茲卡病毒(ZIKV)能造成部分感染者一些嚴重的症狀，包括新生兒缺陷小頭症和其他神經相關疾病，傳染途徑包括蚊蟲叮咬、孕婦垂直傳染、性行為、輸血，而且已經在亞洲、非洲與美洲地區證實有病例感染，因此，在疫苗的發展上提供安全有效的黏膜性疫苗來保護胎兒有其重要性。在此論文中，我使用茲卡病毒套膜第三區域重組蛋白 (ZDIII) 作為抗原，並搭配鞭毛重組蛋白 (FliC) 、第二型b型忌熱型腸毒素B次單元重組蛋白 (LTIIb-B5) 以及靈芝多醣體 (PS-G) 作為疫苗佐劑來進行比較和探討。我以八組BALB/c小鼠進行三次的鼻腔免疫 (i.n.) ，結果顯示以鞭毛蛋白融合茲卡病毒套膜第三區域重組蛋白 (FliC-ZDIII) 搭配靈芝多醣體 (PS-G) 最能增加血清中IgG與IgA的含量。而鞭毛蛋白融合茲卡病毒套膜第三區域重組蛋白 (FliC-ZDIII) 搭配第二型b型忌熱型腸毒素B次單元重組蛋白 (LTIIb-B5) 或靈芝多醣體 (PS-G) 有較高的中和性抗體。關於黏膜系統免疫，鞭毛蛋白融合茲卡病毒套膜第三區域重組蛋白 (FliC-ZDIII) 搭配第二型b型忌熱型腸毒素B次單元重組蛋白 (LTIIb-B5) 或靈芝多醣體 (PS-G) 在呼吸道中能有較多的IgA分泌，而在陰道中，鞭毛蛋白融合茲卡病毒套膜第三區域重組蛋白 (FliC-ZDIII) 搭配靈芝多醣體 (PS-G) 最能增加IgA的分泌。鞭毛蛋白融合茲卡病毒套膜第三區域重組蛋白 (FliC-ZDIII) 搭配靈芝多醣體 (PS-G) 在脾臟中都能檢測出高量的細胞因子 (IFN-r, IL-4, IL-17A and IL-22)。在第一次免疫實驗中，鞭毛蛋白融合茲卡病毒套膜第三區域重組蛋白 (FliC-ZDIII) 搭配靈芝多醣體 (PS-G) 取得了最佳的實驗結果。於是我們試圖以提高一倍抗原劑量來得到更好的免疫結果，使用四組BALB/c小鼠進行提高劑量的三次鼻腔免疫 (i.n.) ，測量血清及陰道沖洗液中的抗體、中和性抗體含量並進行攻毒實驗。在中和性抗體含量及攻毒實驗中，我們可以看到鞭毛蛋白融合茲卡病毒套膜第三區域重組蛋白 (FliC-ZDIII) 搭配靈芝多醣體 (PS-G) 擁有最高的中和性抗體、最高的生存率及最低的感染指標 (clinical score)。

Zika virus (ZIKV) can cause some of significant symptoms, including birth defects Microcephaly and other neuro-related diseases, such as Guillain-Barré syndrome. ZIKV is transmitted by mosquito bites, pregnancy, sex and blood transfusion. ZIKV infections in humans have been confirmed by sero-surveillance in Asia, Africa and America. And because of the transmission route, the vaccine with mucosal route is better than other route for ZIKV vaccine. So, it is necessary to provide a safe and effective mucosal vaccine for protection of new born. In my study, I used ZIKV E protein domain III (ZDIII) as antigen, and I used FliC, LTIIb-B5 and PS-G as adjuvants for vaccine development. Three-dose immunizations were conducted in eight groups of BALB/c mice through intranasal (i.n.) route. My results show that FliC-ZDIII+PS-G triggered the highest amount of IgG and IgA in sera. FliC-ZDIII+PS-G and FliC-ZDIII+LTIIb-B5 induced more neutralization antibodies in sera. The secreting IgA in mucosal system, FliC-ZDIII+PS-G and FliC-ZDIII+LTIIb-B5 induced more secreting IgA in respiratory route. FliC-ZDIII+PS-G induced the highest amount of secreting IgA in vagina. In T cell response, ZDIII+PS-G and FliC-ZDIII+PS-G induced higher Th2 and Th17 response. Three-dose immunizations with two-fold FliC-ZDIII were conducted in four groups of BALB/c mice through intranasal (i.n.) route. In PRNT50, immunizations with two-fold FliC-ZDIII induced more neutralization antibodies than the first immunized experiment. FliC-ZDIII and FliC-ZDIII+PS-G induced higher neutralization antibodies. In challenge data, FliC-ZDIII+PS-G had the highest survival rate and the lowest clinical score in these four groups.

