Vaccine 32 (2014) 949–956
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Recombinant chimeric Japanese encephalitis virus/tick-borne encephalitis virus is attenuated and protective in mice Hong-Jiang Wang a,1 , Xiao-Feng Li a,1 , Qing Ye a , Shi-Hua Li a , Yong-Qiang Deng a , Hui Zhao a , Yan-Peng Xu a , Jie Ma a , E-De Qin a , Cheng-Feng Qin a,b,∗ a b
Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China Graduate School, Anhui Medical University, Hefei, China
a r t i c l e
i n f o
Article history: Received 9 July 2013 Received in revised form 14 November 2013 Accepted 18 December 2013 Available online 4 January 2014 Keywords: Tick-borne encephalitis virus Japanese encephalitis virus Vaccine Chimeric flavivirus
a b s t r a c t Tick-borne encephalitis virus (TBEV) represents one of the most dangerous human pathogens circulating in Europe and East Asia. No effective treatment for TBEV infection currently exists, and vaccination is the primary preventive measure. Although several inactivated vaccines have been licensed, the development of novel vaccines against TBEV remains a high priority in disease-endemic countries. In the present study, a live chimeric recombinant TBEV (ChinTBEV) was created by substituting the major structural genes of TBEV for the corresponding regions of Japanese encephalitis virus (JEV) live vaccine strain SA1414-2. The resulting chimera had a small-plaque phenotype, replicated efficiently in both mammalian and mosquito cells. The preliminary data from in vitro passaging indicated the potential for stability of ChinTBEV. ChinTBEV also exhibited significantly attenuated neuroinvasiveness in mice upon either intraperitoneal or subcutaneous inoculation in comparison with its parental TBEV. Importantly, a single immunisation with ChinTBEV elicited TBEV-specific IgG and neutralising antibody responses in a dose-dependent manner, providing significant protection against lethal TBEV challenge in mice. Taken together, the results of this proof-of-concept study indicate that ChinTBEV can be further developed as a potential vaccine candidate against TBEV infection. Moreover, the construction of this type of flavivirus chimera using a JEV vaccine strain as the genetic backbone represents a universal vaccine approach. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Tick-borne encephalitis (TBE) is the most serious ticktransmitted human infection in Europe and Asia, accounting for approximately 8500 human cases annually [1–5]. During the last few decades, new endemic foci and a great increase in TBE morbidity have been reported in many European and Asian countries [6,7]. This infection is characterised by typical neurological complications, such as meningitis, meningoencephalitis and encephalomyelitis/radiculitis, in both children and adults [3]. The causative agent, TBE virus (TBEV), belongs to the genus Flavivirus within the family Flaviviridae, which also contains other important human pathogens, including yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV) and West Nile virus (WNV). TBEV contains an approximately 11-kb positive-sense single-stranded RNA genome flanked by 5 - and 3 -untranslated regions (UTRs) and encodes a polyprotein that is proteolytically
∗ Corresponding author at: Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China. Tel.: +86 1066948604. E-mail addresses: [email protected], chengfeng [email protected] (C.-F. Qin). 1 These authors contribute equally to this work. 0264-410X/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.12.050
cleaved into three structural proteins (C, prM and E) and seven nonstructural proteins. TBEV can be divided into three genetically related subtypes: the European, Siberian and Far Eastern subtypes [8]. No effective treatment for TBEV infection is currently available. Currently, vaccination offers the most effective protection against TBE, and the vaccine has been introduced into many disease-endemic countries. There are at least four inactivated, cell culture-derived vaccines currently available, including Encepur® and TBE-Immun® , which are manufactured in Western Europe, as well as TBE-Moscow vaccine® and EnceVir® , which are manufactured in Russia [9,10]. Although the current vaccination schedules using these inactivated vaccines have led to a dramatic decline in the annual incidence of the disease [11], the requirement of multiple doses for primary and booster immunisations and the relatively high cost influence the success of TBEV immunisation programmes. Moreover, post-vaccination fever has been well documented, particularly in young children [12,13]. Although rare, vaccination failures have also been reported with use of the current inactivated TBEV vaccines [14–20]. In addition to killed vaccines, live attenuated vaccines have been demonstrated to induce long-term immunity at low cost. Two prominent flavivirus vaccines, the yellow fever live vaccine (strain
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YF-17D) and the Japanese encephalitis (JE) live vaccine (strain SA14-14-2), have been used globally and have saved the lives of millions suffering from flavivirus infections. In recent years, much effort has been made to rationally design a live attenuated vaccine against TBEV using a reverse genetics approach. Kofler et al. engineered an attenuated TBEV with a small deletion in the central portion of its C protein [21]. In other studies, a recombinant TBEV bearing a complementary target for mir-9 or mir-124a was significantly attenuated, and this virus induced strong humoral immune responses in monkeys [22,23]. A well-known strategy for generating attenuated flavivirus strains is to construct a chimeric flavivirus by replacing its structural proteins with the corresponding regions of another attenuated flavivirus strain. Using this strategy, a chimeric TBEV based on DENV-4 has been created and shown to be safe and efficacious in mice and non-human primates [24–27]. The JE live vaccine has been used in most JE-endemic countries in Asia, and more than 300 million doses have been administered since its licensure [28]. Large-scale vaccination programmes in various countries have shown that the vaccine possesses an excellent safety profile and remarkable effectiveness and efficacy [29–33]. The phenotypic and genotypic characteristics that correlate with attenuation are highly stable [28]. These properties make the JE vaccine strain an attractive genetic backbone with which to create a chimeric TBEV vaccine. However, no chimeric flavivirus based on JEV SA14-14-2 has been described to date. In this study, a live chimeric TBEV (ChinTBEV) was generated using a molecular clone of the JE vaccine strain SA14-14-2 as the genetic backbone. This novel flavivirus chimera, ChinTBEV, represents a potential vaccine candidate for TBEV infection that deserves further development.
2.3. Transcription and transfection Plasmid pChinTBEV was linearised with Xho I and used as a template for SP6 RNA polymerase transcription in the presence of an m7GpppA cap analogue (Promega). In vitro transcription was performed using the RiboMAX Large Scale RNA Production System (Promega) according to the manufacturer’s protocols. The yield and integrity of the RNA transcripts were analysed by gel electrophoresis under non-denaturing conditions. RNA transcripts (5 g) were then transfected into BHK-21 cells using Lipofectamine 2000 (Invitrogen). 2.4. Growth curves Growth curves were performed in BHK-21, Vero and C6/36 cells in a 12-well plate at a multiplicity of infection (MOI) of 0.01. Culture supernatants were collected at successive 24-h intervals, and progeny viral titres were then quantitated by plaque assay on BHK21 cells. 2.5. Indirect immunofluorescence assay (IFA) IFAs were performed as previously described. Briefly, BHK21 cells were infected with each virus for 48 h, after which the cells were fixed with ice-cold acetone and incubated with primary monoclonal antibodies (mAbs) against the JEV E protein (4AD5F5D5D6), the TBEV E protein (4A4) or the JEV NS1 protein (JN1). The cells were then incubated with secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen). Positive cells were examined using a fluorescent microscope (Olympus). 2.6. Genetic stability assay
2. Materials and methods 2.1. Cells and viruses Mammalian BHK-21 and Vero cells were maintained in Dulbecco’s minimal essential medium (DMEM; Invitrogen) supplemented with 10% foetal bovine serum (FBS), 100 U/ml penicillin and 100 g/ml streptomycin. Aedes albopictus C6/36 cells were cultured in RPMI 1640 supplemented with nonessential amino acids (Invitrogen), and 10% FBS. All cell lines were cultured at 37 ◦ C in 5% CO2 , except for the C6/36 cells, which were maintained at 28 ◦ C. TBEV strain Senzhang (GenBank no. JQ650523.1) and the JEV vaccine strain SA14-14-2 (GenBank no. D90195) were stored in our laboratory and prepared in BHK-21 cells. Stocks of each virus were titrated by performing a standard plaque-forming assay, as previously described [34,35].
2.2. Plasmid construction All plasmids were constructed using standard molecular cloning protocols. The sequences of the primers used in this study are shown in Table S1. Genetic construction of the full-length infectious clone of JEV has been described [36]. Briefly, the plasmid pACYC-Linker was first created from the plasmid pACYC177 (Fermentas) and the plasmid pSP64 (Promega). Further details about the construction of pACYC-Linker are available from the authors on request. Cloning sites were engineered to permit replacement of the entire pre-M and E coding sequences of JEV with the corresponding sequences of TBEV. Sites for posttranslational cleavage of the capsid and pre-M proteins and the E and NS1 proteins were preserved (Fig. 1A). The resulting plasmid contained the full-length cDNA of the JEV/TBEV chimera and was named pChinTBEV.
