Cellular & Molecular Immunology (2016) 13, 36–46 ß 2015 CSI and USTC. All rights reserved 1672-7681/15 $32.00 www.nature.com/cmi REVIEW Guiding dengue vaccine development using knowledge gained from the success of the yellow fever vaccine Huabin Liang, Min Lee and Xia Jin Flaviviruses comprise approximately 70 closely related RNA viruses. These include several mosquito-borne pathogens, such as yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), which can cause significant human diseases and thus are of great medical importance. Vaccines against both YFV and JEV have been used successfully in humans for decades; however, the development of a DENV vaccine has encountered considerable obstacles. Here, we review the protective immune responses elicited by the vaccine against YFV to provide some insights into the development of a protective DENV vaccine. Cellular & Molecular Immunology (2016) 13, 36–46; doi:10.1038/cmi.2015.76 Keywords: dengue virus; protective immunity; vaccine; yellow fever INTRODUCTION from a viremic patient and is capable of delivering viruses to its Flaviviruses belong to the family Flaviviridae, which includes offspring, thereby amplifying the number of carriers of infec- mosquito-borne pathogens such as yellow fever virus (YFV), tion.6,7 Because international travel has become more frequent, Japanese encephalitis virus (JEV), dengue virus (DENV), and infected vectors can be transported much more easily from an West Nile virus (WNV). Flaviviruses can cause human diseases endemic region to other areas of the world, rendering vector- and thus are considered medically important. According to borne diseases such as dengue fever a global health problem. WHO statistics, approximately 200,000 cases of YF occur The use of vaccines to prevent viral infection is the most cost- annually, leading to 30,000 deaths worldwide.1,2 Although 3 effective public health strategy. Vaccines against YFV (the YF- billion people are at risk of JEV infection, only 20,000 clinical 17D vaccine) and JEV have been effectively used in humans for cases and 6,000 deaths occur annually. However, the case fat- decades. However, a licensed DENV vaccine has not been suc- ality rate of JEV infection ranges from 5 to 30%, and approxi- cessfully developed using similar strategies. Because the DENV mately 30–50% of patients who recover from the infection have vaccine that is in the most advanced stage of clinical develop- permanent neuropsychiatric sequelae, while only one-third of ment is based on a chimera between YF-17D and DENV com- patients have complete remission without lingering complica- ponents, we closely examine the immunological factors that tions.3 Human cases of WNV infection are relatively rare, and have been associated with the protection conferred by the the most recent cases have largely been reported in the USA. YF-17D vaccine in the hopes of elucidating a strategy for the DENV infection has dramatically increased since the Second development of an efficacious DENV vaccine. World War, and 2.5 billion people in more than 100 countries across the tropical and subtropical regions are currently at risk STRUCTURE AND REPLICATION STRATEGY for DENV infection. An estimated 390 million dengue infec- OF FLAVIVIRUSES tions occur every year, of which 96 million manifest clinically as All flaviviruses share similar structural features and replication 4,5 dengue fever. strategies (Figure 1). The mature virion consists of an icosahed- Flaviviruses are transmitted by arthropod vectors, princip- ral particle comprising three structural proteins: the capsid ally represented by the Aedes aegypti and Aedes albopictus mos- protein (C), the membrane protein (M), and the envelope quitoes. The mosquito becomes infected when it feeds on blood protein (E). The viral genome is composed of an approximately Viral Disease and Vaccine Translational Research Unit, and Vaccine Centre, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China. Correspondence: X Jin, M.D., Ph.D., Professor, Principal Investigator, Chief, Viral Disease and Vaccine Translational Research Unit, B507 Life Science Research Building, 320 Yueyang Road, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China. E-mail: [email protected] Received: 5 April 2015; Revised 15 June 2015; Accepted 14 July 2015 Guiding dengue vaccine development H Liang et al 37 a Structural Non-Structural 5'UTR C prIM E NS1 NS2ANS3 NS4B NS5 3'UTR NS2B NS4A Protease RNA-dependent RNA polymetase NTPase Methyltransfetase RTPase Helicase Host cell signalase cleavage site Furin cleavage site Viral NS2B/NS3 protease cleavage site Unidentified protease cleavege site b DENV-1 DENV-3 DENV-2 DENV-4 JEV YFV 0.1 Figure 1 Structure of the flavivirus genome and genetic distance of selected flaviviruses.