摘要 ..................I
Abstract ..............III
Acknowledgements .......V
Contents .....VI
1. Introduction .....1
1.1 Zika virus(ZIKV) .....1
1.2 Mucosal immunity and mucosal adjuvants .....3
1.3 Flagellin (FliC) .....6
1.4 E.coli Heat-labile enterotoxin (LT) .....7
1.5 Ganoderma lucidum polysaccharide (PS-G) .....9
1.6 Study goals of this research .....10
2. Materials and methods .....12
2.1 Expression and purification of heat-labile enterotoxin IIb5 (LTIIb5) protein …………………………………………………………………………………………………………12
2.2 Expression and purification of ZIKV E protein domain III (ZDIII) protein ……..... 13
2.3 Expression and purification of FliC-ZIKV E protein domain III (FliC-ZDIII) protein .......14
2.4 Mice immunization and sample preparation .....16
2.5 Vaginal secretion and blood sampling preparation ......16
2.6 Cervical lymph nodes、spleen sampling preparation ......17
2.7 Bronchoalveolar lavage fluid (BALF) sampling and preparation ......18
2.8 Anti-ZDIII IgA/IgG enzyme-linked immunosorbent assay (ELISA) .......18
2.9 PRNT .....19
2.10 Total IgA ELISA assay of brochoalveolar lavage fluid (BALF) ......20
2.11 T cell cytokines ELISA assay ......21
2.12 Challenge .....21
3. Results .......23
3.1 ZIKV E DIII、FliC- ZIKV E DIII and LTIIb-B5 construction、expression and purification .....23
3.2 Immunization of ZDIII recombinant proteins combined with different adjuvants into mouse by intranasal route .....24
3.3 Analysis the systemic immunity against ZDIII after immunization in sera ………………………………………………………………………………………………………….24
3.4 Detection of the neutralizing antibodies against ZIKV in sera ..........25
3.5 Analysis the mucosal immunity against ZDIII after immunization in bronchoalveolar lavage fluids and vaginal secretions ..........26
3.6 Detection of the T cells response in spleens and cervical lymph nodes by T cell cytokines ELISA assay ........27
3.7 Injection of twice higher dosage FliC-ZDIII combined with LTIIb-B5 /PS-G into mouse by intranasal route ..........28
3.8 Analysis the mucosal immunity against ZDIII after immunization in vaginal secretions in second immunized experiment ................29
3.9 Analysis the systemic immunity against ZDIII after immunization in sera in second immunized experiment .........29
3.10 Detection of the neutralization antibodies in sera in second immunized experiment .............30
3.11 The mice do the challenge experiment with ZIKV(PRVABC59) after immunization .........30
4. Discussion .........32
5. References .........34
6. Figures 43
Figure1. World map of area with risk of Zika virus (CDC, 2017) .....43
Figure2. ZIKV vaccine candidates in clinical trial (WHO, 2018) .....44
Figure3. ZIKV E DIII、FliC- ZIKV E DIII and LTIIb-B5 construction and purification. .....45
Figure4. First intranasal immunized experiment .....46
Figure5. Analysis the humoral immunity in sera of each groups ......47
Figure6. Analysis the neutralization antibodies in sera of each groups .....50
Figure7. The secretory IgA titer in mucosal system of each groups .....51
Figure8. T cell relative cytokines response in spleen cells .....53
Figure9. T cell relative cytokines response in CLN cells .....55
Figure10. Second intranasal immunized experiment .....56
Figure11. The secretory IgA titer in vaginal fluids in second immunized experiment .....57
Figure12. The anti-ZDIII IgG titer in sera in second immunized experiment .....58
Figure13. The anti-ZDIII IgA titer in sera in second immunized experiment .....59
Figure14. Analysis the neutralization antibodies in sera in second immunized experiment .....61
Figure15. Survival rates, clinical scores and body weight loss of challenge experiment .....63

2013. in: Reducing Tobacco-Related Cancer Incidence and Mortality: Workshop Summary. Washington (DC).
