登革熱是一種藉由病媒蚊傳播的疾病，每年估計造成3.9億起感染且尚未具有有效的治療藥物或預防疫苗，是快速加劇中的公眾健康問題。因此，急迫需要開發出能夠有效預防登革病毒感染的疫苗。在過去的研究中，活毒減毒疫苗、腺病毒載體疫苗與重組套膜蛋白第三區融合鞭毛蛋白已經被證實能夠引發對抗登革病毒之免疫反應。此篇研究中，我們利用上述三種疫苗針對第二型及第四型登革病毒組成單價或雙價疫苗，進行homologous prime-boost免疫法和heterologous prime-boost免疫法的實驗，研究不同免疫法所引發的免疫反應。結果顯示，各種免疫法的單價疫苗都能在小鼠體內針對該血清型病毒產生體液免疫反應並產生中和抗體，其中，兩劑重組套膜蛋白第三區融合鞭毛蛋白可針對第二型病毒產生最佳的中和抗體效價，對第四型病毒則以腺病毒載體prime/重組套膜蛋白第三區融合鞭毛蛋白boost為最佳。我們亦證實辨認套膜蛋白第三區之抗體在病毒中和能力中扮演重要的角色。第二、四型雙價疫苗的評估結果顯示使用活毒減毒疫苗或腺病毒載體疫苗prime/重組套膜蛋白第三區融合鞭毛蛋白boost的heterologous prime-boost免疫法能夠引發較佳的體液免疫反應且對第二、四型都能激起最高的中和抗體效價。Heterologous prime-boost免疫法相較於兩劑活毒減毒疫苗或腺病毒載體疫苗，並不會對異型感染產生更嚴重的抗體依賴性增強之現象，對同型感染則僅造成輕微的增強反應。此研究成果顯示以活毒減毒疫苗或腺病毒載體疫苗prime/重組套膜蛋白第三區融合鞭毛蛋白boost的heterologous prime-boost免疫法具有開發登革疫苗之潛力。

Dengue, an arthropod-borne viral disease, is estimated to cause about 390 million infections annually and is a fast emerging public health problem without any licensed anti-dengue drugs for treatment. The need of an effective dengue vaccine to prevent the disease is urgent. Live-attenuated virus vaccine (LAV), adenoviral vector expressing prME vaccine (Ad-prME), flagellin-fused envelop domain III recombinant protein (FliC-EDIII) have been reported to elicit immune responses against dengue viruses (DENV), and are potential for vaccine development. In this study, we evaluated both homologous and heterologous prime-boost strategy using LAV, Ad-prME and FliC-EDIII against DENV2 and DENV4 in monovalent or bivalent formulations. All monovalent regimens were found to trigger humoral immune responses and neutralizing antibodies against homotypic DENV while two-dose FliC-DENV2 EDIII and Ad-DENV4 prME primed/ FliC-DENV4 EDIII boosted group performed best against respective serotypes in mice. We also found that anti-EDIII antibodies played important roles in virus neutralization. In bivalent formulation, using LAV or Ad-prME to prime and FliC-EDIII to boost performed better than those homologous regimens in both humoral immune responses and virus neutralization abilities against both DENV serotypes. Moreover, heterologous regimens wouldn’t trigger severer antibody-dependent enhancement (ADE) in heterotypic infections compared to LAV and Ad-prME homologous regimens, but induce only minor ADE in homotypic infections. These results suggest that heterologous regimens using LAV or Ad-prME to prime and FliC-EDIII to boost have the potential of developing dengue vaccine.

