茲卡病毒是一種屬經由蚊子傳播的黃病毒屬病毒，自從2015年巴西大爆發後便造成急迫的全球健康危機。茲卡病毒在基因上與登革熱病毒緊密相關，登革熱病毒有四個血清型，是一種常見且分布廣泛的黃病毒屬病毒，由於兩種病毒擁有同樣的伊蚊屬傳播媒介，因此在地理上擁有交疊的流行區域。抗茲卡病毒免疫反應在過去研究指出與登革熱病毒具有高度交叉反應，這些交叉反應可能是由茲卡病毒表面套膜蛋白上序列高度保守的融合環(Fusion loop)引發多數的抗體反應，但這些抗體中和能力較差，故很可能透過抗體依賴性免疫加強反應(antibody-dependent enhancement, ADE)導致異型病毒感染增加。目前為止並沒有任何預防型的茲卡病毒疫苗，也尚未有一種針對茲卡融合環區域，能降低對登革熱交叉反應的合理抗原設計。本研究中，我們設計了八種針對茲卡prME上融合區域的醣遮蔽突變株，並經由腺病毒載體表現(AdZ-prME)作為一種疫苗策略。AdZ-prME免疫的小鼠血清進行了套膜蛋白第三區域特異性IgG測試、溶斑減少中和試驗法(Plaque reduction neutralization test，PRNT)以及抗體依賴性免疫加強反應試驗。在醣遮蔽突變株中，有三個突變株引起的抗體不會在K562細胞出現DENV2的ADE，且三株其中的兩株仍然具有ZIKV中和能力。這兩個醣遮蔽突變位或許對醣遮蔽融合環區域的策略來說是十分重要的位點，且在茲卡及登革熱疫苗發展或許有可行的潛力。

Zika virus (ZIKV) is a mosquito-borne flavivirus that has caused urgent global health crisis since Brazil outbreak in 2015. ZIKV is genetically close-related to dengue virus (DENV), a common flavivirus worldwide with four serotypes, and shares geologically overlapping epidemic area due to the same Aedes mosquito transmitters. Anti-ZIKV immunity has been reported with high cross-reactivity to DENV that may enhance heterologous viral infection via antibody-dependent enhancement (ADE). The highly conserved fusion loop (FL) on ZIKV surface envelope(E) protein is responsible for eliciting the major antibodies, while domain III (DIII) in E protein elicits type-specific antibodies. However, so far no prophylactic ZIKV vaccine is available and no rational design aims to dampen the ZIKV cross-reactive FL region(s) toward DENV. In this study, we introduced eight ZIKV prME glycan-masking mutants in FL region(s) that expressed by adenoviral vectors (AdZ-prME) as a vaccine strategy. AdZ-prME immunized mice’s sera were tested for E protein DIII-specific IgG, plaque reduction neutralization test (PRNT) and ADE assay. Among the glycan-masking mutants, three mutants could elicit antibodies that were bereft of ADE in K562 cells and two of them possess neutralizing activity toward ZIKV. The two mutation sites may be important targets for glycan-masking strategy in ZIKV and DENV vaccine development.