Bargieri, D.Y., Rosa, D.S., Braga, C.J., Carvalho, B.O., Costa, F.T., Espindola, N.M., Vaz, A.J., Soares, I.S., Ferreira, L.C., Rodrigues, M.M. 2008. New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin. Vaccine, 26(48), 6132-42.
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(3), 271-83.
Bhatnagar, J., Rabeneck, D.B., Martines, R.B., Reagan-Steiner, S., Ermias, Y., Estetter, L.B., Suzuki, T., Ritter, J., Keating, M.K., Hale, G., Gary, J., Muehlenbachs, A., Lambert, A., Lanciotti, R., Oduyebo, T., Meaney-Delman, D., Bolanos, F., Saad, E.A., Shieh, W.J., Zaki, S.R. 2017. Zika Virus RNA Replication and Persistence in Brain and Placental Tissue. Emerg Infect Dis, 23(3), 405-414.
Campos, G.S., Bandeira, A.C., Sardi, S.I. 2015. Zika Virus Outbreak, Bahia, Brazil. Emerg Infect Dis, 21(10), 1885-6.
Cao-Lormeau, V.M., Blake, A., Mons, S., Lastere, S., Roche, C., Vanhomwegen, J., Dub, T., Baudouin, L., Teissier, A., Larre, P., Vial, A.L., Decam, C., Choumet, V., Halstead, S.K., Willison, H.J., Musset, L., Manuguerra, J.C., Despres, P., Fournier, E., Mallet, H.P., Musso, D., Fontanet, A., Neil, J., Ghawche, F. 2016. Guillain-Barre Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet, 387(10027), 1531-1539.
Cao-Lormeau, V.M., Roche, C., Teissier, A., Robin, E., Berry, A.L., Mallet, H.P., Sall, A.A., Musso, D. 2014. Zika virus, French polynesia, South pacific, 2013. Emerg Infect Dis, 20(6), 1085-6.
Cauchemez, S., Besnard, M., Bompard, P., Dub, T., Guillemette-Artur, P., Eyrolle-Guignot, D., Salje, H., Van Kerkhove, M.D., Abadie, V., Garel, C., Fontanet, A., Mallet, H.P. 2016. Association between Zika virus and microcephaly in French Polynesia, 2013-15: a retrospective study. Lancet, 387(10033), 2125-2132.
Chan, J.F., Choi, G.K., Yip, C.C., Cheng, V.C., Yuen, K.Y. 2016. Zika fever and congenital Zika syndrome: An unexpected emerging arboviral disease. J Infect, 72(5), 507-24.
Chien, C.M., Cheng, J.L., Chang, W.T., Tien, M.H., Tsao, C.M., Chang, Y.H., Chang, H.Y., Hsieh, J.F., Wong, C.H., Chen, S.T. 2004. Polysaccharides of Ganoderma lucidum alter cell immunophenotypic expression and enhance CD56+ NK-cell cytotoxicity in cord blood. Bioorg Med Chem, 12(21), 5603-9.
Christensen, D., Mortensen, R., Rosenkrands, I., Dietrich, J., Andersen, P. 2017. Vaccine-induced Th17 cells are established as resident memory cells in the lung and promote local IgA responses. Mucosal Immunol, 10(1), 260-270.
Czerkinsky, C., Holmgren, J. 2010. Topical immunization strategies. Mucosal Immunol, 3(6), 545-55.
D'Ortenzio, E., Matheron, S., Yazdanpanah, Y., de Lamballerie, X., Hubert, B., Piorkowski, G., Maquart, M., Descamps, D., Damond, F., Leparc-Goffart, I. 2016. Evidence of Sexual Transmission of Zika Virus. N Engl J Med, 374(22), 2195-8.
Devriendt, B., De Geest, B.G., Goddeeris, B.M., Cox, E. 2012. Crossing the barrier: Targeting epithelial receptors for enhanced oral vaccine delivery. J Control Release, 160(3), 431-9.
Duffy, M.R., Chen, T.H., Hancock, W.T., Powers, A.M., Kool, J.L., Lanciotti, R.S., Pretrick, M., Marfel, M., Holzbauer, S., Dubray, C., Guillaumot, L., Griggs, A., Bel, M., Lambert, A.J., Laven, J., Kosoy, O., Panella, A., Biggerstaff, B.J., Fischer, M., Hayes, E.B. 2009. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med, 360(24), 2536-43.