中文摘要 I
Abstract II
Acknowledgements III
Contents IV
1. Introduction 1
1.1 Overview of Dengue virus 1
1.2 Dengue vaccine development 3
1.2.1 Live-attenuated virus vaccines 4
1.2.2 Adenovirus-vectored vaccines 7
1.2.3 Recombinant Subunit vaccines 9
1.3 Heterologous prime-boost strategy 11
1.4 Study aims 12
2. Materials and methods 14
2.1 Cells and media 14
2.2 Preparation of wild-type dengue viruses and live-attenuated virus vaccines 14
2.2.1 DENV1, DENV2 and DENV3 wild-type viruses 14
2.2.2 Production of DENV4, DENV4 Δ30 and DENV2/4 Δ30 from infectious cDNA clones 15
2.2.3 Determination of dengue virus titer 16
2.3 Preparation of recombinant adenoviral-vector vaccines 16
2.3.1 Construction and production of adenoviral vectors expressing DENV prM and E 16
2.3.2 Determination of recombinant adenovirus titers 17
2.4 Preparation of recombinant Flic, EDIII and Flic-EDIII proteins 18
2.4.1 Construction of recombinant protein expression vectors 18
2.4.2 Expression of recombinant proteins 18
2.4.3 Purification of recombinant proteins 19
2.5 Replication kinetics of live-attenuated virus 19
2.6 Immunofluorescence assay 20
2.7 SDS-PAGE, coomassie blue staining and westernblotting 20
2.8 TLR5-dependent functional assay 21
2.9 Mice immunization 22
2.10 Enzyme-linked immunosorbent assay (ELISA) 22
2.11 Focus reduction neutralizing test (FRNT) 23
2.12 Depletion of EDIII-reactive antibody in mouse immune sera 23
2.13 In vivo neutralizing assay 24
2.14 Antibody-dependent enhancement assay 24
3. Results 26
3.1 Recovery and analysis of infectious DENV4 Δ30 and DENV2/4 Δ30 viruses 26
3.2 Preparation of adenoviral vectors expressing DENV2 or 4 prM and E 26
3.3 Preparation and functional examination of recombinant EDIII and FliC-EDIII proteins 27
3.4 Immune responses induced by DENV2 monovalent vaccines 28
3.5 Immune responses induced by DENV4 monovalent vaccines 29
3.6 Contribution of anti-EDIII antibodies elicited by DENV2 or 4 monovalent vaccines to virus neutralization 30
3.7 Immune responses induced by DENV2 and DENV4 bivalent vaccine in BALB/c or AG129 immunization experiments 30
3.8 Neutralizing antibody responses in BALB/c or AG129 mice immunized with DENV2 and DENV4 bivalent vaccine 32
3.9 Protection of sera from AG129 mice immunized with bivalent vaccines against DENV2 or DENV4 in suckling mice 33
3.10 In vitro antibody-dependent enhancement mediated by the sera from AG129 mice immunized with DENV2 and 4 bivalent vaccines 34
4. Discussion 35
5. References 41
6. Figures 51
Figure 1. Characterization of recombinant live-attenuated DENV vaccine candidates 51
Figure 2. Characterization of recombinant adenoviruses expressed prM and E genes of dengue virus 52
Figure 3. Expression and identification of recombinant proteins 53
Figure 4. Schedule of DENV vaccines immunization 54
Figure 5. The immune responses against DENV2 in BALB/c mice immunized with monovalent vaccines 55
Figure 6. The immune responses against DENV4 in BALB/c mice immunized with monovalent vaccines 56
Figure 7. Neutralization against DENV2 in D2EDIII binding antibodies depleted mice immune sera 57
Figure 8. Neutralization against DENV4 in D4EDIII binding antibodies depleted mice immune sera 58
Figure 9. Total IgG titers against inactivated DENV2 or DENV4 virus in BALB/c and AG129 mice immunized with bivalent vaccines 59
Figure 10. Total IgG titers against D2EDIII or D4EDIII in BALB/c and AG129 mice immunized with bivalent vaccines 60
Figure 11. Neutralization curves against DENV2 or DENV4 in BALB/c or AG129 mice immunized with bivalent vaccines 61
Figure 12. Neutralizing antibody titers against DENV2 or DENV4 in BALB/c or AG129 mice immunized with bivalent vaccines 62
Figure 13. Protection of sera from AG129 mice immunized with bivalent vaccines against DENV2 or DENV4 in suckling mice 63
Figure 14. ADE of DENV replication in K562 cells mediated by sera of AG129 mice immunized with bivalent vaccines 64
7. Tables 65
Table 1. DENV2 monovalent immunization regimens 65
Table 2. DENV4 monovalent immunization regimens 66
Table 3. DENV2 and DENV4 bivalent immunization regimens 67

Allison, S.L., Schalich, J., Stiasny, K., Mandl, C.W., Heinz, F.X., 2001. Mutational evidence for an internal fusion peptide in flavivirus envelope protein E. Journal of virology 75, 4268-4275.
Angsubhakorn, S., Moe, J.B., Marchette, N.J., Palumbo, N.E., Yoksan, S., Bhamarapravati, N., 1987. Neurovirulence effects of dengue-2 viruses on the rhesus (Macaca mulatta) brain and spinal cord. The Southeast Asian journal of tropical medicine and public health 18, 52-55.
Angsubhakorn, S., Yoksan, S., Bhamarapravati, N., Moe, J.B., Marchette, N.J., Pradermwong, A., Sahaphong, S., 1988. Dengue-4 vaccine: neurovirulence, viraemia and immune responses in rhesus and cynomolgus monkeys. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 746-749.
Angsubhakorn, S., Yoksan, S., Pradermwong, A., Nitatpattana, N., Sahaphong, S., Bhamarapravati, N., 1994. Dengue-3 (16562) PGMK 33 vaccine: neurovirulence, viremia and immune responses in Macaca fascicularis. The Southeast Asian journal of tropical medicine and public health 25, 554-559.
