茲卡病毒是一種蚊子傳播的黃病毒，最早在獼猴身上發現，後來成為一種廣泛存在的人類疾病。自2015年爆發以來，茲卡病毒成為全世界關注的健康問題。除了可以通過蚊子傳播傳播給他人外，這種病毒還可以通過性交和母嬰垂直感染。這病毒導致成人嬰兒小頭畸形和格林 - 巴利綜合症的數量增加。考慮到這病毒有多危險和危及生命，設計一種突出，有效，安全和有效的疫苗被視為重要的。在這項研究中，提出了鞭毛素融合蛋白的鼻噴疫苗。我們利用甘氨酸 - 絲氨酸（GS）作為架橋將茲卡病毒包膜蛋白結構域三（ZDIII）與鞭毛蛋白連接來做為抗原設計。利用此設計原理我們設計了5 種不同組別 (FliC-ZDIII，FliC-2ZDIII，FliC-3ZDIII，FliCΔD3-2ZDIII和FliCΔD2ΔD3-3ZDIII)。TLR5活性體外測定顯示觸發FliC-ZDIII和FliCΔD3-2ZDIII的NF-κB活性有顯著的反應，特別是雖然FliCΔD3-2ZDIII已經失去部分鞭毛蛋白，但它仍然保有足夠的NF-κB活性。動物研究結果顯示FliC-ZDIII和FliCΔD3-2ZDIII在B細胞和T細胞上均誘導有效的免疫應答。此外，這兩種蛋白質也顯示出對ZIKV的顯著中和能力。然而，因為與FliCΔD3-2ZDIII相比，FliC-ZDIII誘導針對FliC的顯著更高的免疫應答，它可以引起高的促炎細胞因子反應。因此，FliCΔD3-2ZDIII是該項目的最佳候選者。與LTIIb-B5分子共同施用作為另外的佐劑顯示出免疫應答的輕微增加。這些發現為ZIKV疫苗提供了新的見解和設計思路。

Zika virus is a mosquito borne flavivirus first found in a macaque, which then become a widespread human disease. Besides being able to be transmitted to others by mosquito vector, this virus can also be transferred via sexual intercourse and vertical transmission from mother to child. This virus has caused increasing number of infant microcephaly and Guillain-Barre syndrome in adults. To develop a prominent, effective, safe, and efficient vaccine has been an emergency considering how dangerous and life threatening this virus is. In this study flagellin fuse proteins with intranasal administration is proposed. The protein constructs are designed by fusing Zika virus envelope protein domain III (ZDIII) on to flagellin bridged by Glycine-Serine (GS) linkers. There are 5 candidates on this project, FliC-ZDIII, FliC-2ZDIII, FliC-3ZDIII, FliCΔD3-2ZDIII, and FliCΔD2ΔD3-3ZDIII. TLR5 functional assays shows an impressive response of triggering the NF-κB activity from both FliC-ZDIII and FliCΔD3-2ZDIII, especially although FliCΔD3-2ZDIII has lost part of the flagellin, it still can achieve almost similar NF-κB activity. Animal study results shows that FliC-ZDIII and FliCΔD3-2ZDIII induced potent immune responses both on B cells and T cells. Moreover, these two proteins also showed a prominent neutralizing ability against ZIKV. However, because FliC-ZDIII induce significantly higher immune responses against FliC compared to FliCΔD3-2ZDIII, it can cause high proinflammatory cytokine response. Therefore, FliCΔD3-2ZDIII is the best candidate on this project. Co-administration with LTIIb-B5 molecule as an additional adjuvant shows a slight increase on the immune responses. These findings provide a new insight and design idea on vaccine against ZIKV.