Evrard, B., Balestrino, D., Dosgilbert, A., Bouya-Gachancard, J.L., Charbonnel, N., Forestier, C., Tridon, A. 2010. Roles of capsule and lipopolysaccharide O antigen in interactions of human monocyte-derived dendritic cells and Klebsiella pneumoniae. Infect Immun, 78(1), 210-9.
Fauci, A.S., Morens, D.M. 2016. Zika Virus in the Americas--Yet Another Arbovirus Threat. N Engl J Med, 374(7), 601-4.
Ferguson, N.M., Cucunuba, Z.M., Dorigatti, I., Nedjati-Gilani, G.L., Donnelly, C.A., Basanez, M.G., Nouvellet, P., Lessler, J. 2016. EPIDEMIOLOGY. Countering the Zika epidemic in Latin America. Science, 353(6297), 353-4.
Franca, G.V., Schuler-Faccini, L., Oliveira, W.K., Henriques, C.M., Carmo, E.H., Pedi, V.D., Nunes, M.L., Castro, M.C., Serruya, S., Silveira, M.F., Barros, F.C., Victora, C.G. 2016. Congenital Zika virus syndrome in Brazil: a case series of the first 1501 livebirths with complete investigation. Lancet, 388(10047), 891-7.
Franchi, L., Munoz-Planillo, R., Nunez, G. 2012. Sensing and reacting to microbes through the inflammasomes. Nat Immunol, 13(4), 325-32.
Freytag, L.C., Clements, J.D. 2005. Mucosal adjuvants. Vaccine, 23(15), 1804-13.
Ganglioside – Wikipedia. http://en.wikipedia.org/wiki/Ganglioside
Gargili, A., Estrada-Pena, A., Spengler, J.R., Lukashev, A., Nuttall, P.A., Bente, D.A. 2017. The role of ticks in the maintenance and transmission of Crimean-Congo hemorrhagic fever virus: A review of published field and laboratory studies. Antiviral Res, 144, 93-119.
Giddings, O.K., Eickhoff, C.S., Sullivan, N.L., Hoft, D.F. 2010. Intranasal vaccinations with the trans-sialidase antigen plus CpG Adjuvant induce mucosal immunity protective against conjunctival Trypanosoma cruzi challenges. Infect Immun, 78(3), 1333-8.
Gopal, R., Rangel-Moreno, J., Slight, S., Lin, Y., Nawar, H.F., Fallert Junecko, B.A., Reinhart, T.A., Kolls, J., Randall, T.D., Connell, T.D., Khader, S.A. 2013. Interleukin-17-dependent CXCL13 mediates mucosal vaccine-induced immunity against tuberculosis. Mucosal Immunol, 6(5), 972-84.
Guo, Q., Chan, J.F., Poon, V.K., Wu, S., Chan, C.C., Hou, L., Yip, C.C., Ren, C., Cai, J.P., Zhao, M., Zhang, A.J., Song, X., Chan, K.H., Wang, B., Kok, K.H., Wen, Y., Yuen, K.Y., Chen, W. 2018. Immunization With a Novel Human Type 5 Adenovirus-Vectored Vaccine Expressing the Premembrane and Envelope Proteins of Zika Virus Provides Consistent and Sterilizing Protection in Multiple Immunocompetent and Immunocompromised Animal Models. J Infect Dis, 218(3), 365-377.
Hajishengallis, G., Connell, T.D. 2013. Type II heat-labile enterotoxins: structure, function, and immunomodulatory properties. Vet Immunol Immunopathol, 152(1-2), 68-77.
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.
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 Toll-like receptor 5. Nature, 410(6832), 1099-103.
Hou, Y., Ding, X., Hou, W., Zhong, J., Zhu, H., Ma, B., Xu, T., Li, J. 2013. Anti-microorganism, anti-tumor, and immune activities of a novel polysaccharide isolated from Tricholoma matsutake. Pharmacogn Mag, 9(35), 244-9.
Hsu, H.Y., Hua, K.F., Lin, C.C., Lin, C.H., Hsu, J., Wong, C.H. 2004. Extract of Reishi polysaccharides induces cytokine expression via TLR4-modulated protein kinase signaling pathways. J Immunol, 173(10), 5989-99.
Hsu, M.J., Lee, S.S., Lin, W.W. 2002. Polysaccharide purified from Ganoderma lucidum inhibits spontaneous and Fas-mediated apoptosis in human neutrophils through activation of the phosphatidylinositol 3 kinase/Akt signaling pathway. J Leukoc Biol, 72(1), 207-16.