Back, A.T., Lundkvist, A., 2013. Dengue viruses - an overview. Infection ecology & epidemiology 3.
Beckett, C.G., Tjaden, J., Burgess, T., Danko, J.R., Tamminga, C., Simmons, M., Wu, S.J., Sun, P., Kochel, T., Raviprakash, K., Hayes, C.G., Porter, K.R., 2011. Evaluation of a prototype dengue-1 DNA vaccine in a Phase 1 clinical trial. Vaccine 29, 960-968.
Bellanti, J.A., Bourke, A.T., Buescher, E.L., Cadigan, F.C., Cole, G.A., El Batawi, Y., Hatgi, J.N., McCown, J.M., Negron, H., Ordonez, J.V., Scheider, F.G., Smith, T., Warram, J.H., Wisseman, C.L., 1966. Report of dengue vaccine field trial in the Caribbean, 1963: a collaborative study. Bulletin of the World Health Organization 35, 93.
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.
Bernardo, L., Izquierdo, A., Alvarez, M., Rosario, D., Prado, I., Lopez, C., Martinez, R., Castro, J., Santana, E., Hermida, L., Guillen, G., Guzman, M.G., 2008. Immunogenicity and protective efficacy of a recombinant fusion protein containing the domain III of the dengue 1 envelope protein in non-human primates. Antiviral research 80, 194-199.
Bhamarapravati, N., Yoksan, S., 1989. Study of bivalent dengue vaccine in volunteers. Lancet 1, 1077.
Bhamarapravati, N., Yoksan, S., Chayaniyayothin, T., Angsubphakorn, S., Bunyaratvej, A., 1987. Immunization with a live attenuated dengue-2-virus candidate vaccine (16681-PDK 53): clinical, immunological and biological responses in adult volunteers. Bulletin of the World Health Organization 65, 189-195.
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.
Blaney, J.E., Jr., Hanson, C.T., Firestone, C.Y., Hanley, K.A., Murphy, B.R., Whitehead, S.S., 2004. Genetically modified, live attenuated dengue virus type 3 vaccine candidates. The American journal of tropical medicine and hygiene 71, 811-821.
Blaney, J.E., Jr., Sathe, N.S., Goddard, L., Hanson, C.T., Romero, T.A., Hanley, K.A., Murphy, B.R., Whitehead, S.S., 2008. Dengue virus type 3 vaccine candidates generated by introduction of deletions in the 3' untranslated region (3'-UTR) or by exchange of the DENV-3 3'-UTR with that of DENV-4. Vaccine 26, 817-828.
Bray, M., Lai, C.J., 1991. Construction of intertypic chimeric dengue viruses by substitution of structural protein genes. Proceedings of the National Academy of Sciences of the United States of America 88, 10342-10346.
Brewoo, J.N., Kinney, R.M., Powell, T.D., Arguello, J.J., Silengo, S.J., Partidos, C.D., Huang, C.Y., Stinchcomb, D.T., Osorio, J.E., 2012. Immunogenicity and efficacy of chimeric dengue vaccine (DENVax) formulations in interferon-deficient AG129 mice. Vaccine 30, 1513-1520.
Brien, J.D., Austin, S.K., Sukupolvi-Petty, S., O'Brien, K.M., Johnson, S., Fremont, D.H., Diamond, M.S., 2010. Genotype-specific neutralization and protection by antibodies against dengue virus type 3. Journal of virology 84, 10630-10643.
Butrapet, S., Huang, C.Y., Pierro, D.J., Bhamarapravati, N., Gubler, D.J., Kinney, R.M., 2000. Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5' noncoding region and nonstructural proteins 1 and 3. Journal of virology 74, 3011-3019.
Butrapet, S., Rabablert, J., Angsubhakorn, S., Wiriyarat, W., Huang, C., Kinney, R., Punyim, S., Bhamarapravati, N., 2002. Chimeric dengue type 2/type 1 viruses induce immune responses in cynomolgus monkeys. The Southeast Asian journal of tropical medicine and public health 33, 589-599.
Centers for Disease Control and Prevention, 2012. Principles of Vaccination, in: Atkinson W, W.S., Hamborsky J (Ed.), Epidemiology and Prevention of Vaccine-Preventable Diseases, 12 ed. Public Health Foundation, Washington DC.
Chen, H.W., Liu, S.J., Liu, H.H., Kwok, Y., Lin, C.L., Lin, L.H., Chen, M.Y., Tsai, J.P., Chang, L.S., Chiu, F.F., Lai, L.W., Lian, W.C., Yang, C.Y., Hsieh, S.Y., Chong, P., Leng, C.H., 2009. A novel technology for the production of a heterologous lipoprotein immunogen in high yield has implications for the field of vaccine design. Vaccine 27, 1400-1409.