(a). Genomic structure and polyprotein processing of flaviviruses. The positive-sense RNA genome forms a single open-reading frame that is translated into a polyprotein precursor containing three structural proteins (blue) and seven NS proteins (yellow) flanked by the 59 and 39 UTRs. Cleavage sites for the host-cell signalase and furin, and the viral NS2B/NS3 protease as well as an unidentified protease are indicated by arrows. The functions of the NS3 and NS5 proteins that are required for RNA replication and polyprotein processing are also shown. (b). Phylogenetic analysis of selected flaviviruses. A neighbor-joining tree was compiled with complete genome sequences of DENV, JEV, and YFV available in the GenBank database using a Kimura two-parameter model. Sequence alignment and phylogenetic analysis were performed with MEGA 6 software(Oxford, UK). Virus sequences included are DENV-1 Hawaii, DENV-2 16681, DENV-3 H87, DENV-4 H241, JEV JaOArS982, and YFV ASIBI. 11,000-nucleotide single-stranded, positive-sense RNA assemble at the intracellular membranes of infected cells.19 (ssRNA). This ssRNA encodes the three structural proteins The viral C protein and ssRNA form a nucleocapsid structure, and seven non-structural (NS) proteins in a single open-read- probably in association with the membranes of the endoplas- ing frame that is flanked by untranslated regions (UTRs) at mic reticulum (ER). Then, the prM and E proteins are trans- either end, forming the gene order 59UTR-C-prM-E-NS1- ferred into the lumen of the ER to form a heterotrimeric prM/E NS2A-NS2B-NS3-NS4A-NS4B-NS5-39UTR.8,9 complex that surrounds the nucleocapsid. The prM protein is DENV infection is initiated by viral attachment to the recep- further cleaved into the pr and M proteins by the furin protease tors on target cells, followed principally by clathrin-mediated during a pH-dependent virion maturation process. endocytosis or other less predominant modes of entry. The The mechanism by which the immune response confers pro- virus E protein dimers undergo a conformational change with tection against the virus is likely to involve the targeting of a the gradual acidification of the endosome to expose the fusion specific structural component or a particular step of viral rep- loop, leading to fusion between the viral and endosomal mem- lication. Elucidating how successful vaccines have worked in branes.10,11 Then, the uncoated viral ssRNA is released into the the past will provide a useful guide for vaccine research and cytoplasm and translated into a polyprotein that is subse- development in the future. The YFV-17D vaccine for yellow quently cleaved by virus-encoded or host-cell proteases into fever is an ideal example of a successful vaccine that can serve as three structural proteins (C, prM, and E) and seven NS proteins a gold-standard to determine the immune correlates of vac- (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).12 Viral geno- cine-induced protection. mic RNA synthesis is based on a double-stranded RNA (dsRNA) template in the viral replication complex, which is YELLOW FEVER AND YELLOW FEVER VACCINES located within virus-induced membrane structures originating YFV is the prototype member of the flavivirus genus and is the from the trans-Golgi network or the intermediate compart- source of the genus name. YFV rampantly attacked the world ment.13 Most viral NS proteins and the core protein are population (especially in Africa, America, and Europe) for over involved in the replication process.14–18 Flavivirus virions 200 years until the French neurotropic vaccine (FNV) and Cellular & Molecular Immunology Guiding dengue vaccine development H Liang et al 38 Table 1 Comparison between FNV and yellow fever-17D (YF-17D) vaccinea FNV YF-17D vaccine Origin French viscerotropic virus strain Asibi strain Attenuation ,128 times in mouse brain ,17 times in mouse embryo tissue ,58 times in chick embryo tissue ,128 times in chick embryo tissue without nerve tissue Inoculation Scarification Subcutaneous inoculation Side effects Post-vaccination encephalitis YEL-AVD and YEL-ANDb Efficacy ,90%c .90% Amino acid DI & DII 52G, 54V, 56A, 84K, 142R, 153K, 170A, 173T, 52R, 54A, 56V, 84E, 142Q, 153N, 170V, 173I, 200T, 249N differences 200K, 249D DIII 305S, 325P, 380T 305F, 325S, 380R a Adapted from references24,122–125. b YEL-AVD, YF vaccine-associated viscerotropic disease; YEL-AND, YF vaccine-associated neurotropic disease. c Estimated based on seroconversion rates. yellow fever 17D vaccine (YF-17D) were developed in the unwanted adverse events may occur. In contrast, when viremia 1930s. Although highly efficacious, the use of FNV was stopped is absent the viral replication may be too limited to produce in 1981 due to its association with an increased risk of post- sufficient antigens to stimulate the magnitude of immune res- vaccination encephalitis (Table 1).20 The YF-17D vaccine was ponse required for protection. Thus, an appropriate level of developed by successive passages of the Asibi strain of YFV in viremia is crucial to activate multiple types of immune res- mouse embryo tissue culture, chicken embryo tissue culture, ponses after immunization.