ChinTBEV was serially passaged in Vero cells at an initial MOI of 0.01. Viral RNA was extracted from the supernatants of the fourth and eighth passages and sequenced accordingly. The plaque phenotypes of passages 4 and 8 were also assessed. 2.7. Enzyme-linked immunosorbent assay (ELISA) Serum IgG against TBEV was detected by indirect ELISA using the formaldehyde-inactivated form of the TBEV strain Senzhang as the coating antigen and peroxidase-conjugated horse anti-mouse IgG as the secondary antibody. Serial two-fold dilutions of serum were tested in duplicate. Endpoint titres were considered the highest dilution that resulted in a value two-fold greater than the absorption of the control serum, with a cut-off value of 0.05. 2.8. Neutralisation assay Neutralising antibody titres were determined by a standard 50% plaque reduction neutralisation test (PRNT50 ). The endpoint neutralisation titre was calculated according to the method of Reed and Muench [37]. 2.9. Mouse experiments All animal experiments were approved by and conducted in strict accordance with the guidelines of the Experimental Animal Ethics Committee of the Beijing Institute of Microbiology and Epidemiology. Groups of 4-week-old BALB/c mice (n = 5–9) were inoculated by the intraperitoneal (i.p.), subcutaneous (s.c.) or intracerebral (i.c.) route with ChinTBEV, TBEV or JEV at varying doses. The mice were then monitored for clinical symptoms and mortality for 21 days. The 50% lethal dose (LD50 ) of each virus was calculated according to the method of Reed and Muench [37].
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Fig. 1. Construction and characterisation of ChinTBEV. (A) Graphic representation of the genomic constructs of ChinTBEV and its parental viruses. JEV genes are shown in grey, and TBEV is shown in white. (B) IFAs of ChinTBEV and its parental viruses. Green fluorescence represents positive reactivity to mAbs against JEV-E, TBEV-E and JEV-NS1, as indicated at the bottom. (C) Plaque morphologies of ChinTBEV and its parental viruses. Plaques were observed 3 days post-infection after staining with 0.2% crystal violet.
Immunogenicity was assessed by the s.c. or i.p. inoculation of 4-week-old BALB/c mice with various doses of ChinTBEV. The mice were bled by tail vein puncture 1 day prior to and 2 and 4 weeks post-immunisation. TBEV-specific IgG and neutralising antibodies were measured by ELISA and PRNT50 , respectively. For the protection assay, all of the ChinTBEV-immunised mice were challenged by the i.p. route with 500 PFU (100 LD50 ) of TBEV at 4 weeks after the initial immunisation. The mice were monitored for signs of illness and death for at least 21 days. 2.10. Histopathological analysis Mice were inoculated with 104 PFU of TBEV or ChinTBEV by the i.p. route. At 8 days post-inoculation, the brains of three mice were collected, fixed with perfusion fixative (4% formaldehyde) for 48 h and processed according to standard histological methods. All brain tissue sections from each animal were stained with haematoxylin and eosin (HE). 2.11. Statistical analysis For survival analysis, Kaplan–Meier survival curves were analysed by a log-rank test (GraphPad Prism software 5.0, San Diego, CA).
3. Results 3.1. Construction and characterisation of ChinTBEV To generate a chimeric flavivirus carrying the protective antigens of TBEV, the prM and E genes of JEV SA14-14-2 were substituted with the corresponding genes of the TBEV strain Senzhang using standard DNA recombination technology, resulting in the DNA construct ChinTBEV (Fig. 1A). The prM signal peptide of JEV and the last three amino acids of the JEV E protein were retained to ensure efficient cleavage at the C-prM and E-NS1 junctions [38–40]. The full-length cDNA clone of ChinTBEV was then sequenced and compared with the cDNA of the parental viruses JEV strain SA14-142 and TBEV strain Senzhang (Table S2). Only five nucleotides in the prM-E coding region of ChinTBEV differed from those in TBEV, three of which resulted in amino acid changes within the prM protein (Val28 → Met, Thr51 → Ala and Glu87 → Gly). To obtain viable ChinTBEV from BHK-21 cells, in vitrotranscribed RNAs from linearised pChinTBEV were transfected into BHK-21 cells. The recovered ChinTBEV was characterised by IFA, together with its parental viruses JEV and TBEV. As shown in Fig. 1B, both the TBEV E protein and the JEV NS1 protein were detected in ChinTBEV-infected cells. As expected, JEV and TBEV could only
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Fig. 2. Growth curves and phenotype stability of ChinTBEV. (A) Monolayers of BHK-21, Vero and C6/36 cells were infected with the indicated viruses at an MOI of 0.01. Viral titres were determined on BHK-21 cells by plaque assay at the indicated times. The dotted lines represent the limit of sensitivity of the plaque assay. (B) Plaque phenotypes of passages 1, 4 and 8 on BHK-21 cells. The average plaque sizes (mean ± standard deviation) were estimated by counting 40 plaques.
react with JEV- and TBEV-specific mAbs, respectively. Importantly, the plaque morphology of ChinTBEV was different from that of its parental viruses; in particular, the average plaque size was significantly smaller compared with the plaque size of the parental viruses (Fig. 1C). The small-plaque phenotype of ChinTBEV indicates its potential attenuation in comparison with its parental viruses. Next, the growth efficiencies of ChinTBEV and its parental viruses were compared in mammalian and mosquito cells. ChinTBEV replicated slightly less efficiently than TBEV, and both peaked at approximately 48 h post-infection in BHK-21 cells (Fig. 2A). In Vero cells, the certified cell bank intended for vaccine manufacturing, ChinTBEV and TBEV replicated to their highest titres at 96 h post-infection, reaching 107.0 and 108.1 PFU/ml, respectively (Fig. 2A). In contrast, in mosquito C6/36 cells, TBEV failed to replicate, but both JEV and the chimera were capable of replicating, attaining peak titres of 106.4 and 104.1 PFU/ml, respectively (Fig. 2A). Furthermore, to assess the genetic stability of ChinTBEV during sequential passaging in vitro, ChinTBEV recovered from BHK-21 cells (named passage 0) was passaged up to eight times in Vero cells. Viruses harvested at passages 1, 4 and 8 were sequenced and compared with viruses harvested at passage 0. No amino acid substitutions were identified in the prM-E regions and the nonstructural proteins until passage 8. The homogeneous small-plaque phenotype of ChinTBEV in BHK-21 cells was also retained during in vitro passaging (Fig. 2B). Taken together, these results demonstrate that this novel chimera with a small-plaque phenotype is highly replicative and potentially genetically stable in cell culture. 3.2. ChinTBEV demonstrates highly attenuated neuroinvasiveness in mice To further characterise the attenuation phenotype of ChinTBEV, groups of BALB/c mice were inoculated with 10-fold serial dilutions of either ChinTBEV or TBEV. Mice inoculated with varying doses of
the JEV vaccine strain SA14-14-2 were used as controls. Upon s.c. inoculation, ChinTBEV did not cause any clinical symptoms or mortality during the observation period, even at a dose of 105 PFU. The LD50 of ChinTBEV should be higher than the LD50 of the JEV vaccine strain, which was approximately 105 PFU (Table 1). Meanwhile, s.c. inoculation of TBEV resulted in 100% mortality even at the lowest dose, 103 PFU, and the LD50 of TBEV was calculated to be less than 103 PFU (Table 1). Thus, ChinTBEV is more than 100-fold attenuated in comparison with TBEV upon s.c. infection. Upon i.p. injection, ChinTBEV failed to cause any clinical symptoms or mortality in mice at a dose of 103 PFU, whereas higher doses of ChinTBEV injection caused the central nervous system (CNS) manifestations typical of TBEV infection. The LD50 of ChinTBEV was calculated to be 105.5 PFU. As all of the mice that were inoculated with TBEV by the i.p. route died, even at the lowest dose of 10 PFU, the LD50 of TBEV should be less than 10 PFU (Table 1). Thus, ChinTBEV is at least 31,600-fold more attenuated than TBEV upon i.p. infection. Furthermore, the abilities of ChinTBEV and parental TBEV to induce viraemia in mice were compared following i.p. inoculation. As shown in Fig. 3A, TBEV-inoculated mice developed significant viraemia from day 1 to day 4 post-infection, with a peak titre of 104.0 PFU/ml on day 2 post-infection, and no viraemia was observed in the following days. In contrast, viraemia was not detectable in any of the mice inoculated with ChinTBEV throughout the observation period. In particular, no virus was isolated from the brains of mice infected with ChinTBEV by the i.p. route. The histopathological results showed typical focal gliosis and neuronophagia in the temporal cerebral cortex of the TBEV-infected mice, whereas no unusual lesions were observed in the brains of mice inoculated with ChinTBEV (Fig. 3B). To examine neurovirulence, groups of mice were challenged by the i.c. route with various doses of ChinTBEV and TBEV. The results showed that ChinTBEV retained partial neurovirulence in mice, as an i.c. inoculation of 1 PFU caused 60% mortality. Compared with
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Table 1 Attenuation of ChinTBEV in mice in comparison with its parental virusesa . Virus
Route
ChinTBEV
s.c.
i.p.
TBEV
s.c.
i.p.
