imojev ® : a yellow fever virus-based novel japanese encephalitis...

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Vaccine Profile

Japanese encephalitisJapanese encephalitis (JE) is a disease of the CNS in humans caused by the Japanese encephalitis virus (JEV), a member of the flavivirus genus. The disease is characterized by high fever, headache, nausea, dizziness, myalgia, convulsions, focal neu-rological signs and decreased consciousness. JE is generally asymptomatic and clinical manifest-ations are seen only in approximately 0.1–1% of JEV-infected subjects. JE is endemic in most parts of Asia and occasionally a few cases have been reported from the Pacific region. Genetic studies on the origin of JEV suggest that the virus origi-nated from an ancestral virus in the area of Malay Archipelago and then evolved into different geno-types [1]. A clinical case of JE was reported for the first time in Japan in 1871, although the infectious agent was only isolated approximately 50 years later from an infected human brain. The WHO estimates that approximately 50,000 clinical

cases of JE occur annually worldwide, of which 10,000–15,000 cases result in fatal encephali-tis and approximately 30–50% of the survivors experience long-lasting neurological problems. According to these estimates, more than 3 billion people are living in JE-endemic regions and are therefore at risk of contracting the disease [2]. JE transmission is highly dynamic and occurs mostly in the form of small to large outbreaks; hence, there is a huge fluctuation in the annual incidence of JE in different parts of Asia. JE incidence is influenced by several factors that include changes in climatic conditions, socioeconomic standards of the population, sanitation, JE immune status of the population, presence of mosquito vectors, agricultural practices and proximity of mosquito breeding grounds to the human population.

Epidemiological studies on JE incidence sug-gest that temperate regions experience large epi-demics during the summer months, whereas they

Mohan Babu Appaiahgari1 and Sudhanshu Vrati†1,2

1Vaccine and Infectious Disease Research Center, Translational Health Science and Technology Institute, Gurgaon 122 016, India 2National Institute of Immunology, New Delhi 110 067, India †Author for correspondence:Tel.: +91 11 2670 3696 Fax: +91 11 2616 2125 [email protected]

Japanese encephalitis (JE) is a disease of the CNS caused by Japanese encephalitis virus (JEV). The disease appears in the form of frequent outbreaks in most south- and southeast Asian countries and the virus has become endemic in several areas. There is no licensed therapy available and disease control by vaccination is considered to be most effective. Mouse brain-derived inactivated JE vaccines, although immunogenic, have several limitations in terms of safety, availability and requirement for multiple doses. Owing to these drawbacks, the WHO called for the development of novel, safe and more efficacious JE vaccines. Several candidate vaccines have been developed and at least three of them that demonstrated strong immunogenicity after one or two doses of the vaccine in animal models were subsequently tested in various clinical trials. One of these vaccines, IMOJEV® (JE-CV and previously known as ChimeriVax™-JE), is a novel recombinant chimeric virus vaccine, developed using the Yellow fever virus (YFV) vaccine vector YFV17D, by replacing the cDNA encoding the envelope proteins of YFV with that of an attenuated JEV strain SA14-14-2. IMOJEV was found to be safe, highly immunogenic and capable of inducing long-lasting immunity in both preclinical and clinical trials. Moreover, a single dose of IMOJEV was sufficient to induce protective immunity, which was similar to that induced in adults by three doses of JE-VAX®, a mouse brain-derived inactivated JE vaccine. Recently, Phase III trials evaluating the immunogenicity and safety of the chimeric virus vaccine have been successfully completed in some JE-endemic countries and the vaccine manufacturers have filed an application for vaccine registration. IMOJEV may thus be licensed for use in humans as an improved alternative to the currently licensed JE vaccines.

Keywords: immunogenicity • JEV • JE-VAX® • MBDV • SA14-14-2 • YFV17D

IMOJEV®: a Yellow fever virus-based novel Japanese encephalitis vaccineExpert Rev. Vaccines 9(12), 1371–1384 (2010)

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Vaccine Profile Appaiahgari & Vrati

occur during or immediately after the rainy season in tropical regions [3]. Importantly, JE-endemic regions are generally rice-cultivating areas and the mosquito vectors implicated in JE trans-mission are known to breed preferentially in irrigated rice paddies and large pools densely filled with masses of algae. Although JEV has been isolated from different mosquito species, members of the Culex genus have been primarily implicated in the transmission of the virus. The virus is now spreading to new territories, like the Torres Strait islands in Northern Australia, probably assisted by bird migration and various environmental factors such as climate change and jet streams of wind carrying infected mosquitoes [4,5]. Primarily, the Culicine mosquitoes are zoophilic and feed on their reservoir vertebrate hosts such as wading birds, bats, pigs, ducks and hibernating lizards. However, extreme climatic conditions, such as floods, result in the transfer of virus from animals into the human population, causing outbreaks [5]. Although the factors responsible for the occurrence and spread of JE are not well under-stood, field data from various outbreaks suggest that JE epidemics are usually preceded by severe drought, followed by heavy rains, wind storms and floods. Furthermore, an entomological study found that the Culicine mosquitoes can harbor the virus for up to 40 days after infection, suggesting that they may play a major role in the maintenance and transmission of the virus for longer peri-ods in nature [6]. In addition, travelers from nonendemic regions are also at risk of contracting the disease, depending on the time and place of visit, as people coming from these regions are not expected to possess immunity to JE [7,8]. These epidemiological studies highlight the complex interplay of the various factors listed above in the maintenance and spread of JEV in nature.