Chen, L., Ewing, D., Subramanian, H., Block, K., Rayner, J., Alterson, K.D., Sedegah, M., Hayes, C., Porter, K., Raviprakash, K., 2007. A heterologous DNA prime-Venezuelan equine encephalitis virus replicon particle boost dengue vaccine regimen affords complete protection from virus challenge in cynomolgus macaques. Journal of virology 81, 11634-11639.
Chmielewska, A.M., Naddeo, M., Capone, S., Ammendola, V., Hu, K., Meredith, L., Verhoye, L., Rychlowska, M., Rappuoli, R., Ulmer, J.B., Colloca, S., Nicosia, A., Cortese, R., Leroux-Roels, G., Balfe, P., Bienkowska-Szewczyk, K., Meuleman, P., McKeating, J.A., Folgori, A., 2014. Combined adenovirus vector and hepatitis C virus envelope protein prime-boost regimen elicits T cell and neutralizing antibody immune responses. Journal of virology 88, 5502-5510.
Choi, Y., Chang, J., 2013. Viral vectors for vaccine applications. Clinical and experimental vaccine research 2, 97-105.
Clements, D.E., Coller, B.A., Lieberman, M.M., Ogata, S., Wang, G., Harada, K.E., Putnak, J.R., Ivy, J.M., McDonell, M., Bignami, G.S., Peters, I.D., Leung, J., Weeks-Levy, C., Nakano, E.T., Humphreys, T., 2010. Development of a recombinant tetravalent dengue virus vaccine: immunogenicity and efficacy studies in mice and monkeys. Vaccine 28, 2705-2715.
Coller, B.A., Barrett, A.D., Thomas, S.J., 2011. The development of Dengue vaccines. Introduction. Vaccine 29, 7219-7220.
Crill, W.D., Chang, G.J., 2004. Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes. Journal of virology 78, 13975-13986.
Crill, W.D., Roehrig, J.T., 2001. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. Journal of virology 75, 7769-7773.
Durbin, A.P., Karron, R.A., Sun, W., Vaughn, D.W., Reynolds, M.J., Perreault, J.R., Thumar, B., Men, R., Lai, C.J., Elkins, W.R., Chanock, R.M., Murphy, B.R., Whitehead, S.S., 2001. Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3'-untranslated region. The American journal of tropical medicine and hygiene 65, 405-413.
Durbin, A.P., Kirkpatrick, B.D., Pierce, K.K., Schmidt, A.C., Whitehead, S.S., 2011. Development and clinical evaluation of multiple investigational monovalent DENV vaccines to identify components for inclusion in a live attenuated tetravalent DENV vaccine. Vaccine 29, 7242-7250.
Durbin, A.P., McArthur, J., Marron, J.A., Blaney, J.E., Jr., Thumar, B., Wanionek, K., Murphy, B.R., Whitehead, S.S., 2006a. The live attenuated dengue serotype 1 vaccine rDEN1Delta30 is safe and highly immunogenic in healthy adult volunteers. Human vaccines 2, 167-173.
Durbin, A.P., McArthur, J.H., Marron, J.A., Blaney, J.E., Thumar, B., Wanionek, K., Murphy, B.R., Whitehead, S.S., 2006b. rDEN2/4Delta30(ME), a live attenuated chimeric dengue serotype 2 vaccine is safe and highly immunogenic in healthy dengue-naive adults. Human vaccines 2, 255-260.
Durbin, A.P., Whitehead, S.S., 2010. Dengue vaccine candidates in development. Current topics in microbiology and immunology 338, 129-143.
Feighny, R., Burrous, J., Putnak, R., 1994. Dengue type-2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge. The American journal of tropical medicine and hygiene 50, 322-328.
Fonseca, B.A., Pincus, S., Shope, R.E., Paoletti, E., Mason, P.W., 1994. Recombinant vaccinia viruses co-expressing dengue-1 glycoproteins prM and E induce neutralizing antibodies in mice. Vaccine 12, 279-285.
Gabitzsch, E.S., Xu, Y., Yoshida, L.H., Balint, J., Amalfitano, A., Jones, F.R., 2009. Novel Adenovirus type 5 vaccine platform induces cellular immunity against HIV-1 Gag, Pol, Nef despite the presence of Ad5 immunity. Vaccine 27, 6394-6398.
Gromowski, G.D., Barrett, N.D., Barrett, A.D., 2008. Characterization of dengue virus complex-specific neutralizing epitopes on envelope protein domain III of dengue 2 virus. Journal of virology 82, 8828-8837.
Guirakhoo, F., Kitchener, S., Morrison, D., Forrat, R., McCarthy, K., Nichols, R., Yoksan, S., Duan, X., Ermak, T.H., Kanesa-Thasan, N., Bedford, P., Lang, J., Quentin-Millet, M.J., Monath, T.P., 2006. Live attenuated chimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vaccine: Phase I clinical trial for safety and immunogenicity: effect of yellow fever pre-immunity in induction of cross neutralizing antibody responses to all 4 dengue serotypes. Human vaccines 2, 60-67.