Table of Contents
Abstract i
中文摘要 ii
Acknowledgements iii
1. Introduction 1
1.1. Zika Virus 1
1.2. Mucosal immunity. 3
1.3. Flagellin. 3
1.4. Heat-labile toxin subunit B as an adjuvant. 5
1.5. Research aims and hypothesis. 6
2. Materials and Methods 8
2.1. Cell line culture. 8
2.2. Virus 8
2.3. Recombinant proteins construction. 8
2.4. Recombinant Protein Production and Purification 9
2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE and Western Blot Analysis. 10
2.6. Toll-like Receptor 5 (TLR5)-dependent activity assay. 11
2.7. In vivo vaccination. 12
2.8. Splenocytes sampling preparation. 12
2.9. Bronchoalveolar lavage fluid (BALF) sampling preparation. 13
2.10. Enzyme-linked immunosorbent assay (ELISA). 13
2.11. Plaque Reduction Neutralization Test (PRNT) 14
2.12. T cell cytokines determination assay. 14
3. Results 17
3.1. Cloning, expression, and purification of FliC, ZDIII, LTIIb-B5, recombinant FliC and ZDIII fusion protein. 17
3.2. FliC-ZDIII, FliC-2ZDIII, FliC-3ZDIII recombinant protein functional analysis and vaccination via intranasal route. 18
3.2.1. TLR5-dependent functional assay of FliC-ZDIII, FliC-2ZDIII, FliC-3ZDIII recombinant protein. 19
3.2.2. Systemic immunity titers analysis towards Zika virus envelope domain III. 20
3.2.3. Mucosal immunity titers analysis in vaginal fluid towards Zika virus envelope domain III. 21
3.2.4. Neutralizing antibody ability against ZIKV. 21
3.3. Domain-substitution FliCΔD3-2ZDIII, FliCΔD2ΔD3-3ZDIII recombinant proteins vaccination via intranasal route. 22
3.3.1. TLR-agonist activity of FliCΔD3-2ZDIII and FliCΔD2ΔD3-3ZDIII recombinant protein. 22
3.3.2. Systemic immunity response analysis towards Zika virus envelope domain III. 23
3.3.3. Mucosal immunity response analysis towards Zika virus envelope domain III. 24
3.3.4. Neutralizing antibody ability against ZIKV. 25
3.3.5. T cells response in spleen. 25
4. Discussion 27
5. References 29
6. Figures 35
Figure 1. 36
Figure 2. 38
Figure 3. 40
Figure 4. 42
7. Tables 43
Table 1. 43
8. Supplementary Figure 44
Supplementary Figure 1. 45

(WHO), World Health Organization. 2017. WHO. April 7. Accessed April 8, 2019. https://apps.who.int/iris/bitstream/handle/10665/204961/zikasitrep_7Apr2016_eng.pdf;jsessionid=701D4C4347D30821F42CE14892C63C34?sequence=1.
—. 2016. WHO. February 26. Accessed January 20, 2019. https://www.who.int/emergencies/zika-virus/situation-report-26-02-2016.pdf.
Almeida A. J., Alpar H. O. 1996. "Nasal delivery of vaccines ." J Drug Target 3(6):455-467.
Andrew D. Haddow, Amy J. Schuh, Chadwick Y. Yasuda, Matthew R. Kasper, Vireak Heang, Rekol Huy, Hilda Guzman, Robert B. Tesh, Scott C. Weaver. 2012. "Genetic Characterization of Zika Virus Strains: Geographic Expansion of the Asian Lineage." PLOS Negl Trop Dis 6(2): e1477.
Bennett M. K., Ronald D. G., Veronica G., Lien T., Dimitrios M., David D. L. 2015. "Hybrid flagellin as a T cell independent vaccine scaffold." BMC Biotechnol 15:71.
Boyaka P.N., Tafaro A., Fischer R., Leppla S. H., Fujihashi K., McGhee J. R. 2003. "Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen." J Immunol 170(11): 5636-5643.
Branchett J. W., Lloyd C. M. 2019. "Regulatory cytokine function in the respitratory tract." Mucosal Immunol 12: 589-600.
Brandtzaeg. 2010. "Function of mucosa-associated lymphoid tissue in antibody formation." Immunol Invest 39(4-5): 303-355.
Cauchemez S, Besnard M, Bompard P, Dub T, Guillemette-Artur P, Eyrolle-Guignot D, et al. 2016. "Association between Zika virus and microcephaly in French Polynesia, 2013-15: A retrospective study." Lancet 387(10033): 2125–2132.