Although immunization practice and improvements in living conditions have significantly helped to reduce the incidence, JE is still a major health problem in most Asian countries. Since most of the people living in JE-endemic areas are concentrated in India and China, the disease burden in these two countries would influ-ence the JE disease burden on a global scale. Extensive use of the live-attenuated SA14-14-2 JE vaccine has resulted in reduced JE incidence in China [9]. This vaccine has recently been used in some parts of India, however, its impact on JE incidence is not known. Interestingly, a study conducted in Taiwan found that routine immunization of children below 15 years of age resulted in a shift in age preference for JE occurrence from children to adults. These data advocate vaccine coverage for people of all ages to effectively control JE in the population as a whole [10].

JE virusJapanese encephalitis virus, belonging to the Flaviviridae family of animal viruses, is an enveloped, spherical virus of approximately 50 nm diameter. The virus has an approximately 11-kb long, single-stranded, positive-sense RNA genome, which is capped at the 5́ end but is without a poly(A) tail at the 3´ end. The genome contains two noncoding regions, with one each at the 5́ and the 3´ ends, which play a major role in viral genome replication. The viral genome codes for an approximately 3400-amino acid poly-protein, which is co- and post-translationally cleaved into three structural (capsid [C], premembrane [prM] and envelope [E]) and

seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins (Figure 1). The non structural proteins have roles at various stages of viral replication and, hence, are important for virus survival and spread in the host. Apart from having possible roles in viral replication, the viral C protein plays a major role in virus assembly. The prM protein contains a presignal peptide at its N-terminus, which is important for the translocation of the prM–E complex to the cell membrane and is also crucial for maturation of the E protein. The presignal peptide is cleaved in the late-Golgi complex to release the mature membrane (M) protein. The mature M protein together with the E protein forms the viral envelope. Thus, the mature virion contains a single copy of viral genome tightly packaged inside the viral coat comprising multiple copies of C protein arranged in an icosahedral symmetry, which is surrounded by approximately 180 copies of two viral glycopro-teins, M and E, arranged in a T = 3 quasi-symmetry inside the host-derived lipid bilayer.

Of the three structural proteins, the E protein plays various important roles in infection such as receptor binding, membrane fusion and cell entry. The E protein is the principal neutraliza-tion antigen that induces JEV-neutralizing antibodies in a virus-infected host [11–14]. It has been demonstrated that protection against JEV is mainly antibody dependent and virus-neutralizing antibodies alone are sufficient to impart protection [15,16]. Based on the immunization data in humans and experimental data in passively immunized mice, a neutralizing antibody titer of 1:10 is considered protective in humans [2,17].

Vaccines against JEThere are no approved drugs for JE therapy and prevention by vaccination as well as by mosquito control are the most effec-tive measures. Mouse brain-derived vaccines (MBDVs) have been in use for many years due to their strong protective efficacy. However, these vaccines have limitations in terms of safety, avail-ability and requirement for multiple doses. Several novel can-didate JE vaccines have been developed in recent years, which include cell culture-based inactivated vaccines, chimeric virus vaccines, recombinant adenovirus-based vaccines, plasmid DNA-based vaccines and peptide vaccines [14,18–24]. Some of these vac-cine candidates have been tested in both animal models and in human clinical trials. In the next section, a review of the status of JE vaccines that are presently in use and those in advanced stages of development is provided, with a particular emphasis on the development of the novel chimeric JE vaccine IMOJEV®.