中文摘要 I
ABSTRACT II
ABBREVIATIONS III
致謝 IV
CONTENTS V
1. INTRODUCTION 1
1.1 Overview of zika virus 1
1.2 ZIKV vaccine development 6
1.3 Adenovirus-vector vaccine 7
1.4 Study goals 8
2. MATERIALS AND METHODS 9
2.1 Cell line culture 9
2.2 Viruses 9
2.3 Construction of recombinant adenoviral vector with full-length prME and glycan masking mutants 10
2.4 Preparation of recombinant prME adenovirus 10
2.5 Adenovirus titer determination 11
2.6 Preparation of adenoviral infected cells lysates 11
2.7 Glycosylation analysis by PNGaseF 12
2.8 SDS-PAGE and western blotting 12
2.9 Mice immunization 13
2.10 Enzyme-Linked ImmunoSorbent Assay (ELISA) 14
2.11 Plaque reduction neutralization test (PRNT) 15
2.12 Antibody-dependent enhancement (ADE) assay 15
3. RESULTS AND DISCUSSION 17
3.1 Adenovirus-backbone with glycan masking ZIKV prME 17
3.2 PNGaseF treatment revealed glycan-masking evidence in AdZ-prME derivatives 18
3.3 Total IgG of AdZ-prME immunized mice resum recognized recombinant ZIKV DIII in ELISA while barely bind to DENV DIII 18
3.4 Sera of AdZ-prME-wt and its derivatives, except -74 mutants, possess activity of neutralizing ZIKV 19
3.5 ADE assay indicated infection enhancement in three glycan-masking mutants while lack of ADE were observed in the others 20
3.6 Conclusion and discussion 21
4. REFERENCE 25
5. FIGURES 30
Figure 1. Designs of glycan masking on ZIKV E protein 30
Figure 2. AdZ-prME constructs and mutants 31
Figure 3. Western blotting labels ZIKV E protein in AdZ-prME infected 293A cells 32
Figure 4. Mice immunization regimen 33
Figure 5. ZDIII and D2DIII binding ability of AdZ-prME immunized sera in ELISA 34
Figure 6. Blocking infectivity of ZIKV by AdZ-prME immunized sera 35
Figure 7. DENV2 infection enhancement with AdZ-prME immunized sera in FcR-bearing K562 cells 36
Figure 8. Comparison of PRNT and ADE intensity in AdZ-prME and its derivatives 37
6. TABLES 38
Table 1. Primer sequence for AdZ-prME construction 38
Table 2. N-Glycosylation mutant sites of ZIKV Envelope protein 39
Table 3. Groups of mice immunization 40

Abbink, P., Larocca, R.A., De La Barrera, R.A., Bricault, C.A., Moseley, E.T., Boyd, M., Kirilova, M., Li, Z., Ng'ang'a, D., Nanayakkara, O., Nityanandam, R., Mercado, N.B., Borducchi, E.N., Agarwal, A., Brinkman, A.L., Cabral, C., Chandrashekar, A., Giglio, P.B., Jetton, D., Jimenez, J., Lee, B.C., Mojta, S., Molloy, K., Shetty, M., Neubauer, G.H., Stephenson, K.E., Peron, J.P., Zanotto, P.M., Misamore, J., Finneyfrock, B., Lewis, M.G., Alter, G., Modjarrad, K., Jarman, R.G., Eckels, K.H., Michael, N.L., Thomas, S.J., Barouch, D.H., 2016. Protective efficacy of multiple vaccine platforms against Zika virus challenge in rhesus monkeys. Science 353, 1129-1132.
Barba-Spaeth, G., Dejnirattisai, W., Rouvinski, A., Vaney, M.C., Medits, I., Sharma, A., Simon-Loriere, E., Sakuntabhai, A., Cao-Lormeau, V.M., Haouz, A., England, P., Stiasny, K., Mongkolsapaya, J., Heinz, F.X., Screaton, G.R., Rey, F.A., 2016. Structural basis of potent Zika-dengue virus antibody cross-neutralization. Nature 536, 48-53.
Bardina, S.V., Bunduc, P., Tripathi, S., Duehr, J., Frere, J.J., Brown, J.A., Nachbagauer, R., Foster, G.A., Krysztof, D., Tortorella, D., Stramer, S.L., Garcia-Sastre, A., Krammer, F., Lim, J.K., 2017. Enhancement of Zika virus pathogenesis by preexisting antiflavivirus immunity. Science 356, 175-180.