Ciacci-Woolwine F., Blomfield I. C., Richardson S. H., Mizel S. B.,. 1998. "Salmonella flagellin induces tumor necrosis factor alpha in a human promonocytic cell line." Infect immun 66: 1127-1134.
Corona-Rivera J. R., Corona-RIvera E., Romero-Velarde E., Hernandez-Rocha J., Bobadilla-Morales L., Corona-Rivera A. 2001. "Report and review of the fetal brain disruption sequence." Eur J Pediatr 160(11):664-667.
D., Connell T. 2007. "Cholera toxin, LT-I, LT-IIa, LT-IIb: the critical role of ganglioside binding in immunomodulation by type I and type II heat-labile enterotoxins." Expert Rev Vaccines 6(5): 821-34.
Eaves-Pyles T. D., Wong H. R., Odoms K., Pyles R. B. 2001. "Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein." J Immunol 167: 7009-7016.
Elson C. O., Dertzbaugh M. T. 2005. "Mucosal adjuvants." In Mucosal immunology 3, by Bienenstock J., Lamm M. E., Mayer L., Strober W., McGhee J. R. Mestecky J., 967-86. San Diego: Elsevier/Academic Press.
F., Cesta M. 2006. "Normal structure, function, and histology of mucosa-associated lymphoid tissue." Toxicol Pathol 34(5): 599-608.
G. W. A. Dick, S. F. Kitchen, A. J. Haddow. 1952. "ZIka Virus (I). Isolations and Serological Specificity." Communication.
Gill D. M., Clements J. D., Robertson D. C., Finkelstein R. A. 1981. "Subunit number and arrangement in Escherichia coli heat-labile enterotoxin." Infect Immun 33(3): 677-82.
Hajam I. A., Dar P. A., Chandrasekar S., Nanda R. K., Kishore S., Bhanuprakash V., et.al. 2013. "Co-administration of flagellin augments immune responses to inactivated foot and mouth disease virus (FMDV) antigen." Res Vet Sci 95:936-941.
Hajishengallis G., Arce S., Gockel C. M., Connell T. D., Russell M. W. 2005. "Immunomodulation with enterotoxins for the generation of secretory immunity or tolerance: applications for oral infections." J Dent Res 6(5): 821-34.
Hajishengallis G., Tapping R. I., Martin M. H., Nawar H., Lyle E. A., Russell M. W., Connell T. D. 2005. "Toll-like receptor 2 mediates cellular activation by the B subunits of type II heat-labile enterotoxins." Infect Immun 73(3): 1343-9.
Hamel R., Dejarnac O., Wichit S., Ekchariyawat P., Neynet A., Luplertlop N., Perera-Lecoin M., Surasombatpattana P., Talignani L., Thomas F.,. 2015. "Biology of ZIka virus infection in huma skin cells." J. Virol. 89: 8880-8896.
Hayashi F., Means T.K., Luster A. D. Toll-like receptor stimulate human neutrophil function. 2003. "Toll-like receptor stimulate human neutrophil function." Blood 102:2660-2669.
Hayashi F., Smith K. D., Ozinsky A., Hawn T. R., Yi E. C., Goodlett D. R., Eng J. K., Akira S., Underhill D. M., Aderem A. 2001. "The innate immune response to bacterial flagellin is mediated by oll-like receptor 5." Nature 410(6832): 1099-103.
He B., Xu W., Santini P. A., Polydorides A. D., Chiu A., Estrella J., SHan M., Chadburn A., Villanacci V., Plebani A., Knowles D. M., Rescigno M., Cerutti A. 2007. "Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing ephotelial-cell secretion of the cytokine APRIL." Immunity 26(6): 812-26.
Hiroi T., Iwatani K., Iijima H., Kodama S., Yanagita M., Kiyono H. 1998. "Nasal immune system: distinctive Th0 and Th1/Th2 type environments in murine nasal-associated lymphoid tissues and nasal passage, respectively." Eur J Immunol 28(10) : 3346-3353.