Mouse brain-derived JE vaccinesA number of MBDVs for JE with different brand names are licensed for use in different countries. Essentially, they are made from the Nakayama and/or Beijing strains of JEV grown in mouse brain. The inactivated virus is purified and formulated as vaccine. Most studies on MBDV immunogenicity and efficacy have been carried out using JE-VAX®, which is a mouse brain-derived inactivated JE vaccine manufactured by BIKEN (Osaka, Japan), a Japanese com-pany, and distributed by Sanofi Pasteur (Toronto, ON, Canada). A monovalent JE-VAX is based on the Nakayama-NIH strain of JEV,

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Vaccine ProfileIMOJEV®: a Yellow fever virus-based novel Japanese encephalitis vaccine

whereas the bivalent JE-VAX contains both the Nakayama-NIH strain and Beijing-1 strain of JEV. In both cases, the virus is inac-tivated using formaldehyde and then purified by ultracentrifuga-tion through 40% (weight/volume) sucrose gradient to remove the mouse brain proteins. The purified virus is then mixed with gelatin and thimerosal, used as stabilizer and preservative, respectively, to prepare the vaccine formulation, which is then lyophilized in vials to prepare the final vaccine dose that is supplied with sterile water as a diluent for injection. Besides the inactivated JEV, the JE-VAX contains less than 50 ng mouse serum protein, approxi-mately 500 µg gelatin, less than 2 ng myelin basic protein, less than 100 µg formaldehyde and 0.007% (weight/volume) thimerosal per milliliter of the vaccine dose. Three doses of vaccine administered subcutaneously on days 0, 7 and 14 or 30 are recommended for optimal protection. A dose of 1 ml per administration in children above 3 years of age and adults is recommended, whereas chil-dren between 1 and 3 years are administered a dose of 0.5 ml. An optional dose, 2 years after the primary immunization, is recom-mended to boost the immune response. Although available since 1955, the first-ever efficacy trial for a Nakayama strain-derived MBDV was conducted in Thai children between the age of 1 and 14 years during the mid-1980s [25]. This study demonstrated an efficacy of 91% with few side effects, which were similar to those seen in the placebo groups. Later studies conducted in US military volunteers demonstrated that three doses of vaccine were important for the induction of long-lasting immunity and these volunteers had detectable neutralizing antibodies even 12 months after vaccination [26].

For many years, MBDVs were used extensively in the expanded program for immunization and this resulted in a significant reduc-tion in the number of JE cases in many of the JE-endemic counties

like Japan and Taiwan. However, there have been some safety-related limitations of these vaccines. For example, use of JE-VAX is not recommended for children less than 1 year of age, for those hypersensitive to thimerosal and in immunocompromised indi-viduals. That aside, mild-to-severe adverse reactions have been recorded in vaccinees. Severe hypersensitivity events included generalized urticaria, angioedema, respiratory distress and hos-pitalization due to disseminated encephalomyelitis. In addition, concerns of possible vaccine-related neurologic reactions have been suspected due to the mouse brain origin of MBDVs [27–30]. Several countries, including Japan, have recently suspended MBDV from their routine immunization campaigns due to the occurrence of severe adverse events in some of the vaccinees. Although the mouse brain-derived inactivated JE vaccine is being produced in limited quantities by several manufacturers for their local needs, BIKEN stopped JE-VAX production in 2007.

SA14-14-2 JE vaccineThe live-attenuated cell culture-based SA14-14-2 JE vaccine has been developed by Chengdu Institute of Biological Products, People’s Republic of China. The vaccine virus, SA14-14-2 strain of JEV, was generated from its wild-type SA14 strain by serial passage in certified, pathogen-free primary hamster kidney cells and animal species, followed by plaque purification on primary chick embryo cells [31]. The vaccine is now being produced in accordance with the WHO guidelines for production of live JE vaccines for human use [32]. Millions of doses have been prepared by several companies for distribution within China, although Chengdu Institute of Biological Products is the sole export manufacturer. The vaccine has been in use for almost 20 years in different parts of China without any reports of severe side

5´-NCR 3´-NCR

C

C

prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5

Host signalase Unknown protease Viral serine protease Golgi–Furin protease

Structural region Nonstructural region

~3400 aa long polyprotein

Figure 1. Japanese encephalitis virus genome organization and the polyprotein processing. The approximately 11-kilobase genomic RNA contains a type I cap at the 5´-end but no polyA tail at the 3’-end. The RNA has a single open-reading frame that is flanked by noncoding regions at the 5´- and 3´-ends. The genome codes for an approximately 3400 amino acid polyprotein, which is processed into three structural (C, M, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins. The ‘C’ at the end of the structural region indicates the 5´-cap and the sites for different proteases involved in polyprotein processing are marked. aa: Amino acid; C: Capsid; E: Envelope; NCR: Noncoding region; NS: Nonstructural; prM: Premembrane.



effects in vaccinees [33]. The main advantage of this vaccine is the requirement of a single dose to generate long-lasting neutralizing immunity in 85–100% of nonimmune vaccine recipients [34]. Several studies within and outside of China have demonstrated a protective efficacy of greater than 95% and a recent follow-up study conducted in Nepal, a JE-endemic area in Asia, reported an efficacy of 98.5% even at 12 months postimmunization (p.i.) [21,34–36]. In addition, the vaccine induced cross-protective immunity, a major attribute of any live vaccine, against a broad spectrum of genotypes circulating in Asia [37]. Moreover, no major vaccine-induced side effects or hypersensitivity reactions were recorded in a study conducted in more than 1 million chil-dren and, accordingly, the WHO Global Advisory Committee on Vaccine Safety recently acknowledged its excellent safety and efficacy [38]. A recent study in children in the Philippines demon-strated that administration of SA14-14-2 JE vaccine along with measles vaccine had no side effect on the immunogenicity of either of the vaccines [39]. Although approval from the WHO is awaited, national regulatory authorities of China, South Korea, India, Nepal and Sri Lanka have already licensed the vaccine for use in their respective countries.