JEV
s.c.
i.p.
a b
Dose (PFU) 5
10 104 103 106 105 104 103 105 104 103 103 102 10 105 104 103 106 105 104
Mortality (no. dead/no. tested, %)
AST (day)b
LD50 (PFU)
0 (0/8) 0 (0/8) 0 (0/8) 77.8 (7/9) 22.2 (2/9) 25.0 (2/8) 0 (0/8) 100 (6/6) 100 (6/6) 100 (6/6) 100 (9/9) 100 (9/9) 88.9 (8/9) 0 (0/8) 0 (0/8) 0 (0/7) 0 (0/8) 0 (0/8) 0 (0/8)
N/A N/A N/A 11.3 16.5 16.0 N/A N/A N/A 8.83 N/A 9.0 10.0 N/A N/A N/A N/A N/A N/A
>105
105.5
106
Groups of 4-week-old BALB/c female mice were s.c. or i.p. inoculated with the indicated doses of viruses. The mortality was monitored for 21 days after inoculation. AST for mice that died; N/A, not applicable.
mice infected with the parental TBEV, the survival rate among mice infected with varying doses of ChinTBEV was higher, indicating slightly attenuated neurovirulence for ChinTBEV compared with its parental TBEV (Fig. 3C). Together, these results demonstrate that the peripheral virulence of ChinTBEV is highly attenuated in mice and that this chimeric virus possesses slightly attenuated neurovirulence compared with TBEV.
3.3. ChinTBEV elicits neutralising antibodies To evaluate the immunogenicity of ChinTBEV, groups of 4week-old BALB/c mice were immunised once with varying doses of ChinTBEV by the s.c. or i.p. route. Mice immunised with PBS were used as controls. It should be noted that i.p. immunisation with 105 PFU of ChinTBEV caused 20% mortality in mice, and only
Fig. 3. ChinTBEV was attenuated in mice. (A) Viraemia profile of ChinTBEV in mice following i.p. challenge. Mice were inoculated with 104 PFU of the indicated viruses, and viraemia was determined on days 1 to 4 post-inoculation by plaque assay. (B) Histopathological analysis for the virus-infected mice brain. Mice were inoculated with 104 PFU of TBEV or ChinTBEV by the i.p. route, and brains were collected at 8 days post-infection and subjected to HE analysis. The arrow indicates focal gliosis and neuronophagia in temporal cerebral cortex. (C) Neurovirulence tests of ChinTBEV. Mice received either ChinTBEV or TBEV at the indicated doses, and mortality was recorded for 21 days.
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Fig. 4. Immunogenicity of ChinTBEV in mice. IgG and neutralising antibody responses in mice after s.c. or i.p. inoculation with the indicated doses of ChinTBEV. Sera were collected from animals at 28 days post-inoculation to measure IgG and neutralising antibody responses against TBEV by ELISA and PRNT50 , respectively. The dotted lines represent the limits of detection of the ELISA and PRNT50 , which were assigned values of 100 and 5, respectively.
the survival were subjected to subsequent antibody and challenge assay. All of the immunised mice were bled at 4 weeks after immunisation, and IgG and neutralising antibody titres against TBEV were tested by ELISA and PRNT50 , respectively. As shown in Fig. 4A, both s.c. and i.p. immunisation with ChinTBEV induced high titres of TBEV-specific IgG antibodies in a dose-dependent manner. Most importantly, TBEV-specific neutralising antibodies were elicited following a single immunisation with varying doses of ChinTBEV (Fig. 4B). As expected, no TBEV-specific neutralising antibodies were detected in sera from control mice. Collectively, these results show that a single immunisation with ChinTBEV induces a significant humoral immune response to TBEV. 3.4. ChinTBEV confers protection against TBEV challenge in mice Finally, to determine the in vivo protection elicited by ChinTBEV vaccination, all of the above-mentioned mice immunised with ChinTBEV were challenged with 100 LD50 of TBEV by the i.p. route at 28 days post-immunisation. As shown in Fig. 5, all mice immunised with PBS developed symptoms of CNS infection and died within 11 to 15 days after i.p. challenge. Importantly, i.p. immunisation with a high dose (105 PFU) of ChinTBEV provided full protection against lethal TBEV challenge; all of these animals survived challenge and exhibited no signs of CNS disease during the 21-day observation period. In contrast, the low dose (104 PFU) of ChinTBEV provided 60% protection. In the s.c. immunisation group, both a high dose and a low dose of ChinTBEV provided significant protection, with 71% and 50% of the immunised mice, respectively, surviving the lethal challenge. ChinTBEV immunisation significantly decreased mortality and extended the AST (P < 0.05). These data indicate
that a single immunisation with ChinTBEV confers solid protection against lethal TBEV challenge in mice. 4. Discussion This proof-of-concept study was to provide a vaccine approach using chimeric JEV/TBEV for further development. Chimeric flaviviruses created using an attenuated strain as the backbone have been well explored in the development of DENV, WNV, JEV and TBEV vaccines [24,41–43]. In the present study, we successfully transferred the prM-E gene of TBEV into the genome of the JEV vaccine strain SA14-14-2, resulting in a viable chimeric flavivirus that we called ChinTBEV. ChinTBEV produced significantly smaller plaques than did both of its parental viruses (Fig. 1C), indicating the potentially attenuated phenotype of ChinTBEV. The peak titre of ChinTBEV reached 107.0 PFU in Vero cells, indicating the possibility of large-scale production at low cost. Although TBEV failed to replicate in mosquito cells, a delayed and restricted growth pattern was observed for ChinTBEV compared with JEV in mosquito C6/36 cells (Fig. 2A), which is in agreement with previous findings [26,44]. Additionally, ChinTBEV exhibited potential genetic and plaque morphology stability after serial passaging in Vero cells (Fig. 2B), and this preliminary data indicate the potential for stability of ChinTBEV. Whatever, a detailed investigation of stability at the gene and population levels should be warranted in the near future. Our data showed that the neuroinvasiveness of ChinTBEV was highly attenuated in mice in comparison with TBEV. The LD50 of ChinTBEV was approximately 100- and 31,600-fold higher that of TBEV upon s.c. or i.p. inoculation, respectively (Table 1). Antigenic
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Fig. 5. Protection against lethal TBEV challenge in ChinTBEV-immunised mice. All of the immunised mice were challenged with 500 PFU of TBEV by the i.p. route. The statistical significance of differences in mortality and AST between the two groups was determined using the log-rank test in GraphPad Prism 5.0.
chimerisation between flavivirus members has been proven to lead to the attenuation of neurotropic viruses [26,41,45]. Additionally, the presence of specific mutations in certain virulence determinants within the genetic background of JEV may contribute to attenuation in mice [42,46,47]. Previously, chimeric TBEV vaccine candidates including Langat/DENV-4 and TBEV/DENV-4 have been well evidenced to be highly attenuated in neurovirulence in mice and non-human primates [26,27]. However, ChinTBEV was only slightly attenuated in neurovirulence compared with its parental TBEV (Fig. 3C). This result is in agreement with previous findings [48–50] and is most likely due to the presence of critical neurovirulence determinants in the E protein of parental TBEV. Future experiments using reverse genetics are warranted to mutate these amino acid residues to achieve an attenuated neurovirulence phenotype in mice. A single immunisation with ChinTBEV induced a protective antibody response in mice (Fig. 4). Both the IgG and the neutralising antibody responses in mice were dose dependent. Compared with other TBEV vaccine candidates and commercial products [25–27], the peak titre of neutralising antibody induced by a single immunisation was relatively low. This is probably due to the restricted replication of ChinTBEV in mice. Nevertheless, a neutralising antibody titre >1:10 is considered as seroconversion and represents the best surrogate marker for protection. Challenge experiments showed that immunisation with a high dose (105 PFU) of ChinTBEV (i.p. and s.c. routes) provided solid protection (100% and 71%) against lethal TBEV challenge. Immunisation with the lower dose (104 PFU) of ChinTBEV conferred only 60% and 50% protection (Fig. 5), which was most likely due to the relatively low neutralising antibody titre and the relatively high challenge dose (Fig. 4B). In our experiments, mice were challenged with 100 LD50 of TBEV, and decreasing this challenge dose would most likely increase the survival rate. A second booster immunisation may be needed that is common for the previously described chimeric DENV vaccine based on YFV 17D [51]. Conflict of interests statement None. Acknowledgments This work was supported by the National Basic Research Project (no. 2012CB518904), the National Natural Science Foundation of China (no. 81101243, no. U1132002 and no. 31270974) and the National Major Special Program of Science and Technology of China (2008ZX10004-015 and 2013ZX10004001). C.F. Qin was
supported by the Beijing Nova Program of Science and Technology (no. 2010B041).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine. 2013.12.050.