Guy, B., Barrere, B., Malinowski, C., Saville, M., Teyssou, R., Lang, J., 2011. From research to phase III: preclinical, industrial and clinical development of the Sanofi Pasteur tetravalent dengue vaccine. Vaccine 29, 7229-7241.
Guy, B., Nougarede, N., Begue, S., Sanchez, V., Souag, N., Carre, M., Chambonneau, L., Morrisson, D.N., Shaw, D., Qiao, M., Dumas, R., Lang, J., Forrat, R., 2008. Cell-mediated immunity induced by chimeric tetravalent dengue vaccine in naive or flavivirus-primed subjects. Vaccine 26, 5712-5721.
Hahn, C.S., Dalrymple, J.M., Strauss, J.H., Rice, C.M., 1987. Comparison of the virulent Asibi strain of yellow fever virus with the 17D vaccine strain derived from it. Proceedings of the National Academy of Sciences of the United States of America 84, 2019-2023.
Halstead, S.B., 2003. Neutralization and antibody-dependent enhancement of dengue viruses. Advances in virus research 60, 421-467.
Halstead, S.B., 2007. Dengue. Lancet 370, 1644-1652.
Hartman, Z.C., Black, E.P., Amalfitano, A., 2007. Adenoviral infection induces a multi-faceted innate cellular immune response that is mediated by the toll-like receptor pathway in A549 cells. Virology 358, 357-372.
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, 1099-1103.
Hensley, S.E., Giles-Davis, W., McCoy, K.C., Weninger, W., Ertl, H.C., 2005. Dendritic cell maturation, but not CD8+ T cell induction, is dependent on type I IFN signaling during vaccination with adenovirus vectors. Journal of immunology 175, 6032-6041.
Hermida, L., Bernardo, L., Martin, J., Alvarez, M., Prado, I., Lopez, C., Sierra Bde, L., Martinez, R., Rodriguez, R., Zulueta, A., Perez, A.B., Lazo, L., Rosario, D., Guillen, G., Guzman, M.G., 2006. A recombinant fusion protein containing the domain III of the dengue-2 envelope protein is immunogenic and protective in nonhuman primates. Vaccine 24, 3165-3171.
Hermida, L., Rodriguez, R., Lazo, L., Silva, R., Zulueta, A., Chinea, G., Lopez, C., Guzman, M.G., Guillen, G., 2004. A dengue-2 Envelope fragment inserted within the structure of the P64k meningococcal protein carrier enables a functional immune response against the virus in mice. Journal of virological methods 115, 41-49.
Holman, D.H., Wang, D., Raviprakash, K., Raja, N.U., Luo, M., Zhang, J., Porter, K.R., Dong, J.Y., 2007. Two complex, adenovirus-based vaccines that together induce immune responses to all four dengue virus serotypes. Clinical and vaccine immunology : CVI 14, 182-189.
Huang, C.Y., Butrapet, S., Tsuchiya, K.R., Bhamarapravati, N., Gubler, D.J., Kinney, R.M., 2003. Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development. Journal of virology 77, 11436-11447.
Ishikawa, T., Yamanaka, A., Konishi, E., 2014. A review of successful flavivirus vaccines and the problems with those flaviviruses for which vaccines are not yet available. Vaccine 32, 1326-1337.
Kanesa-thasan, N., Sun, W., Kim-Ahn, G., Van Albert, S., Putnak, J.R., King, A., Raengsakulsrach, B., Christ-Schmidt, H., Gilson, K., Zahradnik, J.M., Vaughn, D.W., Innis, B.L., Saluzzo, J.F., Hoke, C.H., Jr., 2001. Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers. Vaccine 19, 3179-3188.
Khanam, S., Pilankatta, R., Khanna, N., Swaminathan, S., 2009. An adenovirus type 5 (AdV5) vector encoding an envelope domain III-based tetravalent antigen elicits immune responses against all four dengue viruses in the presence of prior AdV5 immunity. Vaccine 27, 6011-6021.
Khanam, S., Rajendra, P., Khanna, N., Swaminathan, S., 2007. An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes. BMC biotechnology 7, 10.
Kinney, R.M., Butrapet, S., Chang, G.J., Tsuchiya, K.R., Roehrig, J.T., Bhamarapravati, N., Gubler, D.J., 1997. Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53. Virology 230, 300-308.
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.
Kyle, J.L., Balsitis, S.J., Zhang, L., Beatty, P.R., Harris, E., 2008. Antibodies play a greater role than immune cells in heterologous protection against secondary dengue virus infection in a mouse model. Virology 380, 296-303.