Hwang, I., Ahn, G., Park, E., Ha, D., Song, J.Y., Jee, Y. 2011. An acidic polysaccharide of Panax ginseng ameliorates experimental autoimmune encephalomyelitis and induces regulatory T cells. Immunol Lett, 138(2), 169-78.
Joguet, G., Mansuy, J.M., Matusali, G., Hamdi, S., Walschaerts, M., Pavili, L., Guyomard, S., Prisant, N., Lamarre, P., Dejucq-Rainsford, N., Pasquier, C., Bujan, L. 2017. Effect of acute Zika virus infection on sperm and virus clearance in body fluids: a prospective observational study. Lancet Infect Dis, 17(11), 1200-1208.
Kim, H.S., Shin, B.R., Lee, H.K., Park, Y.S., Liu, Q., Kim, S.Y., Lee, M.K., Hong, J.T., Kim, Y., Han, S.B. 2013. Dendritic cell activation by polysaccharide isolated from Angelica dahurica. Food Chem Toxicol, 55, 241-7.
Kiyono, H., Fukuyama, S. 2004. NALT- versus Peyer's-patch-mediated mucosal immunity. Nat Rev Immunol, 4(9), 699-710.
Kofoed, E.M., Vance, R.E. 2011. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature, 477(7366), 592-5.
Koosakulnirand, S., Phokrai, P., Jenjaroen, K., Roberts, R.A., Utaisincharoen, P., Dunachie, S.J., Brett, P.J., Burtnick, M.N., Chantratita, N. 2018. Immune response to recombinant Burkholderia pseudomallei FliC. PLoS One, 13(6), e0198906.
Labastida-Conde, R.G., Ramirez-Pliego, O., Peleteiro-Olmedo, M., Lopez-Guerrero, D.V., Badillo-Godinez, O.D., Gutierrez-Xicotencatl, M.L., Rosas-Salgado, G., Gonzalez-Fernandez, A., Esquivel-Guadarrama, F.R., Santana, M.A. 2018. Flagellin is a Th1 polarizing factor for human CD4(+) T cells and induces protection in a murine neonatal vaccination model of rotavirus infection. Vaccine, 36(29), 4188-4197.
Le Moigne, V., Robreau, G., Mahana, W. 2008. Flagellin as a good carrier and potent adjuvant for Th1 response: study of mice immune response to the p27 (Rv2108) Mycobacterium tuberculosis antigen. Mol Immunol, 45(9), 2499-507.
Liang, S., Hajishengallis, G. 2010. Heat-labile enterotoxins as adjuvants or anti-inflammatory agents. Immunol Invest, 39(4-5), 449-67.
Liang, S., Hosur, K.B., Lu, S., Nawar, H.F., Weber, B.R., Tapping, R.I., Connell, T.D., Hajishengallis, G. 2009. Mapping of a microbial protein domain involved in binding and activation of the TLR2/TLR1 heterodimer. J Immunol, 182(5), 2978-85.
Lin, J.J., Chin, T.Y., Chen, C.P., Chan, H.L., Wu, T.Y. 2017. Zika virus: An emerging challenge for obstetrics and gynecology. Taiwan J Obstet Gynecol, 56(5), 585-592.
Lin, K.I., Kao, Y.Y., Kuo, H.K., Yang, W.B., Chou, A., Lin, H.H., Yu, A.L., Wong, C.H. 2006. Reishi polysaccharides induce immunoglobulin production through the TLR4/TLR2-mediated induction of transcription factor Blimp-1. J Biol Chem, 281(34), 24111-23.
Lin, Y.L., Liang, Y.C., Lee, S.S., Chiang, B.L. 2005. Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and p38 mitogen-activated protein kinase pathways. J Leukoc Biol, 78(2), 533-43.
Lindenbach, B.D., Rice, C.M. 2003. Molecular biology of flaviviruses. Adv Virus Res, 59, 23-61.
Liu, G., Song, L., Reiserova, L., Trivedi, U., Li, H., Liu, X., Noah, D., Hou, F., Weaver, B., Tussey, L. 2012. Flagellin-HA vaccines protect ferrets and mice against H5N1 highly pathogenic avian influenza virus (HPAIV) infections. Vaccine, 30(48), 6833-8.