References [1] Poponnikova TV. Specific clinical and epidemiological features of tick-borne encephalitis in Western Siberia. Int J Med Microbiol 2006;296(Suppl 40):59–62. [2] Gritsun TS, Lashkevich VA, Gould EA. Tick-borne encephalitis. Antiviral Res 2003;57:129–46. [3] Lindquist L, Vapalahti O. Tick-borne encephalitis. Lancet 2008;371:1861–71. [4] Mandl CW. Steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis. Virus Res 2005;111:161–74. [5] Kaiser R. Tick-borne encephalitis. Infect Dis Clin North Am 2008;22:561–75, x. [6] Suss J. Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in Europe and Asia-an overview. Ticks Tick Borne Dis 2011;2:2–15. [7] Mansfield KL, Johnson N, Phipps LP, Stephenson JR, Fooks AR, Solomon T. Tick-borne encephalitis virus—a review of an emerging zoonosis. J Gen Virol 2009;90:1781–94. [8] Ecker M, Allison SL, Meixner T, Heinz FX. Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia. J Gen Virol 1999;80(Pt 1):179–85. [9] Zent O, Broker M. Tick-borne encephalitis vaccines: past and present. Expert Rev Vaccines 2005;4:747–55. [10] Leonova GN, Pavlenko EV. Characterization of neutralizing antibodies to Far Eastern of tick-borne encephalitis virus subtype and the antibody avidity for four tick-borne encephalitis vaccines in human. Vaccine 2009;27:2899–904. [11] Heinz FX, Holzmann H, Essl A, Kundi M. Field effectiveness of vaccination against tick-borne encephalitis. Vaccine 2007;25:7559–67. [12] Pavlova BG, Loew-Baselli A, Fritsch S, Poellabauer EM, Vartian N, Rinke I, et al. Tolerability of modified tick-borne encephalitis vaccine FSME-IMMUN “NEW” in children: results of post-marketing surveillance. Vaccine 2003;21:742–5. [13] Zent O, Banzhoff A, Hilbert AK, Meriste S, Sluzewski W, Wittermann C. Safety, immunogenicity and tolerability of a new pediatric tick-borne encephalitis (TBE) vaccine, free of protein-derived stabilizer. Vaccine 2003;21:3584–92. [14] Bender A, Jager G, Scheuerer W, Feddersen B, Kaiser R, Pfister HW. Two severe cases of tick-borne encephalitis despite complete active vaccination—the significance of neutralizing antibodies. J Neurol 2004;251:353–4. [15] Kleiter I, Jilg W, Bogdahn U, Steinbrecher A. Delayed humoral immunity in a patient with severe tick-borne encephalitis after complete active vaccination. Infection 2007;35:26–9. [16] Kunze U, Baumhackl U, Bretschneider R, Chmelik V, Grubeck-Loebenstein B, Haglund M, et al. The golden agers and tick-borne encephalitis. Conference report and position paper of the International Scientific Working Group on Tick-borne encephalitis. Wien Med Wochenschr 2005;155:289–94. [17] Stiasny K, Holzmann H, Heinz FX. Characteristics of antibody responses in tickborne encephalitis vaccination breakthroughs. Vaccine 2009;27:7021–6. [18] Grgic-Vitek M, Avsic-Zupanc T, Klavs I. Tick-borne encephalitis after vaccination: vaccine failure or misdiagnosis. Vaccine 2010;28:7396–400. [19] Andersson CR, Vene S, Insulander M, Lindquist L, Lundkvist A, Gunther G. Vaccine failures after active immunisation against tick-borne encephalitis. Vaccine 2010;28:2827–31.
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[20] Plisek S, Honegr K, Beran J. TBE infection in an incomplete immunized person at-risk who lives in a high-endemic area—impact on current recommendations for immunization of high-risk groups. Vaccine 2008;26:301–4. [21] Kofler RM, Heinz FX, Mandl CW. Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence. J Virol 2002;76:3534–43. [22] Heiss BL, Maximova OA, Thach DC, Speicher JM, Pletnev AG. MicroRNA targeting of neurotropic flavivirus: effective control of virus escape and reversion to neurovirulent phenotype. J Virol 2012;86:5647–59. [23] Heiss BL, Maximova OA, Pletnev AG. Insertion of microRNA targets into the flavivirus genome alters its highly neurovirulent phenotype. J Virol 2011;85:1464–72. [24] Wright PF, Ankrah S, Henderson SE, Durbin AP, Speicher J, Whitehead SS, et al. Evaluation of the Langat/dengue 4 chimeric virus as a live attenuated tick-borne encephalitis vaccine for safety and immunogenicity in healthy adult volunteers. Vaccine 2008;26:882–90. [25] Rumyantsev AA, Chanock RM, Murphy BR, Pletnev AG. Comparison of live and inactivated tick-borne encephalitis virus vaccines for safety, immunogenicity and efficacy in rhesus monkeys. Vaccine 2006;24:133–43. [26] Pletnev AG, Men R. Attenuation of the Langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4. PNAS 1998;95:1746–51. [27] Pletnev AG, Karganova GG, Dzhivanyan TI, Lashkevich VA, Bray M. Chimeric Langat/Dengue viruses protect mice from heterologous challenge with the highly virulent strains of tick-borne encephalitis virus. Virology 2000;274:26–31. [28] Yu Y. Phenotypic and genotypic characteristics of Japanese encephalitis attenuated live vaccine virus SA14-14-2 and their stabilities. Vaccine 2010;28:3635–41. [29] Tandan JB, Ohrr H, Sohn YM, Yoksan S, Ji M, Nam CM, et al. Single dose of SA 14-14-2 vaccine provides long-term protection against Japanese encephalitis: a case-control study in Nepalese children 5 years after immunization. Vaccine 2007;25:5041–5. [30] Kumar R, Tripathi P, Rizvi A. Effectiveness of one dose of SA 14-14-2 vaccine against Japanese encephalitis. N Engl J Med 2009;360:1465–6. [31] Hennessy S, Liu Z, Tsai TF, Strom BL, Wan CM, Liu HL, et al. Effectiveness of liveattenuated Japanese encephalitis vaccine (SA14-14-2): a case-control study. Lancet 1996;347:1583–6. [32] Gatchalian S, Yao Y, Zhou B, Zhang L, Yoksan S, Kelly K, et al. Comparison of the immunogenicity and safety of measles vaccine administered alone or with live, attenuated Japanese encephalitis SA 14-14-2 vaccine in Philippine infants. Vaccine 2008;26:2234–41. [33] Bista MB, Banerjee MK, Shin SH, Tandan JB, Kim MH, Sohn YM, et al. Efficacy of single-dose SA 14-14-2 vaccine against Japanese encephalitis: a case control study. Lancet 2001;358:791–5. [34] Li SH, Dong H, Li XF, Xie X, Zhao H, Deng YQ, et al. Rational design of a flavivirus vaccine by abolishing viral RNA 2 -O methylation. J Virol 2013;87:5812–9. [35] Si BY, Jiang T, Zhang Y, Deng YQ, Huo QB, Zheng YC, et al. Complete genome sequence analysis of tick-borne encephalitis viruses isolated in northeastern China. Arch Virol 2011;156:1485–8.
[36] Ye Q, Li XF, Zhao H, Li SH, Deng YQ, Cao RY, et al. A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1 formation and contributes to attenuation. J Gen Virol 2012;93:1959–64. [37] Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg 1938;27:493–7. [38] Amberg SM, Rice CM. Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing. J Virol 1999;73:8083–94. [39] Yamshchikov VF, Compans RW. Formation of the flavivirus envelope: role of the viral NS2B-NS3 protease. J Virol 1995;69:1995–2003. [40] Lobigs M, Lee E. Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes. J Virol 2003;78:178–86. [41] Pletnev AG, Putnak R, Speicher J, Wagar EJ, Vaughn DW. West Nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective efficacy. PNAS 2002;99:3036–41. [42] Chambers TJ, Nestorowicz A, Mason PW, Rice CM. Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties. J Virol 1999;73:3095–101. [43] Guirakhoo F, Weltzin R, Chambers TJ, Zhang ZX, Soike K, Ratterree M, et al. Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates. J Virol 2000;74:5477–85. [44] Engel AR, Mitzel DN, Hanson CT, Wolfinbarger JB, Bloom ME, Pletnev AG. Chimeric tick-borne encephalitis/dengue virus is attenuated in Ixodes scapularis ticks and Aedes aegypti mosquitoes. Vector Borne Zoonotic Dis 2011;11: 665–74. [45] Whitehead SS, Hanley KA, Blaney Jr JE, Gilmore LE, Elkins WR, Murphy BR. Substitution of the structural genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes, mice, and rhesus monkeys. Vaccine 2003;21:4307–16. [46] Durbin AP, Karron RA, Sun W, Vaughn DW, Reynolds MJ, Perreault JR, et al. Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3 -untranslated region. Am J Trop Med Hyg 2001;65:405–13. [47] Huang CY, Butrapet S, Pierro DJ, Chang GJ, Hunt AR, Bhamarapravati N, et al. Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine. J Virol 2000;74:3020–8. [48] Pletnev AG, Bray M, Huggins J, Lai CJ. Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses. PNAS 1992;89:10532–6. [49] Arroyo J, Miller C, Catalan J, Myers GA, Ratterree MS, Trent DW, et al. ChimeriVax-West Nile virus live-attenuated vaccine: preclinical evaluation of safety, immunogenicity, and efficacy. J Virol 2004;78:12497–507. [50] Huang CY, Silengo SJ, Whiteman MC, Kinney RM. Chimeric dengue 2 PDK53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus. J Virol 2005;79:7300–10. [51] Morrison D, Legg TJ, Billings CW, Forrat R, Yoksan S, Lang J. A novel tetravalent dengue vaccine is well tolerated and immunogenic against all 4 serotypes in flavivirus-naive adults. J Infect Dis 2010;201:370–7.