Lai, C.J., Zhao, B.T., Hori, H., Bray, M., 1991. Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus. Proceedings of the National Academy of Sciences of the United States of America 88, 5139-5143.
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. Journal of virology 82, 6631-6643.
Leng, C.H., Liu, S.J., Tsai, J.P., Li, Y.S., Chen, M.Y., Liu, H.H., Lien, S.P., Yueh, A., Hsiao, K.N., Lai, L.W., Liu, F.C., Chong, P., Chen, H.W., 2009. A novel dengue vaccine candidate that induces cross-neutralizing antibodies and memory immunity. Microbes and infection / Institut Pasteur 11, 288-295.
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. Journal of virology 80, 10237-10247.
Lindenbach BD, T.H.-J., Rice CM, 2007. Flaviviridae: TheViruses and Their Replication, in: D. M. Knipe, P.M.H. (Ed.), Fields Virology, 5 ed. Lippincott-Raven, pp. 1101-1152.
Lu, S., 2009. Heterologous prime-boost vaccination. Current opinion in immunology 21, 346-351.
Mantel, N., Girerd, Y., Geny, C., Bernard, I., Pontvianne, J., Lang, J., Barban, V., 2011. Genetic stability of a dengue vaccine based on chimeric yellow fever/dengue viruses. Vaccine 29, 6629-6635.
Mason, P.W., Zugel, M.U., Semproni, A.R., Fournier, M.J., Mason, T.L., 1990. The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli. The Journal of general virology 71 ( Pt 9), 2107-2114.
Men, R., Bray, M., Clark, D., Chanock, R.M., Lai, C.J., 1996. Dengue type 4 virus mutants containing deletions in the 3' noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. Journal of virology 70, 3930-3937.
Men, R., Wyatt, L., Tokimatsu, I., Arakaki, S., Shameem, G., Elkins, R., Chanock, R., Moss, B., Lai, C.J., 2000. Immunization of rhesus monkeys with a recombinant of modified vaccinia virus Ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge. Vaccine 18, 3113-3122.
Midgley, C.M., Bajwa-Joseph, M., Vasanawathana, S., Limpitikul, W., Wills, B., Flanagan, A., Waiyaiya, E., Tran, H.B., Cowper, A.E., Chotiyarnwong, P., Grimes, J.M., Yoksan, S., Malasit, P., Simmons, C.P., Mongkolsapaya, J., Screaton, G.R., 2011. An in-depth analysis of original antigenic sin in dengue virus infection. Journal of virology 85, 410-421.
Modis, Y., Ogata, S., Clements, D., Harrison, S.C., 2003. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proceedings of the National Academy of Sciences of the United States of America 100, 6986-6991.
Modis, Y., Ogata, S., Clements, D., Harrison, S.C., 2004. Structure of the dengue virus envelope protein after membrane fusion. Nature 427, 313-319.
Monath, T.P., Myers, G.A., Beck, R.A., Knauber, M., Scappaticci, K., Pullano, T., Archambault, W.T., Catalan, J., Miller, C., Zhang, Z.X., Shin, S., Pugachev, K., Draper, K., Levenbook, I.S., Guirakhoo, F., 2005. Safety testing for neurovirulence of novel live, attenuated flavivirus vaccines: infant mice provide an accurate surrogate for the test in monkeys. Biologicals : journal of the International Association of Biological Standardization 33, 131-144.
Morrison, D., Legg, T.J., Billings, C.W., Forrat, R., Yoksan, S., Lang, J., 2010. A novel tetravalent dengue vaccine is well tolerated and immunogenic against all 4 serotypes in flavivirus-naive adults. The Journal of infectious diseases 201, 370-377.
Mota, J., Acosta, M., Argotte, R., Figueroa, R., Mendez, A., Ramos, C., 2005. Induction of protective antibodies against dengue virus by tetravalent DNA immunization of mice with domain III of the envelope protein. Vaccine 23, 3469-3476.
Murphy, B.R., Whitehead, S.S., 2011. Immune response to dengue virus and prospects for a vaccine. Annual review of immunology 29, 587-619.
Osorio, J.E., Brewoo, J.N., Silengo, S.J., Arguello, J., Moldovan, I.R., Tary-Lehmann, M., Powell, T.D., Livengood, J.A., Kinney, R.M., Huang, C.Y., Stinchcomb, D.T., 2011. Efficacy of a tetravalent chimeric dengue vaccine (DENVax) in Cynomolgus macaques. The American journal of tropical medicine and hygiene 84, 978-987.
Paranjape, S.M., Harris, E., 2010. Control of dengue virus translation and replication. Current topics in microbiology and immunology 338, 15-34.
Powell, T.J., Nakaar, V., Song, L., Huleatt, J.W., McDonald, W.F., Hewitt, D.D., 2013. Fusion proteins comprising flagellin and dengue viral envelope proteins. Google Patents.