Beltramello, M., Williams, K.L., Simmons, C.P., Macagno, A., Simonelli, L., Quyen, N.T., Sukupolvi-Petty, S., Navarro-Sanchez, E., Young, P.R., de Silva, A.M., Rey, F.A., Varani, L., Whitehead, S.S., Diamond, M.S., Harris, E., Lanzavecchia, A., Sallusto, F., 2010. The human immune response to Dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity. Cell Host Microbe 8, 271-283.
Bhatt, S., Gething, P.W., Brady, O.J., Messina, J.P., Farlow, A.W., Moyes, C.L., Drake, J.M., Brownstein, J.S., Hoen, A.G., Sankoh, O., Myers, M.F., George, D.B., Jaenisch, T., Wint, G.R., Simmons, C.P., Scott, T.W., Farrar, J.J., Hay, S.I., 2013. The global distribution and burden of dengue. Nature 496, 504-507.
Castanha, P.M.S., Nascimento, E.J.M., Braga, C., Cordeiro, M.T., de Carvalho, O.V., de Mendonca, L.R., Azevedo, E.A.N., Franca, R.F.O., Dhalia, R., Marques, E.T.A., 2017. Dengue Virus-Specific Antibodies Enhance Brazilian Zika Virus Infection. J Infect Dis 215, 781-785.
Choi, Y., Chang, J., 2013. Viral vectors for vaccine applications. Clin Exp Vaccine Res 2, 97-105.
Collins, M.H., McGowan, E., Jadi, R., Young, E., Lopez, C.A., Baric, R.S., Lazear, H.M., de Silva, A.M., 2017. Lack of Durable Cross-Neutralizing Antibodies Against Zika Virus from Dengue Virus Infection. Emerg Infect Dis 23, 773-781.
Crill, W.D., Chang, G.J., 2004. Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes. J Virol 78, 13975-13986.
Dai, L., Song, J., Lu, X., Deng, Y.Q., Musyoki, A.M., Cheng, H., Zhang, Y., Yuan, Y., Song, H., Haywood, J., Xiao, H., Yan, J., Shi, Y., Qin, C.F., Qi, J., Gao, G.F., 2016. Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody. Cell Host Microbe 19, 696-704.
Dejnirattisai, W., Supasa, P., Wongwiwat, W., Rouvinski, A., Barba-Spaeth, G., Duangchinda, T., Sakuntabhai, A., Cao-Lormeau, V.M., Malasit, P., Rey, F.A., Mongkolsapaya, J., Screaton, G.R., 2016. Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nat Immunol 17, 1102-1108.
Fernandez, E., Diamond, M.S., 2017. Vaccination strategies against Zika virus. Curr Opin Virol 23, 59-67.
Halstead, S.B., 2003. Neutralization and antibody-dependent enhancement of dengue viruses. Adv Virus Res 60, 421-467.
Harrison, S.C., 2016. Immunogenic cross-talk between dengue and Zika viruses. Nat Immunol 17, 1010-1012.
Heinz, F.X., Stiasny, K., 2017. The Antigenic Structure of Zika Virus and Its Relation to Other Flaviviruses: Implications for Infection and Immunoprophylaxis. Microbiol Mol Biol Rev 81.
Keasey, S.L., Pugh, C.L., Jensen, S.M., Smith, J.L., Hontz, R.D., Durbin, A.P., Dudley, D.M., O'Connor, D.H., Ulrich, R.G., 2017. Antibody Responses to Zika Virus Infections in Environments of Flavivirus Endemicity. Clin Vaccine Immunol 24.
Kostyuchenko, V.A., Lim, E.X., Zhang, S., Fibriansah, G., Ng, T.S., Ooi, J.S., Shi, J., Lok, S.M., 2016. Structure of the thermally stable Zika virus. Nature 533, 425-428.
Kuhn, R.J., Dowd, K.A., Beth Post, C., Pierson, T.C., 2015. Shake, rattle, and roll: Impact of the dynamics of flavivirus particles on their interactions with the host. Virology 479-480, 508-517.