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Recombinant chimeric Japanese encephalitis virus/tick-borne encephalitis virus is attenuated and protective in mice Hong-Jiang Wang a,1 , Xiao-Feng Li a,1 , Qing Ye a , Shi-Hua Li a , Yong-Qiang Deng a , Hui Zhao a , Yan-Peng Xu a , Jie Ma a , E-De Qin a , Cheng-Feng Qin a,b,∗ a b
Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China Graduate School, Anhui Medical University, Hefei, China
a r t i c l e
i n f o
Article history: Received 9 July 2013 Received in revised form 14 November 2013 Accepted 18 December 2013 Available online 4 January 2014 Keywords: Tick-borne encephalitis virus Japanese encephalitis virus Vaccine Chimeric flavivirus
a b s t r a c t Tick-borne encephalitis virus (TBEV) represents one of the most dangerous human pathogens circulating in Europe and East Asia. No effective treatment for TBEV infection currently exists, and vaccination is the primary preventive measure. Although several inactivated vaccines have been licensed, the development of novel vaccines against TBEV remains a high priority in disease-endemic countries. In the present study, a live chimeric recombinant TBEV (ChinTBEV) was created by substituting the major structural genes of TBEV for the corresponding regions of Japanese encephalitis virus (JEV) live vaccine strain SA1414-2. The resulting chimera had a small-plaque phenotype, replicated efficiently in both mammalian and mosquito cells. The preliminary data from in vitro passaging indicated the potential for stability of ChinTBEV. ChinTBEV also exhibited significantly attenuated neuroinvasiveness in mice upon either intraperitoneal or subcutaneous inoculation in comparison with its parental TBEV. Importantly, a single immunisation with ChinTBEV elicited TBEV-specific IgG and neutralising antibody responses in a dose-dependent manner, providing significant protection against lethal TBEV challenge in mice. Taken together, the results of this proof-of-concept study indicate that ChinTBEV can be further developed as a potential vaccine candidate against TBEV infection. Moreover, the construction of this type of flavivirus chimera using a JEV vaccine strain as the genetic backbone represents a universal vaccine approach. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Tick-borne encephalitis (TBE) is the most serious ticktransmitted human infection in Europe and Asia, accounting for approximately 8500 human cases annually [1–5]. During the last few decades, new endemic foci and a great increase in TBE morbidity have been reported in many European and Asian countries [6,7]. This infection is characterised by typical neurological complications, such as meningitis, meningoencephalitis and encephalomyelitis/radiculitis, in both children and adults [3]. The causative agent, TBE virus (TBEV), belongs to the genus Flavivirus within the family Flaviviridae, which also contains other important human pathogens, including yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV) and West Nile virus (WNV). TBEV contains an approximately 11-kb positive-sense single-stranded RNA genome flanked by 5 - and 3 -untranslated regions (UTRs) and encodes a polyprotein that is proteolytically
∗ Corresponding author at: Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China. Tel.: +86 1066948604. E-mail addresses: [email protected], chengfeng [email protected] (C.-F. Qin). 1 These authors contribute equally to this work. 0264-410X/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.12.050
cleaved into three structural proteins (C, prM and E) and seven nonstructural proteins. TBEV can be divided into three genetically related subtypes: the European, Siberian and Far Eastern subtypes [8]. No effective treatment for TBEV infection is currently available. Currently, vaccination offers the most effective protection against TBE, and the vaccine has been introduced into many disease-endemic countries. There are at least four inactivated, cell culture-derived vaccines currently available, including Encepur® and TBE-Immun® , which are manufactured in Western Europe, as well as TBE-Moscow vaccine® and EnceVir® , which are manufactured in Russia [9,10]. Although the current vaccination schedules using these inactivated vaccines have led to a dramatic decline in the annual incidence of the disease [11], the requirement of multiple doses for primary and booster immunisations and the relatively high cost influence the success of TBEV immunisation programmes. Moreover, post-vaccination fever has been well documented, particularly in young children [12,13]. Although rare, vaccination failures have also been reported with use of the current inactivated TBEV vaccines [14–20]. In addition to killed vaccines, live attenuated vaccines have been demonstrated to induce long-term immunity at low cost. Two prominent flavivirus vaccines, the yellow fever live vaccine (strain
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YF-17D) and the Japanese encephalitis (JE) live vaccine (strain SA14-14-2), have been used globally and have saved the lives of millions suffering from flavivirus infections. In recent years, much effort has been made to rationally design a live attenuated vaccine against TBEV using a reverse genetics approach. Kofler et al. engineered an attenuated TBEV with a small deletion in the central portion of its C protein [21]. In other studies, a recombinant TBEV bearing a complementary target for mir-9 or mir-124a was significantly attenuated, and this virus induced strong humoral immune responses in monkeys [22,23]. A well-known strategy for generating attenuated flavivirus strains is to construct a chimeric flavivirus by replacing its structural proteins with the corresponding regions of another attenuated flavivirus strain. Using this strategy, a chimeric TBEV based on DENV-4 has been created and shown to be safe and efficacious in mice and non-human primates [24–27]. The JE live vaccine has been used in most JE-endemic countries in Asia, and more than 300 million doses have been administered since its licensure [28]. Large-scale vaccination programmes in various countries have shown that the vaccine possesses an excellent safety profile and remarkable effectiveness and efficacy [29–33]. The phenotypic and genotypic characteristics that correlate with attenuation are highly stable [28]. These properties make the JE vaccine strain an attractive genetic backbone with which to create a chimeric TBEV vaccine. However, no chimeric flavivirus based on JEV SA14-14-2 has been described to date. In this study, a live chimeric TBEV (ChinTBEV) was generated using a molecular clone of the JE vaccine strain SA14-14-2 as the genetic backbone. This novel flavivirus chimera, ChinTBEV, represents a potential vaccine candidate for TBEV infection that deserves further development.
2.3. Transcription and transfection Plasmid pChinTBEV was linearised with Xho I and used as a template for SP6 RNA polymerase transcription in the presence of an m7GpppA cap analogue (Promega). In vitro transcription was performed using the RiboMAX Large Scale RNA Production System (Promega) according to the manufacturer’s protocols. The yield and integrity of the RNA transcripts were analysed by gel electrophoresis under non-denaturing conditions. RNA transcripts (5 g) were then transfected into BHK-21 cells using Lipofectamine 2000 (Invitrogen). 2.4. Growth curves Growth curves were performed in BHK-21, Vero and C6/36 cells in a 12-well plate at a multiplicity of infection (MOI) of 0.01. Culture supernatants were collected at successive 24-h intervals, and progeny viral titres were then quantitated by plaque assay on BHK21 cells. 2.5. Indirect immunofluorescence assay (IFA) IFAs were performed as previously described. Briefly, BHK21 cells were infected with each virus for 48 h, after which the cells were fixed with ice-cold acetone and incubated with primary monoclonal antibodies (mAbs) against the JEV E protein (4AD5F5D5D6), the TBEV E protein (4A4) or the JEV NS1 protein (JN1). The cells were then incubated with secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen). Positive cells were examined using a fluorescent microscope (Olympus). 2.6. Genetic stability assay
2. Materials and methods 2.1. Cells and viruses Mammalian BHK-21 and Vero cells were maintained in Dulbecco’s minimal essential medium (DMEM; Invitrogen) supplemented with 10% foetal bovine serum (FBS), 100 U/ml penicillin and 100 g/ml streptomycin. Aedes albopictus C6/36 cells were cultured in RPMI 1640 supplemented with nonessential amino acids (Invitrogen), and 10% FBS. All cell lines were cultured at 37 ◦ C in 5% CO2 , except for the C6/36 cells, which were maintained at 28 ◦ C. TBEV strain Senzhang (GenBank no. JQ650523.1) and the JEV vaccine strain SA14-14-2 (GenBank no. D90195) were stored in our laboratory and prepared in BHK-21 cells. Stocks of each virus were titrated by performing a standard plaque-forming assay, as previously described [34,35].
2.2. Plasmid construction All plasmids were constructed using standard molecular cloning protocols. The sequences of the primers used in this study are shown in Table S1. Genetic construction of the full-length infectious clone of JEV has been described [36]. Briefly, the plasmid pACYC-Linker was first created from the plasmid pACYC177 (Fermentas) and the plasmid pSP64 (Promega). Further details about the construction of pACYC-Linker are available from the authors on request. Cloning sites were engineered to permit replacement of the entire pre-M and E coding sequences of JEV with the corresponding sequences of TBEV. Sites for posttranslational cleavage of the capsid and pre-M proteins and the E and NS1 proteins were preserved (Fig. 1A). The resulting plasmid contained the full-length cDNA of the JEV/TBEV chimera and was named pChinTBEV.