Raviprakash, K., Wang, D., Ewing, D., Holman, D.H., Block, K., Woraratanadharm, J., Chen, L., Hayes, C., Dong, J.Y., Porter, K., 2008. A tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus. Journal of virology 82, 6927-6934.
Robert Putnak, J., Coller, B.A., Voss, G., Vaughn, D.W., Clements, D., Peters, I., Bignami, G., Houng, H.S., Chen, R.C., Barvir, D.A., Seriwatana, J., Cayphas, S., Garcon, N., Gheysen, D., Kanesa-Thasan, N., McDonell, M., Humphreys, T., Eckels, K.H., Prieels, J.P., Innis, B.L., 2005. An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus macaque model. Vaccine 23, 4442-4452.
Roehrig, J.T., Bolin, R.A., Kelly, R.G., 1998. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology 246, 317-328.
Sabchareon, A., Wallace, D., Sirivichayakul, C., Limkittikul, K., Chanthavanich, P., Suvannadabba, S., Jiwariyavej, V., Dulyachai, W., Pengsaa, K., Wartel, T.A., Moureau, A., Saville, M., Bouckenooghe, A., Viviani, S., Tornieporth, N.G., Lang, J., 2012. Protective efficacy of the recombinant, live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial. Lancet 380, 1559-1567.
Sabin, A.B., 1952. Research on dengue during World War II. The American journal of tropical medicine and hygiene 1, 30-50.
Sabin, A.B., Schlesinger, R.W., 1945. Production of Immunity to Dengue with Virus Modified by Propagation in Mice. Science 101, 640-642.
Schmitz, J., Roehrig, J., Barrett, A., Hombach, J., 2011. Next generation dengue vaccines: a review of candidates in preclinical development. Vaccine 29, 7276-7284.
Shahin, S., Proll, D., 2006. Heterologous Prime-Boost Immunisation Regimens Against
Infectious Diseases.
Shott, J.P., McGrath, S.M., Pau, M.G., Custers, J.H., Ophorst, O., Demoitie, M.A., Dubois, M.C., Komisar, J., Cobb, M., Kester, K.E., Dubois, P., Cohen, J., Goudsmit, J., Heppner, D.G., Stewart, V.A., 2008. Adenovirus 5 and 35 vectors expressing Plasmodium falciparum circumsporozoite surface protein elicit potent antigen-specific cellular IFN-gamma and antibody responses in mice. Vaccine 26, 2818-2823.
Shresta, S., Sharar, K.L., Prigozhin, D.M., Beatty, P.R., Harris, E., 2006. Murine model for dengue virus-induced lethal disease with increased vascular permeability. Journal of virology 80, 10208-10217.
Simasathien, S., Thomas, S.J., Watanaveeradej, V., Nisalak, A., Barberousse, C., Innis, B.L., Sun, W., Putnak, J.R., Eckels, K.H., Hutagalung, Y., Gibbons, R.V., Zhang, C., De La Barrera, R., Jarman, R.G., Chawachalasai, W., Mammen, M.P., Jr., 2008. Safety and immunogenicity of a tetravalent live-attenuated dengue vaccine in flavivirus naive children. The American journal of tropical medicine and hygiene 78, 426-433.
Simmons, M., Burgess, T., Lynch, J., Putnak, R., 2010. Protection against dengue virus by non-replicating and live attenuated vaccines used together in a prime boost vaccination strategy. Virology 396, 280-288.
Simmons, M., Nelson, W.M., Wu, S.J., Hayes, C.G., 1998. Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice. The American journal of tropical medicine and hygiene 58, 655-662.
Simon, R., Samuel, C.E., 2007. Activation of NF-kappaB-dependent gene expression by Salmonella flagellins FliC and FljB. Biochemical and biophysical research communications 355, 280-285.
Small, J.C., Ertl, H.C., 2011. Viruses - from pathogens to vaccine carriers. Current opinion in virology 1, 241-245.
Sun, W., Cunningham, D., Wasserman, S.S., Perry, J., Putnak, J.R., Eckels, K.H., Vaughn, D.W., Thomas, S.J., Kanesa-Thasan, N., Innis, B.L., Edelman, R., 2009. Phase 2 clinical trial of three formulations of tetravalent live-attenuated dengue vaccine in flavivirus-naive adults. Human vaccines 5, 33-40.
Valdes, I., Bernardo, L., Gil, L., Pavon, A., Lazo, L., Lopez, C., Romero, Y., Menendez, I., Falcon, V., Betancourt, L., Martin, J., Chinea, G., Silva, R., Guzman, M.G., Guillen, G., Hermida, L., 2009a. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice. Virology 394, 249-258.