IXIARO® (IC51) vaccineTo date, Vero is the only WHO-certified cell line for produc-tion of human vaccines. IXIARO® (also known as IC51), is a recently developed JE vaccine based on the SA14-14-2 strain of JEV adapted to grow on Vero cells [22,40]. The vaccine is manu-factured by Intercell Biomedical (Livington, UK) and distributed by Novartis Vaccines and Diagnostics Inc. (Cambridge, MA, USA). The Vero cell-derived vaccine virus is purified on sucrose gradient and inactivated with formaldehyde. The virus is then adsorbed onto aluminum hydroxide adjuvant to prepare the final vaccine formulation. Each dose of the vaccine is a 0.5-ml suspen-sion supplied in syringes and formulated to contain approximately 6 µg purified, inactivated JEV and 250 µg aluminum hydroxide, without any chemical stabilizers and preservatives. The vaccine, licensed in 2009 in Europe, the USA and Australia for the adult population, is recommended to be given intramuscularly in a two-dose regimen, administered 28 days apart.

The efficacy trials conducted in adult populations using the two-dose regimen observed higher seroconversion rates as well as higher geometric mean neutralizing antibody titers in groups who received IXIARO JE vaccine compared with those who received the regular regimen of JE-VAX. Moreover, the vaccine was capa-ble of inducing long-term immunity, and approximately 95 and 83.4% of vaccinees seroconverted at 6 and 12 months p.i., respec-tively. However, one-time administration of 6 µg or a higher dose of 12 µg vaccine virus showed poor seroconversion rates (37.9 and 58.8%, respectively) compared with a two-dose regimen (97.3%) on day 35 p.i. [8]. These studies warrant the use of a two-dose regimen for higher immunogenicity of IXIARO JE vaccine. In addition, the vaccine was found to be compatible for administra-tion concomitantly with other childhood vaccines such as hepa-titis A vaccine, and no significant cross-vaccine interference was observed, both in terms of vaccine efficacy as well as safety [41]. In

a large-scale, placebo-controlled field trial of more than 2000 sub-jects to address the safety of IXIARO, no significant difference in the adverse events between the vaccinated and placebo-treated groups were recorded, further demonstrating the excellent safety of IXIARO JE vaccine [8]. Together, these studies demonstrate that IXIARO is either superior to or on a par with JE-VAX and just two doses of vaccine can induce nearly 100% seroconversion in vaccinees. However, owing to the absence of detailed clinical safety and immunogenicity data in children, IXIARO is not yet licensed for use in children. In this direction, a recent clinical trial conducted in 60 healthy Indian children between 1 and 3 years of age, administered 3 or 6 µg of the vaccine virus per dose using a regular two-dose regimen, reported seroconversion rates of 95.7% (3 µg/dose) and 95.2% (6 µg/dose) on day 56 p.i. (28 days after the administration of the second dose). Importantly, these rates were higher compared with that obtained with MBDV (90.9%). The study found the safety of IXIARO JE vaccine to be compara-ble to that of the currently licensed JE vaccine, without any severe vaccine-related side effects. The data from this study are promising and recommend the development of a 3-µg dose of IXIARO JE vaccine for children below the age of 3 years [42].

IMOJEV vaccineThe 17D204 strain of yellow fever virus (YFD; YFV17D) has been extensively used as a live-attenuated vaccine against yellow fever (YF) in humans for over 60 years with an excellent record of safety and immunogenicity. The vaccine virus was derived from the wild-type YFV Asibi strain through several rounds of passages in monkeys, mosquito cells, mouse and chicken embryo cells [43]. Inoculation of a single dose of this vaccine generates neutralizing antibodies and protective immunity in nearly 100% of the vaccinees [44]. The vaccine manufacturing process is very well established and the WHO-recommended vaccine is licensed for human use worldwide. Although YFV17D vaccine is associ-ated with very rare cases of viscerotropic disease in the vaccinees that are mostly related to host factors, YFV17D is an ideal vector for making recombinant chimeric virus vaccines due to its long history of safe and effective vaccination [45–48].