ChinTBEV was serially passaged in Vero cells at an initial MOI of 0.01. Viral RNA was extracted from the supernatants of the fourth and eighth passages and sequenced accordingly. The plaque phenotypes of passages 4 and 8 were also assessed. 2.7. Enzyme-linked immunosorbent assay (ELISA) Serum IgG against TBEV was detected by indirect ELISA using the formaldehyde-inactivated form of the TBEV strain Senzhang as the coating antigen and peroxidase-conjugated horse anti-mouse IgG as the secondary antibody. Serial two-fold dilutions of serum were tested in duplicate. Endpoint titres were considered the highest dilution that resulted in a value two-fold greater than the absorption of the control serum, with a cut-off value of 0.05. 2.8. Neutralisation assay Neutralising antibody titres were determined by a standard 50% plaque reduction neutralisation test (PRNT50 ). The endpoint neutralisation titre was calculated according to the method of Reed and Muench [37]. 2.9. Mouse experiments All animal experiments were approved by and conducted in strict accordance with the guidelines of the Experimental Animal Ethics Committee of the Beijing Institute of Microbiology and Epidemiology. Groups of 4-week-old BALB/c mice (n = 5–9) were inoculated by the intraperitoneal (i.p.), subcutaneous (s.c.) or intracerebral (i.c.) route with ChinTBEV, TBEV or JEV at varying doses. The mice were then monitored for clinical symptoms and mortality for 21 days. The 50% lethal dose (LD50 ) of each virus was calculated according to the method of Reed and Muench [37].
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Fig. 1. Construction and characterisation of ChinTBEV. (A) Graphic representation of the genomic constructs of ChinTBEV and its parental viruses. JEV genes are shown in grey, and TBEV is shown in white. (B) IFAs of ChinTBEV and its parental viruses. Green fluorescence represents positive reactivity to mAbs against JEV-E, TBEV-E and JEV-NS1, as indicated at the bottom. (C) Plaque morphologies of ChinTBEV and its parental viruses. Plaques were observed 3 days post-infection after staining with 0.2% crystal violet.
Immunogenicity was assessed by the s.c. or i.p. inoculation of 4-week-old BALB/c mice with various doses of ChinTBEV. The mice were bled by tail vein puncture 1 day prior to and 2 and 4 weeks post-immunisation. TBEV-specific IgG and neutralising antibodies were measured by ELISA and PRNT50 , respectively. For the protection assay, all of the ChinTBEV-immunised mice were challenged by the i.p. route with 500 PFU (100 LD50 ) of TBEV at 4 weeks after the initial immunisation. The mice were monitored for signs of illness and death for at least 21 days. 2.10. Histopathological analysis Mice were inoculated with 104 PFU of TBEV or ChinTBEV by the i.p. route. At 8 days post-inoculation, the brains of three mice were collected, fixed with perfusion fixative (4% formaldehyde) for 48 h and processed according to standard histological methods. All brain tissue sections from each animal were stained with haematoxylin and eosin (HE). 2.11. Statistical analysis For survival analysis, Kaplan–Meier survival curves were analysed by a log-rank test (GraphPad Prism software 5.0, San Diego, CA).
3. Results 3.1. Construction and characterisation of ChinTBEV To generate a chimeric flavivirus carrying the protective antigens of TBEV, the prM and E genes of JEV SA14-14-2 were substituted with the corresponding genes of the TBEV strain Senzhang using standard DNA recombination technology, resulting in the DNA construct ChinTBEV (Fig. 1A). The prM signal peptide of JEV and the last three amino acids of the JEV E protein were retained to ensure efficient cleavage at the C-prM and E-NS1 junctions [38–40]. The full-length cDNA clone of ChinTBEV was then sequenced and compared with the cDNA of the parental viruses JEV strain SA14-142 and TBEV strain Senzhang (Table S2). Only five nucleotides in the prM-E coding region of ChinTBEV differed from those in TBEV, three of which resulted in amino acid changes within the prM protein (Val28 → Met, Thr51 → Ala and Glu87 → Gly). To obtain viable ChinTBEV from BHK-21 cells, in vitrotranscribed RNAs from linearised pChinTBEV were transfected into BHK-21 cells. The recovered ChinTBEV was characterised by IFA, together with its parental viruses JEV and TBEV. As shown in Fig. 1B, both the TBEV E protein and the JEV NS1 protein were detected in ChinTBEV-infected cells. As expected, JEV and TBEV could only
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Fig. 2. Growth curves and phenotype stability of ChinTBEV. (A) Monolayers of BHK-21, Vero and C6/36 cells were infected with the indicated viruses at an MOI of 0.01. Viral titres were determined on BHK-21 cells by plaque assay at the indicated times. The dotted lines represent the limit of sensitivity of the plaque assay. (B) Plaque phenotypes of passages 1, 4 and 8 on BHK-21 cells. The average plaque sizes (mean ± standard deviation) were estimated by counting 40 plaques.
react with JEV- and TBEV-specific mAbs, respectively. Importantly, the plaque morphology of ChinTBEV was different from that of its parental viruses; in particular, the average plaque size was significantly smaller compared with the plaque size of the parental viruses (Fig. 1C). The small-plaque phenotype of ChinTBEV indicates its potential attenuation in comparison with its parental viruses. Next, the growth efficiencies of ChinTBEV and its parental viruses were compared in mammalian and mosquito cells. ChinTBEV replicated slightly less efficiently than TBEV, and both peaked at approximately 48 h post-infection in BHK-21 cells (Fig. 2A). In Vero cells, the certified cell bank intended for vaccine manufacturing, ChinTBEV and TBEV replicated to their highest titres at 96 h post-infection, reaching 107.0 and 108.1 PFU/ml, respectively (Fig. 2A). In contrast, in mosquito C6/36 cells, TBEV failed to replicate, but both JEV and the chimera were capable of replicating, attaining peak titres of 106.4 and 104.1 PFU/ml, respectively (Fig. 2A). Furthermore, to assess the genetic stability of ChinTBEV during sequential passaging in vitro, ChinTBEV recovered from BHK-21 cells (named passage 0) was passaged up to eight times in Vero cells. Viruses harvested at passages 1, 4 and 8 were sequenced and compared with viruses harvested at passage 0. No amino acid substitutions were identified in the prM-E regions and the nonstructural proteins until passage 8. The homogeneous small-plaque phenotype of ChinTBEV in BHK-21 cells was also retained during in vitro passaging (Fig. 2B). Taken together, these results demonstrate that this novel chimera with a small-plaque phenotype is highly replicative and potentially genetically stable in cell culture. 3.2. ChinTBEV demonstrates highly attenuated neuroinvasiveness in mice To further characterise the attenuation phenotype of ChinTBEV, groups of BALB/c mice were inoculated with 10-fold serial dilutions of either ChinTBEV or TBEV. Mice inoculated with varying doses of
the JEV vaccine strain SA14-14-2 were used as controls. Upon s.c. inoculation, ChinTBEV did not cause any clinical symptoms or mortality during the observation period, even at a dose of 105 PFU. The LD50 of ChinTBEV should be higher than the LD50 of the JEV vaccine strain, which was approximately 105 PFU (Table 1). Meanwhile, s.c. inoculation of TBEV resulted in 100% mortality even at the lowest dose, 103 PFU, and the LD50 of TBEV was calculated to be less than 103 PFU (Table 1). Thus, ChinTBEV is more than 100-fold attenuated in comparison with TBEV upon s.c. infection. Upon i.p. injection, ChinTBEV failed to cause any clinical symptoms or mortality in mice at a dose of 103 PFU, whereas higher doses of ChinTBEV injection caused the central nervous system (CNS) manifestations typical of TBEV infection. The LD50 of ChinTBEV was calculated to be 105.5 PFU. As all of the mice that were inoculated with TBEV by the i.p. route died, even at the lowest dose of 10 PFU, the LD50 of TBEV should be less than 10 PFU (Table 1). Thus, ChinTBEV is at least 31,600-fold more attenuated than TBEV upon i.p. infection. Furthermore, the abilities of ChinTBEV and parental TBEV to induce viraemia in mice were compared following i.p. inoculation. As shown in Fig. 3A, TBEV-inoculated mice developed significant viraemia from day 1 to day 4 post-infection, with a peak titre of 104.0 PFU/ml on day 2 post-infection, and no viraemia was observed in the following days. In contrast, viraemia was not detectable in any of the mice inoculated with ChinTBEV throughout the observation period. In particular, no virus was isolated from the brains of mice infected with ChinTBEV by the i.p. route. The histopathological results showed typical focal gliosis and neuronophagia in the temporal cerebral cortex of the TBEV-infected mice, whereas no unusual lesions were observed in the brains of mice inoculated with ChinTBEV (Fig. 3B). To examine neurovirulence, groups of mice were challenged by the i.c. route with various doses of ChinTBEV and TBEV. The results showed that ChinTBEV retained partial neurovirulence in mice, as an i.c. inoculation of 1 PFU caused 60% mortality. Compared with
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Table 1 Attenuation of ChinTBEV in mice in comparison with its parental virusesa . Virus
Route
ChinTBEV
s.c.
i.p.
TBEV
s.c.
i.p.