Holmes R., Jobling M. G., Connell T. 1995. "Cholera toxin and related enterotoxins of Gram-negative bacteria." In Bacterial toxins and virulence factors in disease. Handbook of Natural toxins, by Iglewski B., Vaughn M., Tu A. T. Moss J., 225-55. New York: Marcel Dekker, Inc.
Iwasaki A, , Medzhitov R.,. 2004. "Toll-like receptor control of the adaptive immune responses." Nat Immunol 5(10): 987-95.
KC., Smithburn. 1952. "Neutralizing Antibodies against Certain Recently Isolated Viruses in The Sera of Human Beings Residing in East Africa." J Immunol 69(2): 223-234.
Kuno G., and Chang G. J.,. 2007. "Full length sequencing and genomic characterization of Bagaza, Kedougou and ZIka viruses." Arch. Virol. 152:687-696.
Lazear H. M., Diamond M. S. 2016. "Zika Virus: New Clinical Syndromes and Its Emergence in the Western Hemisphere." J Virol. 90(10): 4864-4875.
Lee E., Leang S. K., Davidson A., Lobigs M.,. 2012. "Both E proteins glycans adversely affect dengue virus inefectivity but are beneficial for virion release." J. Virol 86:3501-3512.
Li Song, Dan X., Xilong K., Yang J., Xiaohui Z., Yi Z., Xinan J., Zhiming P. 2019. "The optimized fusion protein HA1-2-FliCΔD2D3 promotes mixed Th1/Th2 immune responses to influenza H7N9 with low induction of systemic proinflammatory cytokines in mice." Antivir. Res. 10-19.
Liang S., Wang M., Tapping R. I., Stepensky V., Nawar H. F., Triantafilou M., Triantafilou K., Connell T. D., Hajishengallis G. 2007. "Ganglioside GD1a is an essential coreceptor for Toll-like receptor 2 signaling in response to the B subunit of type IIb enterotoxin." J Biol Chem 282(10): 7532-42.
Liu G., Tarbet B., Song L., Reiserova L., Weaver B., Chen Y., et al. 2011. "Immunogenicity and efficacy of flagellin-fused vaccine candidates targeting 2009 pandemic H1N1 influenza in mice." PLoS One 6:e20928.
MacNamara, F. N. 1954. "Zika Virus: A Report on Three Cases of Human Infection During an Epidemic of Jaundice in Nigeria." Transactions of The Royal Society of Tropical Medicine and Hygiene 139-145.
Mantis N. J., Rol N., Corthesy B. 2011. "Secretory IgA's complex roles in immunity and mucosal homeostasis in the gut." Mucosal Immunol 4(6): 603-611.
McDermott P.F., Ciacci-Woolwine F., Snipes J. A., Mizel S. B. 2000. "High-affinity interaction between Gram-negative flagellin and a cell surface polypeptide results in human monocyte activation." Infect Immun 68:5525-5529.
Mizel S. B., Bates J. T. 2010. "Flagellin as an adjuvant: cellular mechanism and potential." J Immunol 185: 5677-5682.
Mlakar J., Korva M., Tul N., Popovic M., Polsak-Prijatelj M., Mraz J.,. 2016. "Zika virus associated with microcephaly." N Engl J Med 5316:1-7.
Muskotal A., Seregelyes C., Sebestyen A., Vonderviszt F. 2010. "Structural basis for stabilization of the hypervariable D3 domain of Salmonella flagellin upon filament formation." J Mol. Biol. 403:607-615.
Nempont C., Cayet D., Rumbo M., Bompard C., Villeret V., Sirard J. C. 2008. "Deletion of flagellin's hypervariable region abrogates antibody-mediated neutralizationand systemic activation of TLR5-dependent immunity." J Immunol 181: 2036-2043.
Newberry R. D., Lorenz R. G. 2005. "Organizing a mucosal defense." Immunol Rev. 206: 6-21.
Newton S. M. C., Wasley R. D., Wilson A., Rosenberg L. T., Miller J. F., Stocker B. A. 1991. "Segment IV of a Salmonella flagellin gene specifies flagellar antigen epitopes." Mol Microbiol 5:419-425.