Kuhn, R.J., Zhang, W., Rossmann, M.G., Pletnev, S.V., Corver, J., Lenches, E., Jones, C.T., Mukhopadhyay, S., Chipman, P.R., Strauss, E.G., Baker, T.S., Strauss, J.H., 2002. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108, 717-725.
Lai, C.Y., Tsai, W.Y., Lin, S.R., Kao, C.L., Hu, H.P., King, C.C., Wu, H.C., Chang, G.J., Wang, W.K., 2008. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. J Virol 82, 6631-6643.
Larocca, R.A., Abbink, P., Peron, J.P., Zanotto, P.M., Iampietro, M.J., Badamchi-Zadeh, A., Boyd, M., Ng'ang'a, D., Kirilova, M., Nityanandam, R., Mercado, N.B., Li, Z., Moseley, E.T., Bricault, C.A., Borducchi, E.N., Giglio, P.B., Jetton, D., Neubauer, G., Nkolola, J.P., Maxfield, L.F., De La Barrera, R.A., Jarman, R.G., Eckels, K.H., Michael, N.L., Thomas, S.J., Barouch, D.H., 2016. Vaccine protection against Zika virus from Brazil. Nature 536, 474-478.
Leopold, P.L., Wendland, R.L., Vincent, T., Crystal, R.G., 2006. Neutralized adenovirus-immune complexes can mediate effective gene transfer via an Fc receptor-dependent infection pathway. J Virol 80, 10237-10247.
Lin, H.H., Huang, L.M., Wu, S.C., 2017. Zika Virus Molecular Biology and Perspectives for Vaccine Development: A Review. J Nurs Res 25, 3-6.
Lin, S.C., Liu, W.C., Jan, J.T., Wu, S.C., 2014. Glycan masking of hemagglutinin for adenovirus vector and recombinant protein immunizations elicits broadly neutralizing antibodies against H5N1 avian influenza viruses. PLoS One 9, e92822.
McArthur, M.A., 2017. Zika Virus: Recent Advances towards the Development of Vaccines and Therapeutics. Viruses 9.
Modis, Y., Ogata, S., Clements, D., Harrison, S.C., 2004. Structure of the dengue virus envelope protein after membrane fusion. Nature 427, 313-319.
Paul, L.M., Carlin, E.R., Jenkins, M.M., Tan, A.L., Barcellona, C.M., Nicholson, C.O., Michael, S.F., Isern, S., 2016. Dengue virus antibodies enhance Zika virus infection. Clin Transl Immunology 5, e117.
Pierson, T.C., Fremont, D.H., Kuhn, R.J., Diamond, M.S., 2008. Structural insights into the mechanisms of antibody-mediated neutralization of flavivirus infection: implications for vaccine development. Cell Host Microbe 4, 229-238.
Priyamvada, L., Quicke, K.M., Hudson, W.H., Onlamoon, N., Sewatanon, J., Edupuganti, S., Pattanapanyasat, K., Chokephaibulkit, K., Mulligan, M.J., Wilson, P.C., Ahmed, R., Suthar, M.S., Wrammert, J., 2016. Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus. Proc Natl Acad Sci U S A 113, 7852-7857.
Roehrig, J.T., 2003. Antigenic structure of flavivirus proteins. Adv Virus Res 59, 141-175.
Rouvinski, A., Guardado-Calvo, P., Barba-Spaeth, G., Duquerroy, S., Vaney, M.C., Kikuti, C.M., Navarro Sanchez, M.E., Dejnirattisai, W., Wongwiwat, W., Haouz, A., Girard-Blanc, C., Petres, S., Shepard, W.E., Despres, P., Arenzana-Seisdedos, F., Dussart, P., Mongkolsapaya, J., Screaton, G.R., Rey, F.A., 2015. Recognition determinants of broadly neutralizing human antibodies against dengue viruses. Nature 520, 109-113.