JEV
s.c.
i.p.
a b
Dose (PFU) 5
10 104 103 106 105 104 103 105 104 103 103 102 10 105 104 103 106 105 104
Mortality (no. dead/no. tested, %)
AST (day)b
LD50 (PFU)
0 (0/8) 0 (0/8) 0 (0/8) 77.8 (7/9) 22.2 (2/9) 25.0 (2/8) 0 (0/8) 100 (6/6) 100 (6/6) 100 (6/6) 100 (9/9) 100 (9/9) 88.9 (8/9) 0 (0/8) 0 (0/8) 0 (0/7) 0 (0/8) 0 (0/8) 0 (0/8)
N/A N/A N/A 11.3 16.5 16.0 N/A N/A N/A 8.83 N/A 9.0 10.0 N/A N/A N/A N/A N/A N/A
>105
105.5
106
Groups of 4-week-old BALB/c female mice were s.c. or i.p. inoculated with the indicated doses of viruses. The mortality was monitored for 21 days after inoculation. AST for mice that died; N/A, not applicable.
mice infected with the parental TBEV, the survival rate among mice infected with varying doses of ChinTBEV was higher, indicating slightly attenuated neurovirulence for ChinTBEV compared with its parental TBEV (Fig. 3C). Together, these results demonstrate that the peripheral virulence of ChinTBEV is highly attenuated in mice and that this chimeric virus possesses slightly attenuated neurovirulence compared with TBEV.
3.3. ChinTBEV elicits neutralising antibodies To evaluate the immunogenicity of ChinTBEV, groups of 4week-old BALB/c mice were immunised once with varying doses of ChinTBEV by the s.c. or i.p. route. Mice immunised with PBS were used as controls. It should be noted that i.p. immunisation with 105 PFU of ChinTBEV caused 20% mortality in mice, and only
Fig. 3. ChinTBEV was attenuated in mice. (A) Viraemia profile of ChinTBEV in mice following i.p. challenge. Mice were inoculated with 104 PFU of the indicated viruses, and viraemia was determined on days 1 to 4 post-inoculation by plaque assay. (B) Histopathological analysis for the virus-infected mice brain. Mice were inoculated with 104 PFU of TBEV or ChinTBEV by the i.p. route, and brains were collected at 8 days post-infection and subjected to HE analysis. The arrow indicates focal gliosis and neuronophagia in temporal cerebral cortex. (C) Neurovirulence tests of ChinTBEV. Mice received either ChinTBEV or TBEV at the indicated doses, and mortality was recorded for 21 days.
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Fig. 4. Immunogenicity of ChinTBEV in mice. IgG and neutralising antibody responses in mice after s.c. or i.p. inoculation with the indicated doses of ChinTBEV. Sera were collected from animals at 28 days post-inoculation to measure IgG and neutralising antibody responses against TBEV by ELISA and PRNT50 , respectively. The dotted lines represent the limits of detection of the ELISA and PRNT50 , which were assigned values of 100 and 5, respectively.
the survival were subjected to subsequent antibody and challenge assay. All of the immunised mice were bled at 4 weeks after immunisation, and IgG and neutralising antibody titres against TBEV were tested by ELISA and PRNT50 , respectively. As shown in Fig. 4A, both s.c. and i.p. immunisation with ChinTBEV induced high titres of TBEV-specific IgG antibodies in a dose-dependent manner. Most importantly, TBEV-specific neutralising antibodies were elicited following a single immunisation with varying doses of ChinTBEV (Fig. 4B). As expected, no TBEV-specific neutralising antibodies were detected in sera from control mice. Collectively, these results show that a single immunisation with ChinTBEV induces a significant humoral immune response to TBEV. 3.4. ChinTBEV confers protection against TBEV challenge in mice Finally, to determine the in vivo protection elicited by ChinTBEV vaccination, all of the above-mentioned mice immunised with ChinTBEV were challenged with 100 LD50 of TBEV by the i.p. route at 28 days post-immunisation. As shown in Fig. 5, all mice immunised with PBS developed symptoms of CNS infection and died within 11 to 15 days after i.p. challenge. Importantly, i.p. immunisation with a high dose (105 PFU) of ChinTBEV provided full protection against lethal TBEV challenge; all of these animals survived challenge and exhibited no signs of CNS disease during the 21-day observation period. In contrast, the low dose (104 PFU) of ChinTBEV provided 60% protection. In the s.c. immunisation group, both a high dose and a low dose of ChinTBEV provided significant protection, with 71% and 50% of the immunised mice, respectively, surviving the lethal challenge. ChinTBEV immunisation significantly decreased mortality and extended the AST (P < 0.05). These data indicate
that a single immunisation with ChinTBEV confers solid protection against lethal TBEV challenge in mice. 4. Discussion This proof-of-concept study was to provide a vaccine approach using chimeric JEV/TBEV for further development. Chimeric flaviviruses created using an attenuated strain as the backbone have been well explored in the development of DENV, WNV, JEV and TBEV vaccines [24,41–43]. In the present study, we successfully transferred the prM-E gene of TBEV into the genome of the JEV vaccine strain SA14-14-2, resulting in a viable chimeric flavivirus that we called ChinTBEV. ChinTBEV produced significantly smaller plaques than did both of its parental viruses (Fig. 1C), indicating the potentially attenuated phenotype of ChinTBEV. The peak titre of ChinTBEV reached 107.0 PFU in Vero cells, indicating the possibility of large-scale production at low cost. Although TBEV failed to replicate in mosquito cells, a delayed and restricted growth pattern was observed for ChinTBEV compared with JEV in mosquito C6/36 cells (Fig. 2A), which is in agreement with previous findings [26,44]. Additionally, ChinTBEV exhibited potential genetic and plaque morphology stability after serial passaging in Vero cells (Fig. 2B), and this preliminary data indicate the potential for stability of ChinTBEV. Whatever, a detailed investigation of stability at the gene and population levels should be warranted in the near future. Our data showed that the neuroinvasiveness of ChinTBEV was highly attenuated in mice in comparison with TBEV. The LD50 of ChinTBEV was approximately 100- and 31,600-fold higher that of TBEV upon s.c. or i.p. inoculation, respectively (Table 1). Antigenic
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Fig. 5. Protection against lethal TBEV challenge in ChinTBEV-immunised mice. All of the immunised mice were challenged with 500 PFU of TBEV by the i.p. route. The statistical significance of differences in mortality and AST between the two groups was determined using the log-rank test in GraphPad Prism 5.0.
chimerisation between flavivirus members has been proven to lead to the attenuation of neurotropic viruses [26,41,45]. Additionally, the presence of specific mutations in certain virulence determinants within the genetic background of JEV may contribute to attenuation in mice [42,46,47]. Previously, chimeric TBEV vaccine candidates including Langat/DENV-4 and TBEV/DENV-4 have been well evidenced to be highly attenuated in neurovirulence in mice and non-human primates [26,27]. However, ChinTBEV was only slightly attenuated in neurovirulence compared with its parental TBEV (Fig. 3C). This result is in agreement with previous findings [48–50] and is most likely due to the presence of critical neurovirulence determinants in the E protein of parental TBEV. Future experiments using reverse genetics are warranted to mutate these amino acid residues to achieve an attenuated neurovirulence phenotype in mice. A single immunisation with ChinTBEV induced a protective antibody response in mice (Fig. 4). Both the IgG and the neutralising antibody responses in mice were dose dependent. Compared with other TBEV vaccine candidates and commercial products [25–27], the peak titre of neutralising antibody induced by a single immunisation was relatively low. This is probably due to the restricted replication of ChinTBEV in mice. Nevertheless, a neutralising antibody titre >1:10 is considered as seroconversion and represents the best surrogate marker for protection. Challenge experiments showed that immunisation with a high dose (105 PFU) of ChinTBEV (i.p. and s.c. routes) provided solid protection (100% and 71%) against lethal TBEV challenge. Immunisation with the lower dose (104 PFU) of ChinTBEV conferred only 60% and 50% protection (Fig. 5), which was most likely due to the relatively low neutralising antibody titre and the relatively high challenge dose (Fig. 4B). In our experiments, mice were challenged with 100 LD50 of TBEV, and decreasing this challenge dose would most likely increase the survival rate. A second booster immunisation may be needed that is common for the previously described chimeric DENV vaccine based on YFV 17D [51]. Conflict of interests statement None. Acknowledgments This work was supported by the National Basic Research Project (no. 2012CB518904), the National Natural Science Foundation of China (no. 81101243, no. U1132002 and no. 31270974) and the National Major Special Program of Science and Technology of China (2008ZX10004-015 and 2013ZX10004001). C.F. Qin was
supported by the Beijing Nova Program of Science and Technology (no. 2010B041).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine. 2013.12.050.