Several chimeric viruses have been developed based on YFV17D using a novel platform known as ChimeriVax technology [49]. The technology was developed by Chambers and coworkers at St. Louis University (MO, USA), where the first YFV17D-based chi-meric virus expressing JEV proteins was constructed in 1999 [50]. This chimeric virus was further developed as a vaccine candidate (ChimeriVax™-JE, now known as IMOJEV) by Guirakhoo and coworkers at Acambis, USA, which has now been taken over by Sanofi Pasteur [23] (Box 1). The ChimeriVax platform uses recom-binant DNA technology to replace the cDNA-encoding prM and E proteins of YFV17D with that of the heterologous flavivi-rus. The prM and E cDNAs are either directly derived from the wild-type/attenuated strain or taken from the wild-type and later genetically modified by mutagenesis.

Yellow fever is caused by YFV-induced pathology in the liver, spleen and kidney, and the critical attenuation event in the YFV17D vaccine is loss of viscero tropism. A number of



studies suggest that the prM–E region of flaviviruses has a role in tissue tropism and its virulence. For example, substitu-tion of YFV17D prM–E with Asibi prM–E resulted in enhanced viremia and virus titers in liver and spleen [51]. Similarly, reversion of certain residues in YFV17D E protein to those in Asibi led to increased visero tropism of YFV17D [52]. In addition, chimeric YFV17D expressing JEV (Nakayama) or Modoc virus prM–E had increased neuro invasiveness than YFV17D [23,53]. Accordingly, the ChimeriVax technology involves replacement of prM–E, a region critical for determining the tissue tro-pism in YFV17D, with that of a second flavivirus; this may or may not affect vis-cerotropism or neuroinvasiveness of the chimeric virus.

Several flavivirus vaccines based on the ChimeriVax technology are in various stages of clinical development. IMOJEV is the first YFV17D recombinant vaccine to be tested in humans. The vaccine virus was constructed by replacing the cDNA encoding the prM and E proteins of YFV17D with that of the live-attenuated SA14-14-2 strain of JEV that is extensively used as JE vaccine in China and more recently in some Asian countries such as India and Nepal [23,50]. During the proc-ess of development of IMOJEV, a series of viruses were constructed and evaluated in animal studies. Two chimeric viruses, pilot JE-ChimeriVax (CV) and large-scale JE-CV, were selected for further evalua-tion in human clinical trials. Pilot JE-CV was not plaque purified and was used in Phase I/II clinical trials [54]. However, for large-scale studies in Phase II and Phase III trials, plaque-purified, large-scale JE-CV produced in serum-free (SF) Vero cells was used. The large-scale JE-CV was produced by transfection of SF Vero cells and pas-saged to establish genetic stability before plaque purification. This virus was found to contain an amino acid mutation at position 60 in the M protein, which was actually identified in the virus after passage 5. It was thought to be an adaptation by the virus to grow on SF Vero cells, since the muta-tion was stable throughout the vaccine development process. Importantly, the selected clone carrying the amino acid muta-tion at position 60 in the M protein grew to high titers, without any loss in neuroattenuation phenotype and immunogenicity compared with the original chimeric virus [55]. IMOJEV is a lyophilized formulation of the large-scale JE-CV.

The E protein of JE-CV differs from its wild-type E protein derived from the SA14 and Nakayama strains of JEV at ten amino acid positions, and at least six of them (107, 138, 176, 279, 315 and 439) were predicted to be the determinants of neuro attenuation of SA14-14-2 [56]. To determine the importance of these residues in the neuroattenuation of JE-CV, attempts were made to system-atically revert the E protein back to the wild type. This resulted in the production of a series of revertants, which were classified into three groups based on their neurovirulence in mouse models: attenuated (zero mortality), sublethal (13–38% mortality) and

Box 1. Preclinical and clinical development of IMOJEV®.

Development

• First constructed by Chamber’s group in 1999 [50] and later developed as vaccine candidate by Guirakhoo’s group [23]

• Constructed by replacing the prM and E-encoding cDNA of YFV with that of the SA14-14-2 strain of JEV

• Contains ten amino acid mutations in E protein compared with wild-type JEV and at least six of them are responsible for neuroattenuation

Preclinical trials

• JE-CV has been tested in the mouse and monkey models of JE

• 100% protection against live JEV challenge was observed in mice

• Passive immunity protected mice from challenge with all the four JEV genotypes

• Cross-protection in chimeric vaccine immunized mice from MVEV challenge

• Low viremia in monkeys after subcutaneous administration

• JE-CV-immunized monkeys protected from live JEV challenge

Clinical trials

• Clinical trials completed in adults in the USA and Australia

• Clinical trials in the pediatric population in India, Thailand and Philippines are ongoing

• The data are consistent with that observed in monkey models, suggesting the utility of monkey models to predict the clinical behavior of chimeric viruses in humans

• Seroconversion rates of >95% are observed in adult populations and children

• JE-CV compared well with MBDV in all the noninferiority comparative trials

• IMOJEV generated rapid neutralizing immunity within 14 days after a single-dose administration in adults