Liu, Z., Dang, J., Wang, Q., Yu, M., Jiang, L., Mei, L., Shao, Y., Tao, Y. 2013. Optimization of polysaccharides from Lycium ruthenicum fruit using RSM and its anti-oxidant activity. Int J Biol Macromol, 61, 127-34.
Lorenzen, E., Follmann, F., Boje, S., Erneholm, K., Olsen, A.W., Agerholm, J.S., Jungersen, G., Andersen, P. 2015. Intramuscular Priming and Intranasal Boosting Induce Strong Genital Immunity Through Secretory IgA in Minipigs Infected with Chlamydia trachomatis. Front Immunol, 6, 628.
Macnab, R.M. 1992. Genetics and biogenesis of bacterial flagella. Annu Rev Genet, 26, 131-58.
Marchette, N.J., Garcia, R. 1969. Isolation of Zika virus from Aedes aegypti mosquitoes in Malaysia. Am. J. Trop. Med. Hyg., 18, 411-415.
Maue, A.C., Mohawk, K.L., Giles, D.K., Poly, F., Ewing, C.P., Jiao, Y., Lee, G., Ma, Z., Monteiro, M.A., Hill, C.L., Ferderber, J.S., Porter, C.K., Trent, M.S., Guerry, P. 2013. The polysaccharide capsule of Campylobacter jejuni modulates the host immune response. Infect Immun, 81(3), 665-72.
Mizel, S.B., Bates, J.T. 2010. Flagellin as an adjuvant: cellular mechanisms and potential. J Immunol, 185(10), 5677-82.
Mizel, S.B., Graff, A.H., Sriranganathan, N., Ervin, S., Lees, C.J., Lively, M.O., Hantgan, R.R., Thomas, M.J., Wood, J., Bell, B. 2009. Flagellin-F1-V fusion protein is an effective plague vaccine in mice and two species of nonhuman primates. Clin Vaccine Immunol, 16(1), 21-8.
Nithichanon, A., Gourlay, L.J., Bancroft, G.J., Ato, M., Takahashi, Y., Lertmemongkolchai, G. 2017. Boosting of post-exposure human T-cell and B-cell recall responses in vivo by Burkholderia pseudomallei-related proteins. Immunology, 151(1), 98-109.
Nutt, C., Adams, P. 2017. Zika in Africa-the invisible epidemic? Lancet, 389(10079), 1595-1596.
Oehler, E., Watrin, L., Larre, P., Leparc-Goffart, I., Lastere, S., Valour, F., Baudouin, L., Mallet, H., Musso, D., Ghawche, F. 2014. Zika virus infection complicated by Guillain-Barre syndrome--case report, French Polynesia, December 2013. Euro Surveill, 19(9).
Olsen, A.W., Follmann, F., Erneholm, K., Rosenkrands, I., Andersen, P. 2015. Protection Against Chlamydia trachomatis Infection and Upper Genital Tract Pathological Changes by Vaccine-Promoted Neutralizing Antibodies Directed to the VD4 of the Major Outer Membrane Protein. J Infect Dis, 212(6), 978-89.
Olsen, A.W., Theisen, M., Christensen, D., Follmann, F., Andersen, P. 2010. Protection against Chlamydia promoted by a subunit vaccine (CTH1) compared with a primary intranasal infection in a mouse genital challenge model. PLoS One, 5(5), e10768.
Pardi, N., Hogan, M.J., Pelc, R.S., Muramatsu, H., Andersen, H., DeMaso, C.R., Dowd, K.A., Sutherland, L.L., Scearce, R.M., Parks, R., Wagner, W., Granados, A., Greenhouse, J., Walker, M., Willis, E., Yu, J.S., McGee, C.E., Sempowski, G.D., Mui, B.L., Tam, Y.K., Huang, Y.J., Vanlandingham, D., Holmes, V.M., Balachandran, H., Sahu, S., Lifton, M., Higgs, S., Hensley, S.E., Madden, T.D., Hope, M.J., Kariko, K., Santra, S., Graham, B.S., Lewis, M.G., Pierson, T.C., Haynes, B.F., Weissman, D. 2017. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature, 543(7644), 248-251.
Peppoloni, S., Ruggiero, P., Contorni, M., Morandi, M., Pizza, M., Rappuoli, R., Podda, A., Del Giudice, G. 2003. Mutants of the Escherichia coli heat-labile enterotoxin as safe and strong adjuvants for intranasal delivery of vaccines. Expert Rev Vaccines, 2(2), 285-93.