Valdes, I., Gil, L., Romero, Y., Castro, J., Puente, P., Lazo, L., Marcos, E., Guzman, M.G., Guillen, G., Hermida, L., 2011. The chimeric protein domain III-capsid of dengue virus serotype 2 (DEN-2) successfully boosts neutralizing antibodies generated in monkeys upon infection with DEN-2. Clinical and vaccine immunology : CVI 18, 455-459.
Valdes, I., Hermida, L., Gil, L., Lazo, L., Castro, J., Martin, J., Bernardo, L., Lopez, C., Niebla, O., Menendez, T., Romero, Y., Sanchez, J., Guzman, M.G., Guillen, G., 2010. Heterologous prime-boost strategy in non-human primates combining the infective dengue virus and a recombinant protein in a formulation suitable for human use. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 14, e377-383.
Valdes, I., Hermida, L., Martin, J., Menendez, T., Gil, L., Lazo, L., Castro, J., Niebla, O., Lopez, C., Bernardo, L., Sanchez, J., Romero, Y., Martinez, R., Guzman, M.G., Guillen, G., 2009b. Immunological evaluation in nonhuman primates of formulations based on the chimeric protein P64k-domain III of dengue 2 and two components of Neisseria meningitidis. Vaccine 27, 995-1001.
van Der Most, R.G., Murali-Krishna, K., Ahmed, R., Strauss, J.H., 2000. Chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific CD8 T-cell response. Journal of virology 74, 8094-8101.
Vaughn, D.W., Hoke, C.H., Jr., Yoksan, S., LaChance, R., Innis, B.L., Rice, R.M., Bhamarapravati, N., 1996. Testing of a dengue 2 live-attenuated vaccine (strain 16681 PDK 53) in ten American volunteers. Vaccine 14, 329-336.
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.
Wallace, D., Canouet, V., Garbes, P., Wartel, T.A., 2013. Challenges in the clinical development of a dengue vaccine. Current opinion in virology 3, 352-356.
Wan, S.W., Lin, C.F., Wang, S., Chen, Y.H., Yeh, T.M., Liu, H.S., Anderson, R., Lin, Y.S., 2013. Current progress in dengue vaccines. Journal of biomedical science 20, 37.
Watanaveeradej, V., Simasathien, S., Nisalak, A., Endy, T.P., Jarman, R.G., Innis, B.L., Thomas, S.J., Gibbons, R.V., Hengprasert, S., Samakoses, R., Kerdpanich, A., Vaughn, D.W., Putnak, J.R., Eckels, K.H., Barrera Rde, L., Mammen, M.P., Jr., 2011. Safety and immunogenicity of a tetravalent live-attenuated dengue vaccine in flavivirus-naive infants. The American journal of tropical medicine and hygiene 85, 341-351.
Westaway, E.G., Brinton, M.A., Gaidamovich, S., Horzinek, M.C., Igarashi, A., Kaariainen, L., Lvov, D.K., Porterfield, J.S., Russell, P.K., Trent, D.W., 1985. Flaviviridae. Intervirology 24, 183-192.
Whitehead, S.S., Blaney, J.E., Durbin, A.P., Murphy, B.R., 2007. Prospects for a dengue virus vaccine. Nature reviews. Microbiology 5, 518-528.
Whitehead, S.S., Falgout, B., Hanley, K.A., Blaney Jr, J.E., Jr., Markoff, L., Murphy, B.R., 2003. A live, attenuated dengue virus type 1 vaccine candidate with a 30-nucleotide deletion in the 3' untranslated region is highly attenuated and immunogenic in monkeys. Journal of virology 77, 1653-1657.
Williams, K.L., Wahala, W.M., Orozco, S., de Silva, A.M., Harris, E., 2012. Antibodies targeting dengue virus envelope domain III are not required for serotype-specific protection or prevention of enhancement in vivo. Virology 429, 12-20.
Woodland, D.L., 2004. Jump-starting the immune system: prime-boosting comes of age. Trends in immunology 25, 98-104.
World Health Organization, 2009. Dengue: guidelines for diagnosis, treatment, prevention and control-New edition. World Health Organization.
World Health Organization, 2012. Global strategy for dengue prevention and control 2012-2020. World Health Organization.
World Health Organization, 2014. Dengue and severe dengue, Factsheet No. 117 World Health Organization, Geneva, p. Factsheet No. 117
Yoksan, S., 2008. WHO's efforts for the development of a dengue vaccine. Dengue Bulletin 32, 1-16.
Zhang, W., Chipman, P.R., Corver, J., Johnson, P.R., Zhang, Y., Mukhopadhyay, S., Baker, T.S., Strauss, J.H., Rossmann, M.G., Kuhn, R.J., 2003. Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nature structural biology 10, 907-912.
Zhu, J., Huang, X., Yang, Y., 2007. Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and -independent pathways. Journal of virology 81, 3170-3180.