al., New insights into the immunopathology and control of dengue virus infection. Nature Reviews Immunology, 2015. 15(12): p. 745-759.
25. Wirawan, M., et al., Mechanism of Enhanced Immature Dengue Virus Attachment to Endosomal Membrane Induced by prM Antibody. Structure, 2019. 27(2): p. 253-267.e8.
26. Yu, I.-M., et al., Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion. Journal of Virology, 2009. 83(23): p. 12101-12107.
27. Junjhon, J., et al., Influence of pr-M Cleavage on the Heterogeneity of Extracellular Dengue Virus Particles. Journal of Virology, 2010. 84(16): p. 8353-8358.
28. Zhang, X., et al., Dengue structure differs at the temperatures of its human and mosquito hosts. Proceedings of the National Academy of Sciences, 2013. 110(17): p. 6795-6799.
29. Zhang, Y., et al., Structures of immature flavivirus particles. The EMBO Journal, 2003. 22(11): p. 2604-2613.
30. Gerold, G., et al., Protein Interactions during the Flavivirus and Hepacivirus Life Cycle*. Molecular & Cellular Proteomics, 2017. 16(4, Supplement 1): p. S75-S91.
31. Pierson, T.C. and M.S. Diamond, Degrees of maturity: the complex structure and biology of flaviviruses. Current Opinion in Virology, 2012. 2(2): p. 168-175.
32. Mukherjee, S., et al., The Infectivity of prM-Containing Partially Mature West Nile Virus Does Not Require the Activity of Cellular Furin-Like Proteases. Journal of Virology, 2011. 85(22): p. 12067-12072.
33. Dejnirattisai, W., et al., A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus. Nature Immunology, 2015. 16(2): p. 170-177.
34. Zellweger, R.M., T.R. Prestwood, and S. Shresta, Enhanced Infection of Liver Sinusoidal Endothelial Cells in a Mouse Model of Antibody-Induced Severe Dengue Disease. Cell Host & Microbe, 2010. 7(2): p. 128-139.
35. Urakami, A., et al., Development of a Novel Virus-Like Particle Vaccine Platform That Mimics the Immature Form of Alphavirus. Clinical and Vaccine Immunology, 2017. 24(7): p. e00090-17.
36. Cimica, V. and J.M. Galarza, Adjuvant formulations for virus-like particle (VLP) based vaccines. Clinical Immunology, 2017. 183: p. 99-108.
37. Schroeder, H.W., Jr. and L. Cavacini, Structure and function of immunoglobulins. The Journal of allergy and clinical immunology, 2010. 125(2 Suppl 2): p. S41-S52.
38. Subclasses, H.I., Useful Diagnostic Markers for Immunocompetence. 2008.
39. Chiu, M.L., et al., Antibody Structure and Function: The Basis for Engineering Therapeutics. Antibodies (Basel, Switzerland), 2019. 8(4): p. 55.
40. Sandin, S., et al., Structure and Flexibility of Individual Immunoglobulin G Molecules in Solution. Structure, 2004. 12(3): p. 409-415.
41. Vidarsson, G., G. Dekkers, and T. Rispens, IgG subclasses and allotypes: from structure to effector functions. Frontiers in immunology, 2014. 5: p. 520-520.
42. Saphire, E.O., et al., Crystal Structure of a Neutralizing Human IgG Against HIV-1: A Template for Vaccine Design. Science, 2001. 293(5532): p. 1155-1159.
43. Roux, K.H., L. Strelets, and T.E. Michaelsen, Flexibility of human IgG subclasses. The Journal of Immunology, 1997. 159(7): p. 3372-3382.
44. Graham, L. and J.M. Orenstein, Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research. Nature Protocols, 2007. 2(10): p. 2439-2450.
45. Mielańczyk, Ł., et al., Transmission Electron Microscopy of Biological Samples. The Transmission Electron Microscope - Theory and Applications, 2015. 9.
46. Bozzola, J.J., Electron Microscopy, in eLS.
47. Böttcher, B., Transmission Electron Microscopy: Preparation of Specimens, in eLS.
48. Singer, A. and F.J. Sigworth, Computational Methods for Single-Particle Electron Cryomicroscopy. Annual Review of Biomedical Data Science, 2020. 3(1): p. 163-190.
49. Mitra, A.K., Visualization of biological macromolecules at near-atomic resolution: cryo-electron microscopy comes of age. Acta Crystallographica Section F-Structural Biology Communications, 2019. 75: p. 3-11.
50. Nogales, E., Cryo-EM. Current Biology, 2018. 28(19): p. R1127-R1128.
51. Boekema, E.J., M. Folea, and R. Kouřil, Single particle electron microscopy. Photosynthesis research, 2009. 102(2-3): p. 189-196.
52. Afsaneh Khetrapal, B. Single Particle Analysis Techniques. 2018 Aug 23, 2018.
53. Galula, J.U., et al., Virus-Like Particle Secretion and Genotype-Dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine. Journal of Virology, 2014. 88(18): p. 10813-10830.
54. Maaß, S. and D. Becher, Methods and applications of absolute protein quantification in microbial systems. Journal of Proteomics, 2016. 136: p. 222-233.