Adverse events

• Approximately eight safety clinical trials have been completed in adults

• Adverse events are mild-to-moderate and no serious adverse events have been recorded

• Common systemic adverse events include headache, fever, loss of appetite, malaise, fatigue, diarrhea and myalgia

• Local reactions include injection-site pain, irritability, injection-site erythema and edema

Safety issues

• The high fidelity of YFV RNA polymerase probably ensures the genetic stability of chimeric viruses and hence their inability to revert to virulent viruses

• JE-CV cannot disseminate in mosquito vectors and hence cannot be transmitted in the environment

• Artificial recombinants were attenuated and were similar to YFV17D

• JE-CV grows locally and was not found to grow in any visceral organs

• JE-CV is highly neuroattenuated in mouse brain and is not neuroinvasive

• Some of the chimeras carrying the envelope sequences from the virulent wild-type flaviviruses were pathogenic

CV: ChimeriVax™; E: Envelope; JE: Japanese encephalitis; JE-CV: Japanese encephalitis ChimeriVax; JEV: Japanese encephalitis virus; MVEV: Murray Valley encephalitis virus; prM: Premembrane; YFV: Yellow fever virus; YFV17D: 17D204 vaccine strain of Yellow fever virus.



lethal (>89% mortality) revertants [57]. Further characterization of these revertants suggested that at least two reversions in the E protein were required to partially restore the neurovirulence, whereas four reversions at specific amino acid positions (107, 138, 176 and 177) made the chimera neurovirulent in suckling mice; however, the neurovirulence was less than that of YFV17D. Moreover, complete reversion of all the amino acids of the E protein to those present in the Nakayama strain did not result in virulence similar to that of the Nakayama strain; rather, it was similar to the parental YFV17D strain. These observations prove that use of the YFV17D backbone and the chimerization proc-ess together contribute to the stabilization of neuroattenuation property of the chimeric viruses and also suggest an extremely low risk of reversion to neurovirulence for the JE-CV used in the formulation of IMOJEV [23,57,58].

Safety & immunogenicity of JE-CV in animal modelsBefore being tested in human trials, JE-CV was tested both in vitro, in mouse and human cells, as well as in vivo, in mouse and monkey models, for its safety and immunogenicity (Box 1). As discussed earlier, the prM–E sequence of flaviviruses is important for tissue tropism. JEV is a neurotropic virus and thus YFV17D carrying the prM–E sequence from JEV must be tested for any enhanced neuro-virulence besides testing its vicerotropism. Although cytotoxicity assays may be performed in vitro in tissue-cultured cells, studies on genetic stability of chimeras, neurovirulence, vicerotropism, immunogenicity and challenge studies need to be performed in animal models, preferably in nonhuman primates to be able to correlate more accurately with the human system. Nonhuman primates, such as monkeys, have been used for testing the neuro-virulence of YFV17D-based recombinant flaviviral vaccines follow-ing the WHO guidelines for safety testing of YFV17D vaccine [59]. However, Monath and coworkers experimentally established the usability of infant imprinting control region mice as a suitable alter-nate for nonhuman primate models for testing the neurovirulence of live-attenuated flavivirus vaccine candidates [60]. This outbred imprinting control region mouse model was relatively more sensi-tive in detecting the differences in viral virulence compared with the monkey models. Mice administerd JE-CV subcutaneously (s.c.) produced low viremia and all mice survived after intracerebral inoc-ulation. In this mouse model, neuroattenuation of JE-CV is more pronounced compared with YFV17D vaccine virus. To further establish the neuroattenuation property of JE-CV, similar experi-ments were carried out in naive rhesus monkeys (Macaca mulatta) using 6.6 log

10 plaque-forming units (PFU) of chimeric virus [61].

Rhesus monkeys are sensitive to JEV infection, resulting in viremia, and were therefore used to assess biodistribution of JE-CV [59]. In these experiments, low viremia was observed for a maximum of 3 days, after which there was no detectable virus, and the virus loads were similar to those obtained with the parent YFV17D strain. Biodistribution studies in rhesus monkeys suggested that JE-CV was detectable only at the site of administration 3 days p.i. and was completely absent by day 21 p.i. However, no signs of infection of JEV target sites were seen in these animals [61]. The aforementioned studies, carried out in mice as well as monkeys, together established

the safety and the stability of JE-CV in preclinical animal models and also established the superior neuroattenuated phenotype of JE-CV compared with its parent YFV17D strain.