Pizza, M., Giuliani, M.M., Fontana, M.R., Monaci, E., Douce, G., Dougan, G., Mills, K.H., Rappuoli, R., Del Giudice, G. 2001. Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants. Vaccine, 19(17-19), 2534-41.
Plourde, A.R., Bloch, E.M. 2016. A Literature Review of Zika Virus. Emerg Infect Dis, 22(7), 1185-92.
Pun, P.B., Bhat, A.A., Mohan, T., Kulkarni, S., Paranjape, R., Rao, D.N. 2009. Intranasal administration of peptide antigens of HIV with mucosal adjuvant CpG ODN coentrapped in microparticles enhances the mucosal and systemic immune responses. Int Immunopharmacol, 9(4), 468-77.
Ramos, H.C., Rumbo, M., Sirard, J.C. 2004. Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa. Trends Microbiol, 12(11), 509-17.
Renegar, K.B., Jackson, G.D., Mestecky, J. 1998. In vitro comparison of the biologic activities of monoclonal monomeric IgA, polymeric IgA, and secretory IgA. J Immunol, 160(3), 1219-23.
Schepetkin, I.A., Kouakou, K., Yapi, A., Kirpotina, L.N., Jutila, M.A., Quinn, M.T. 2013. Immunomodulatory and hemagglutinating activities of acidic polysaccharides isolated from Combretum racemosum. Int Immunopharmacol, 15(3), 628-37.
Schuler-Faccini, L., Ribeiro, E.M., Feitosa, I.M., Horovitz, D.D., Cavalcanti, D.P., Pessoa, A., Doriqui, M.J., Neri, J.I., Neto, J.M., Wanderley, H.Y., Cernach, M., El-Husny, A.S., Pone, M.V., Serao, C.L., Sanseverino, M.T., Brazilian Medical Genetics Society-Zika Embryopathy Task, F. 2016. Possible Association Between Zika Virus Infection and Microcephaly - Brazil, 2015. MMWR Morb Mortal Wkly Rep, 65(3), 59-62.
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(6), 763-767.
Shi, W., Li, Y.H., Liu, F., Yang, J.Y., Zhou, D.H., Chen, Y.Q., Zhang, Y., Yang, Y., He, B.X., Han, C., Fan, M.W., Yan, H.M. 2012. Flagellin enhances saliva IgA response and protection of anti-caries DNA vaccine. J Dent Res, 91(3), 249-54.
Smith, K.D., Andersen-Nissen, E., Hayashi, F., Strobe, K., Bergman, M.A., Barrett, S.L., Cookson, B.T., Aderem, A. 2003. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immunol, 4(12), 1247-53.
Soares de Araujo, J.S., Regis, C.T., Gomes, R.G., Tavares, T.R., Rocha Dos Santos, C., Assuncao, P.M., Nobrega, R.V., Pinto, D.F., Bezerra, B.V., Mattos, S.D. 2016. Microcephaly in north-east Brazil: a retrospective study on neonates born between 2012 and 2015. Bull World Health Organ, 94(11), 835-840.
Song, W.S., Jeon, Y.J., Namgung, B., Hong, M., Yoon, S.I. 2017. A conserved TLR5 binding and activation hot spot on flagellin. Sci Rep, 7, 40878.
Stary, G., Olive, A., Radovic-Moreno, A.F., Gondek, D., Alvarez, D., Basto, P.A., Perro, M., Vrbanac, V.D., Tager, A.M., Shi, J., Yethon, J.A., Farokhzad, O.C., Langer, R., Starnbach, M.N., von Andrian, U.H. 2015. VACCINES. A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells. Science, 348(6241), aaa8205.
Suy, A., Sulleiro, E., Rodo, C., Vazquez, E., Bocanegra, C., Molina, I., Esperalba, J., Sanchez-Seco, M.P., Boix, H., Pumarola, T., Carreras, E. 2016. Prolonged Zika Virus Viremia during Pregnancy. N Engl J Med, 375(26), 2611-2613.
Tai, W., He, L., Wang, Y., Sun, S., Zhao, G., Luo, C., Li, P., Zhao, H., Fremont, D.H., Li, F., Jiang, S., Zhou, Y., Du, L. 2018. Critical neutralizing fragment of Zika virus EDIII elicits cross-neutralization and protection against divergent Zika viruses. Emerg Microbes Infect, 7(1), 7.