References [1] Poponnikova TV. Specific clinical and epidemiological features of tick-borne encephalitis in Western Siberia. Int J Med Microbiol 2006;296(Suppl 40):59–62. [2] Gritsun TS, Lashkevich VA, Gould EA. Tick-borne encephalitis. Antiviral Res 2003;57:129–46. [3] Lindquist L, Vapalahti O. Tick-borne encephalitis. Lancet 2008;371:1861–71. [4] Mandl CW. Steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis. Virus Res 2005;111:161–74. [5] Kaiser R. Tick-borne encephalitis. Infect Dis Clin North Am 2008;22:561–75, x. [6] Suss J. Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in Europe and Asia-an overview. Ticks Tick Borne Dis 2011;2:2–15. [7] Mansfield KL, Johnson N, Phipps LP, Stephenson JR, Fooks AR, Solomon T. Tick-borne encephalitis virus—a review of an emerging zoonosis. J Gen Virol 2009;90:1781–94. [8] Ecker M, Allison SL, Meixner T, Heinz FX. Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia. J Gen Virol 1999;80(Pt 1):179–85. [9] Zent O, Broker M. Tick-borne encephalitis vaccines: past and present. Expert Rev Vaccines 2005;4:747–55. [10] Leonova GN, Pavlenko EV. Characterization of neutralizing antibodies to Far Eastern of tick-borne encephalitis virus subtype and the antibody avidity for four tick-borne encephalitis vaccines in human. Vaccine 2009;27:2899–904. [11] Heinz FX, Holzmann H, Essl A, Kundi M. Field effectiveness of vaccination against tick-borne encephalitis. Vaccine 2007;25:7559–67. [12] Pavlova BG, Loew-Baselli A, Fritsch S, Poellabauer EM, Vartian N, Rinke I, et al. Tolerability of modified tick-borne encephalitis vaccine FSME-IMMUN “NEW” in children: results of post-marketing surveillance. Vaccine 2003;21:742–5. [13] Zent O, Banzhoff A, Hilbert AK, Meriste S, Sluzewski W, Wittermann C. Safety, immunogenicity and tolerability of a new pediatric tick-borne encephalitis (TBE) vaccine, free of protein-derived stabilizer. Vaccine 2003;21:3584–92. [14] Bender A, Jager G, Scheuerer W, Feddersen B, Kaiser R, Pfister HW. Two severe cases of tick-borne encephalitis despite complete active vaccination—the significance of neutralizing antibodies. J Neurol 2004;251:353–4. [15] Kleiter I, Jilg W, Bogdahn U, Steinbrecher A. Delayed humoral immunity in a patient with severe tick-borne encephalitis after complete active vaccination. Infection 2007;35:26–9. [16] Kunze U, Baumhackl U, Bretschneider R, Chmelik V, Grubeck-Loebenstein B, Haglund M, et al. The golden agers and tick-borne encephalitis. Conference report and position paper of the International Scientific Working Group on Tick-borne encephalitis. Wien Med Wochenschr 2005;155:289–94. [17] Stiasny K, Holzmann H, Heinz FX. Characteristics of antibody responses in tickborne encephalitis vaccination breakthroughs. Vaccine 2009;27:7021–6. [18] Grgic-Vitek M, Avsic-Zupanc T, Klavs I. Tick-borne encephalitis after vaccination: vaccine failure or misdiagnosis. Vaccine 2010;28:7396–400. [19] Andersson CR, Vene S, Insulander M, Lindquist L, Lundkvist A, Gunther G. Vaccine failures after active immunisation against tick-borne encephalitis. Vaccine 2010;28:2827–31.
956
H.-J. Wang et al. / Vaccine 32 (2014) 949–956
[20] Plisek S, Honegr K, Beran J. TBE infection in an incomplete immunized person at-risk who lives in a high-endemic area—impact on current recommendations for immunization of high-risk groups. Vaccine 2008;26:301–4. [21] Kofler RM, Heinz FX, Mandl CW. Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence. J Virol 2002;76:3534–43. [22] Heiss BL, Maximova OA, Thach DC, Speicher JM, Pletnev AG. MicroRNA targeting of neurotropic flavivirus: effective control of virus escape and reversion to neurovirulent phenotype. J Virol 2012;86:5647–59. [23] Heiss BL, Maximova OA, Pletnev AG. Insertion of microRNA targets into the flavivirus genome alters its highly neurovirulent phenotype. J Virol 2011;85:1464–72. [24] Wright PF, Ankrah S, Henderson SE, Durbin AP, Speicher J, Whitehead SS, et al. Evaluation of the Langat/dengue 4 chimeric virus as a live attenuated tick-borne encephalitis vaccine for safety and immunogenicity in healthy adult volunteers. Vaccine 2008;26:882–90. [25] Rumyantsev AA, Chanock RM, Murphy BR, Pletnev AG. Comparison of live and inactivated tick-borne encephalitis virus vaccines for safety, immunogenicity and efficacy in rhesus monkeys. Vaccine 2006;24:133–43. [26] Pletnev AG, Men R. Attenuation of the Langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4. PNAS 1998;95:1746–51. [27] Pletnev AG, Karganova GG, Dzhivanyan TI, Lashkevich VA, Bray M. Chimeric Langat/Dengue viruses protect mice from heterologous challenge with the highly virulent strains of tick-borne encephalitis virus. Virology 2000;274:26–31. [28] Yu Y. Phenotypic and genotypic characteristics of Japanese encephalitis attenuated live vaccine virus SA14-14-2 and their stabilities. Vaccine 2010;28:3635–41. [29] Tandan JB, Ohrr H, Sohn YM, Yoksan S, Ji M, Nam CM, et al. Single dose of SA 14-14-2 vaccine provides long-term protection against Japanese encephalitis: a case-control study in Nepalese children 5 years after immunization. Vaccine 2007;25:5041–5. [30] Kumar R, Tripathi P, Rizvi A. Effectiveness of one dose of SA 14-14-2 vaccine against Japanese encephalitis. N Engl J Med 2009;360:1465–6. [31] Hennessy S, Liu Z, Tsai TF, Strom BL, Wan CM, Liu HL, et al. Effectiveness of liveattenuated Japanese encephalitis vaccine (SA14-14-2): a case-control study. Lancet 1996;347:1583–6. [32] Gatchalian S, Yao Y, Zhou B, Zhang L, Yoksan S, Kelly K, et al. Comparison of the immunogenicity and safety of measles vaccine administered alone or with live, attenuated Japanese encephalitis SA 14-14-2 vaccine in Philippine infants. Vaccine 2008;26:2234–41. [33] Bista MB, Banerjee MK, Shin SH, Tandan JB, Kim MH, Sohn YM, et al. Efficacy of single-dose SA 14-14-2 vaccine against Japanese encephalitis: a case control study. Lancet 2001;358:791–5. [34] Li SH, Dong H, Li XF, Xie X, Zhao H, Deng YQ, et al. Rational design of a flavivirus vaccine by abolishing viral RNA 2 -O methylation. J Virol 2013;87:5812–9. [35] Si BY, Jiang T, Zhang Y, Deng YQ, Huo QB, Zheng YC, et al. Complete genome sequence analysis of tick-borne encephalitis viruses isolated in northeastern China. Arch Virol 2011;156:1485–8.
[36] Ye Q, Li XF, Zhao H, Li SH, Deng YQ, Cao RY, et al. A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1 formation and contributes to attenuation. J Gen Virol 2012;93:1959–64. [37] Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg 1938;27:493–7. [38] Amberg SM, Rice CM. Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing. J Virol 1999;73:8083–94. [39] Yamshchikov VF, Compans RW. Formation of the flavivirus envelope: role of the viral NS2B-NS3 protease. J Virol 1995;69:1995–2003. [40] Lobigs M, Lee E. Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes. J Virol 2003;78:178–86. [41] Pletnev AG, Putnak R, Speicher J, Wagar EJ, Vaughn DW. West Nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective efficacy. PNAS 2002;99:3036–41. [42] Chambers TJ, Nestorowicz A, Mason PW, Rice CM. Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties. J Virol 1999;73:3095–101. [43] Guirakhoo F, Weltzin R, Chambers TJ, Zhang ZX, Soike K, Ratterree M, et al. Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates. J Virol 2000;74:5477–85. [44] Engel AR, Mitzel DN, Hanson CT, Wolfinbarger JB, Bloom ME, Pletnev AG. Chimeric tick-borne encephalitis/dengue virus is attenuated in Ixodes scapularis ticks and Aedes aegypti mosquitoes. Vector Borne Zoonotic Dis 2011;11: 665–74. [45] Whitehead SS, Hanley KA, Blaney Jr JE, Gilmore LE, Elkins WR, Murphy BR. Substitution of the structural genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes, mice, and rhesus monkeys. Vaccine 2003;21:4307–16. [46] Durbin AP, Karron RA, Sun W, Vaughn DW, Reynolds MJ, Perreault JR, et al. Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3 -untranslated region. Am J Trop Med Hyg 2001;65:405–13. [47] Huang CY, Butrapet S, Pierro DJ, Chang GJ, Hunt AR, Bhamarapravati N, et al. Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine. J Virol 2000;74:3020–8. [48] Pletnev AG, Bray M, Huggins J, Lai CJ. Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses. PNAS 1992;89:10532–6. [49] Arroyo J, Miller C, Catalan J, Myers GA, Ratterree MS, Trent DW, et al. ChimeriVax-West Nile virus live-attenuated vaccine: preclinical evaluation of safety, immunogenicity, and efficacy. J Virol 2004;78:12497–507. [50] Huang CY, Silengo SJ, Whiteman MC, Kinney RM. Chimeric dengue 2 PDK53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus. J Virol 2005;79:7300–10. [51] Morrison D, Legg TJ, Billings CW, Forrat R, Yoksan S, Lang J. A novel tetravalent dengue vaccine is well tolerated and immunogenic against all 4 serotypes in flavivirus-naive adults. J Infect Dis 2010;201:370–7.