Studies on JE-CV immunogenicity and protective efficacy against wild-type virus challenge were carried out in mice as well as monkeys. Consistent with the high immunogenicity of YFV17D-based vaccines, high levels of immune responses to JEV antigens were observed in mice and, interestingly, a single dose of the vaccine (5 log

10 PFU) was sufficient to induce 100%

protection, whereas three doses of JE-VAX could protect only 87% of mice after challenge with approximately 2000 PFU (~158 LD

50) of virulent IC-37 JEV [23]. In silico ana lysis of JE-CV for

T-helper epitopes using EpiMatrix software, with reference to those present in the circulating JEV strains, suggested that the vaccine virus maintained most of the HLA-binding sites with minor differences, which was due to selection during attenuation of the SA14-14-2 strain [62]. Similarly, mice passively adminis-tered with the serum raised against JE-CV were protected against virulent JEV challenge. Importantly, the protection was against all of the four JEV genotypes circulating in nature [63]. Another study comparing the cross-protective immunity generated by JE-CV and JE-VAX found that JE-CV was able to protect 100% of mice challenged with Murray valley encephalitis (MVE) virus or West Nile virus (Kunjin strain), both belonging to the JEV sero complex, and the cross-protection conferred by JE-CV (100% for 105 PFU) was superior to that induced by three doses of JE-VAX (35%) [64]. These results support the use of live vac-cines, like IMOJEV, to induce cross-protective immunity against different flaviviruses. In addition, in the absence of an effective vaccine against the rarely reported MVE, immunization with JE-CV may help in reducing the encephalitis caused by both JEV and MVE virus in a setting like Australia. Similarly, monkeys immunized with a single dose of JE-CV generated high levels of anti-JEV neutralizing immunity and also protected the monkeys from virulent JEV challenge [61,65]. Thus, these data demonstrate the strong immunogenic potential of JE-CV, its ability to induce cross-protective immunity against different JEV strains as well as against viruses belonging to the JEV serocomplex, and also the genetic stability of the recombinant virus in different animal species. Successful outcome of these safety and immunogenicity studies on JE-CV encouraged the researchers at Acambis, now part of Sanofi Pasteur, in collaboration with other groups, to initiate clinical evaluation of the vaccine in human field trials.

Safety & immunogenicity of IMOJEV in humansWith successful completion of the preclinical studies using a single dose of IMOJEV, clinical studies were carried out, initially in the adult population, and subsequently in children of descending ages (Box 1). Several field trials have been completed in different geo-graphical regions for IMOJEV, including one Phase III safety and immuno genicity trial and one Phase III safety study [54,55,66,101–103]. These studies evaluated various issues related to safety and immu-nogenicity of IMOJEV and correlated the findings with those obtained in preclinical studies to answer some of the concerns over the genetically modified nature of the chimeric virus vaccine.



The most common concerns with respect to live-attenuated YFV-based vaccines are possible reversion to virulent viruses, spread of recombinants in nature and the risk of viscerotropism due to its YFV origin. In addition, issues related to possible recombination events with the virulent virus strains circulating in nature had been a great concern in light of the available evidence of recom-bination in flaviviruses [67–71]. YFV is transmitted by members of the Aedes genus, whereas JEV is transmitted primarily by members of the Culex genus. Following blood feeding by the mosquito, these viruses infect the epithelial cells in the mosquito midgut, replicate and disseminate further. There are several factors that limit human-to-mosquito or mosquito-to-human transmission. In a study to investigate the ability of JE-CV to replicate and dissemi-nate in different mosquito vectors known to transmit members of flaviviridae, the virus was administered through oral or intra-thoracic routes [72,73]. Consistent with YFV17D vaccine and other YFV17D-based chimeras, JE-CV replication was severely inhib-ited and failed to disseminate by the oral route. However, when administered through a thoracic route, the virus did replicate, but again failed to disseminate and transmit the virus. Moreover, s.c. inoculation of the vaccine virus resulted in limited replication in the human host and produced low levels of transient viremia [54,66]. Thus, low-level replication of the chimeric virus in the vac-cinee and the absence of virus replication and dissemination in the mosquito vector make the possibility of transmission of JE-CV in the environment difficult, although this needs further evaluation in long-term clinical trials in the future. Since mosquitoes have proven to be incapable of transmitting the virus, it is unlikely that the reservoir/amplifying hosts will be infected with JE-CV to start the transmission cycle. In fact, pig infection with JE-CV did not result in any detectable viremia, thus providing additional support to the in vitro data discussed above [55].