Takeda, K., Kaisho, T., Akira, S. 2003. Toll-like receptors. Annu Rev Immunol, 21, 335-76.
Vajdy, M., Lycke, N.Y. 1992. Cholera toxin adjuvant promotes long-term immunological memory in the gut mucosa to unrelated immunogens after oral immunization. Immunology, 75(3), 488-92.
van den Akker, F., Sarfaty, S., Twiddy, E.M., Connell, T.D., Holmes, R.K., Hol, W.G. 1996. Crystal structure of a new heat-labile enterotoxin, LT-IIb. Structure, 4(6), 665-78.
van den Berg, B., Bunschoten, C., van Doorn, P.A., Jacobs, B.C. 2013. Mortality in Guillain-Barre syndrome. Neurology, 80(18), 1650-4.
Veazey, R.S., Siddiqui, A., Klein, K., Buffa, V., Fischetti, L., Doyle-Meyers, L., King, D.F., Tregoning, J.S., Shattock, R.J. 2015. Evaluation of mucosal adjuvants and immunization routes for the induction of systemic and mucosal humoral immune responses in macaques. Hum Vaccin Immunother, 11(12), 2913-22.
Wang, Y.Y., Khoo, K.H., Chen, S.T., Lin, C.C., Wong, C.H., Lin, C.H. 2002. Studies on the immuno-modulating and antitumor activities of Ganoderma lucidum (Reishi) polysaccharides: functional and proteomic analyses of a fucose-containing glycoprotein fraction responsible for the activities. Bioorg Med Chem, 10(4), 1057-62.
Wen, J., Elong Ngono, A., Regla-Nava, J.A., Kim, K., Gorman, M.J., Diamond, M.S., Shresta, S. 2017. Dengue virus-reactive CD8(+) T cells mediate cross-protection against subsequent Zika virus challenge. Nat Commun, 8(1), 1459.
Wern, J.E., Sorensen, M.R., Olsen, A.W., Andersen, P., Follmann, F. 2017. Simultaneous Subcutaneous and Intranasal Administration of a CAF01-Adjuvanted Chlamydia Vaccine Elicits Elevated IgA and Protective Th1/Th17 Responses in the Genital Tract. Front Immunol, 8, 569.
WHO, 2016. WHO Director-General summarizes the outcome of the Emergency Committee regarding clusters of microcephaly and Guillain-Barré syndrome, February 1st 2016. http://www.who.int/mediacentre/news/statements/2016/emergency-committee-zika-microcephaly/en/ (Accessed 10 July 2018).
WHO, 2017. Zika virus situation reports, 10 March 2017. http://www.who.int/emergencies/zika-virus/situation-report/10-march-2017/en/ (Accessed 10 July 2018).
WHO, 2018. WHO vaccine pipeline tracker. http://www.who.int/immunization/research/vaccine_pipeline_tracker_spreadsheet/en/ (Accessed 10 July 2018).
Woodrow, K.A., Bennett, K.M., Lo, D.D. 2012. Mucosal vaccine design and delivery. Annu Rev Biomed Eng, 14, 17-46.
Xiao, Z.Q., Wang, Y.L., Yue, Y.D., Zhang, Y.T., Chen, C.P., Wan, L.S., Deng, B., Liu, Z.X., Chen, J.C. 2013. Preventive effects of polysaccharides from Liriope spicata var. prolifera on diabetic nephropathy in rats. Int J Biol Macromol, 61, 114-20.
Yeh, C.H., Chen, H.C., Yang, J.J., Chuang, W.I., Sheu, F. 2010. Polysaccharides PS-G and protein LZ-8 from Reishi (Ganoderma lucidum) exhibit diverse functions in regulating murine macrophages and T lymphocytes. J Agric Food Chem, 58(15), 8535-44.
Yonekura, K., Maki-Yonekura, S., Namba, K. 2002. Growth mechanism of the bacterial flagellar filament. Res Microbiol, 153(4), 191-7.
Zhang, X., Qi, C., Guo, Y., Zhou, W., Zhang, Y. 2016. Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible interaction models. Carbohydr Polym, 149, 186-206.
Zhao, T., Feng, Y., Li, J., Mao, R., Zou, Y., Feng, W., Zheng, D., Wang, W., Chen, Y., Yang, L., Wu, X. 2014. Schisandra polysaccharide evokes immunomodulatory activity through TLR 4-mediated activation of macrophages. Int J Biol Macromol, 65, 33-40.