Sanchez Carnerero, C.I., Alcantara Montero, A., 2017. [Provisional considerations about Zika virus infection in pregnant women: Document for health professionals]. Semergen 43, 252-254.
Saxena, M., Van, T.T., Baird, F.J., Coloe, P.J., Smooker, P.M., 2013. Pre-existing
immunity against vaccine vectors--friend or foe? Microbiology 159, 1-11.
Screaton, G., Mongkolsapaya, J., Yacoub, S., Roberts, C., 2015. New insights into the immunopathology and control of dengue virus infection. Nat Rev Immunol 15, 745-759.
Shan, C., Muruato, A.E., Nunes, B.T.D., Luo, H., Xie, X., Medeiros, D.B.A., Wakamiya, M., Tesh, R.B., Barrett, A.D., Wang, T., Weaver, S.C., Vasconcelos, P.F.C., Rossi, S.L., Shi, P.Y., 2017. A live-attenuated Zika virus vaccine candidate induces sterilizing immunity in mouse models. Nat Med 23, 763-767.
Sirohi, D., Chen, Z., Sun, L., Klose, T., Pierson, T.C., Rossmann, M.G., Kuhn, R.J., 2016. The 3.8 A resolution cryo-EM structure of Zika virus. Science 352, 467-470.
Small, J.C., Ertl, H.C., 2011. Viruses - from pathogens to vaccine carriers. Curr Opin Virol 1, 241-245.
Stettler, K., Beltramello, M., Espinosa, D.A., Graham, V., Cassotta, A., Bianchi, S., Vanzetta, F., Minola, A., 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., Cameroni, E., Hewson, R., Harris, E., Lanzavecchia, A., Sallusto, F., Corti, D., 2016. Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science 353, 823-826.
Sumathy, K., Kulkarni, B., Gondu, R.K., Ponnuru, S.K., Bonguram, N., Eligeti, R., Gadiyaram, S., Praturi, U., Chougule, B., Karunakaran, L., Ella, K.M., 2017. Protective efficacy of Zika vaccine in AG129 mouse model. Sci Rep 7, 46375.
Tatsis, N., Ertl, H.C., 2004. Adenoviruses as vaccine vectors. Mol Ther 10, 616-629.
Wahala, W.M., Donaldson, E.F., de Alwis, R., Accavitti-Loper, M.A., Baric, R.S., de Silva, A.M., 2010. Natural strain variation and antibody neutralization of dengue serotype 3 viruses. PLoS Pathog 6, e1000821.
Wahala, W.M., Kraus, A.A., Haymore, L.B., Accavitti-Loper, M.A., de Silva, A.M., 2009. Dengue virus neutralization by human immune sera: role of envelope protein domain III-reactive antibody. Virology 392, 103-113.
Wahala, W.M., Silva, A.M., 2011. The human antibody response to dengue virus infection. Viruses 3, 2374-2395.
WHO. WHO Director-General summarizes the outcome of the Emergency Committee regarding clusters of microcephaly and Guillain-Barre syndrome (2016a). http://www.who.int/mediacentre/news/statements/2016/emergency-committee-zika-microcephaly/en/
WHO. Zika virus situation reports (2016b). http://www.who.int/entity/emergencies/zika-virus/situation-report/13-october-2016/en/index.html
WHO. WHO vaccine pipeline tracker (2017).
http://www.who.int/immunization/research/vaccine_pipeline_tracker_spreadsheet/en/
Yu, I.M., Zhang, W., Holdaway, H.A., Li, L., Kostyuchenko, V.A., Chipman, P.R., Kuhn, R.J., Rossmann, M.G., Chen, J., 2008. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319, 1834-1837.
Zhang, Y., Zhang, W., Ogata, S., Clements, D., Strauss, J.H., Baker, T.S., Kuhn, R.J., Rossmann, M.G., 2004. Conformational changes of the flavivirus E glycoprotein. Structure 12, 1607-1618.