Oliphant T., Engle M., Nybakken G. E., Doane C., Johnson S>, Huang L., Gorlatov S., Mehlhop E., Marri A., Chung K. M., Ebel G. D., Kramer L. D., Fremont D. H., Diamond M. S. 2005. "Development of a Humanized Monoclonal Antibody with Therapeutic Potential against West Nile Virus." Nat Med 11(5): 522-30.
Organization, World Health. 2018. World Health Organization. July 20. Accessed April 1, 2019. https://www.who.int/news-room/fact-sheets/detail/zika-virus.
Petersen L. R., Jamieson D. J., Powers A.M., Honein M.A. 2016. "Zika Virus." N Engl J Med. 374(16):1552-1563.
Prera-Lecoin M., Meertens L., Carnec X., Amara A. 2013. "Flavivirus entry receptor." Viruses 6:69-88.
Prevention, Centers for Disease Control and. 2019. CDC. May 14. Accessed July 11, 2019. https://www.cdc.gov/zika/healtheffects/birth_defects.html.
Roehrig J. T., Bryant J. E., Calvert A. E., Mesesan K., Crabtree M. B., Volpe K. E., Silengo S., Kinney R. M., Huang C. Y., Miller B. R. 2007. "Glycosylation of the dengue 2 virus E protein at N67 is critical for virus growth in vitro but not for growth in intrathoracically-inoculated Aedes aegepty mosquitoes." Am. J. Trop. Med. Hyg. 77,1.
S., Steiner T. 2007. "How FLagellin and toll-like receptor 5 contribute to enteric infection." Infection and immunity 75(2):545-52.
Stettler K., Beltramello M., Espinosa D. A., Graham V., Cassotta A., Bianchi S., Vanzetta F., Minola A. M., Jaconi S., Mele F., Foglierini M., Pedotti M., Simonelli L., Dowall S., Atkinson B., Percivalle E., Simmons C.P., Varani L., Blum J., Baldanti F. 2016. "Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection." Science 353(6301):823-6.
Taylor D. N., Treanor J. J., STrout C., Johnson C., Fitzgerald T., Kavita U., et.al. 2011. "Induction of a potent immune response in the elderly using the TLR 5 agonist, flagellin, with a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125, STF.HA1 SI)." Vaccine 29:4987-4902.
Tsujimoto H., Uchida T., Efron P. A., Scumpia P. O., Verma A., Matsumoto T., et.al. 2009. "Flagellin enhances NK cell proliferation and activation directly and through dendritic cell-NK cell interactions." Immunol Lett 125: 114-118.
van den Akker F., Sarfaty S., Twiddy E. M., Connell T. D., Holmes R. K., Hol W. G. 1996. "Crystal structure of a new het-labile enterotoxin, LT-IIb." Structure 4(6): 665-78.
Vicente-Suarez I., Brayer J., Villagra A., Cheng F., Sotomayor E. M. 2009. "TLR 5 ligationby flagellin converts tolerogenic dendritic cells into activating antigen-presenting cells that preferentially induce T-helper 1 responses." Immunol Lett 125: 114-118.
WHO. 2018. World Health Organization. July 20. Accessed April 13, 2019. https://www.who.int/news-room/fact-sheets/detail/zika-virus.
Wu H. Y., Nguyen H. H., Russell M. W. 1997. "Nasal lymphoid tissue (NALT) as a mucosal immune inductive site." Scand J Immunol 46(5): 506-513.
Wyant T. L., Tanner M. K., Sztein M. B. 1999. "Salmonella typhi flagella are potent inducers of proinflammatory cytokine secretion by human monocytes." Infect Immun 67: 3619-3624.
Xiao Y., Liu F., Yang J., Zhong M., Zhang E., Li Y., Zhou D., Cao Y., Li W., Yu J., Yang Y., Yan H. 2015. "Over-activation of TLR5 signaling by high-dose flagellin induces liver injury in mice." Cell. Mol. Immunol. 12: 729-742.
Zuercher A. W., Coffin S. E., Thurnheer M. C., Fundova P., Cebra J. J. 2002. "Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses." J Immunol 168(4): 1796-1803.