With respect to concerns regarding reversion to virulence and risk of recombination, considering the high fidelity of YFV RNA polymerase and the absence of virulence-enhancing mutations in the YFV17D genome isolated from the YFV17D vaccinees, it is highly unlikely that the virus would undergo changes at the four amino acid positions required for partial restoration of viru-lence [74–76]. Moreover, as discussed earlier, simultaneous reversion of all the ten amino acids to those found in wild-type SA14 virus also cannot make the chimeric virus neurovirulent and the result-ing virus would have properties similar to the parent YFV17D virus [23,57]. Sanofi Pasteur has experimentally investigated the pos-sibility of recombination events of chimeric viruses with their wild-type viruses and the consequences of such events. For this, the prM and E sequences from the chimeras were artificially replaced with that of wild-type virulent JEV or of highly virulent Asibi strain of YFV, from which the 17D virus was derived [23,77,78]. These viruses were found to be highly attenuated compared with their wild-type viruses both in vitro as well as in vivo, further confirming the nonhazardous nature of the chimeric YFV. However, there are a few exceptions to these observations with respect to recombining the envelope sequences of chimeras with those of the wild-type virulent strains. Thus, replacing the prM and E sequences of SA14-14-2 virus with that of Nakayama or replacing these sequences

of YFV17D with that of the Modoc virus (belonging to the not known vector cluster of the Flaviviridae family) resulted in the transfer of neuroinvasive properties of these wild-type viruses to the chimeric viruses [23,53]. Although the later studies using the enve-lope sequences from virulent strains may raise concerns over the possible threats due to recombination, the absence of such recom-bination events among YFV17D vaccinees for the last 60 years and the stability of the chimeric virus genome through several passages suggest the unlikeliness of a recombination event for JE-CV occur-ring. In addition, the presence of low-level viremia in vaccinated human adults and absence of the chimeric JE virus in any of its target tissues, including the brain, after s.c. inoculation of the virus in monkey models suggest the absence of viscero tropism as well as neurotropism for JE-CV [54,55,66]. Experience with JE-CV and other chimeric viruses indicates that these viruses are highly attenuated and show limited growth in the liver and kidney, a con-cern that is associated with the YFV17D backbone [79–82]. Overall, the historical safety profile of the YFV17D vaccine and the pre-clinical as well as clinical studies using the chimeric YFV vaccines conclusively demonstrate the in vivo safety of chimeric viruses.

Immunogenicity and protective efficacy of IMOJEV has been evaluated in human clinical trials. All of the clinical trials reported to date have been carried out in adult populations aged 18–84 years of age, while safety, immunogenicity and efficacy studies in pediatric populations are ongoing. As mentioned earlier, the first proof-of-concept trial was carried out in 36 healthy vol-unteers with (n = 18) and without (n = 18) immunity to YFV [54]. The seroconversion rates were 100% in both dosage groups, with geometric mean neutralizing titers ranging between 129 and 327 and more than 80% subjects showing low levels of viremia, which is consistent with that observed in YFV17D-vaccinated subjects. Interestingly, higher neutralizing antibody responses to JEV E protein were observed in individuals with immunity to YFV, compared with the YFV-naive population, after vaccination with ChimeriVax-JE. Similarly, viremia in YFV-immune individu-als was high. Whether this was due to the antibody-dependent enhancement of virus replication was not investigated. The safety profile of ChimeriVax-JE was similar to that observed for YFV17D vaccine and no severe adverse events were recorded in this study.

Following the success of the first Phase I/II study, several Phase II trials have been carried out in the USA and Australia to evaluate the safety and immunogenicity of IMOJEV in adults. A Phase II, non-inferiority comparative trial conducted in 99 subjects 18–59 years of age in the USA assessed the safety and immunogenicity of dif-ferent doses of IMOJEV, starting from 1.8 log

10 PFU up to 5.8 log

10

PFU. In this study, the ability of a single dose versus two doses of IMOJEV, administered 30 days apart, to induce durable cross-protective immunity (up to 12 months p.i.) was evaluated [66]. The study found that all the doses tested were well tolerated, with only minor adverse events and no serious adverse events. The low levels of viremia were consistent with that observed in pre clinical and clini-cal studies for YFV17D vaccine and IMOJEV. The sero conversion rates, measured by the 50% plaque reduction neutralization test (PRNT

50), were 100% in almost all the groups. In accordance

with the results obtained in earlier studies, there was no boosting



effect in antibody titers after the second dose of IMOJEV, which was probably due to the induction of strong sterilizing immunity by the first dose. The cross-protective neutralizing antibody responses were higher against the homologous virus compared with those against the heterologous JEV strains [66]. Interestingly, the presence of YFV immunity did not interfere with the immune responses to IMOJEV. Importantly, the study found that the observations were consistent with those obtained in rhesus monkeys and thus sug-gested that responses of nonhuman primates predicted the clinical behavior of IMOJEV in humans [66].

In a Phase III trial to evaluate the safety and tolerability of the vaccine conducted in more than 2000 adult volunteers in 2006, IMOJEV demonstrated excellent safety in the vaccinated subjects with minor adverse events, which were similar to those observed in the placebo group. To further establish the safety and immuno-genicity of IMOJEV, the vaccine was tested in yet another Phase III trial in a randomized, double-blind, multicentered study involving 820 adult volunteers [101–103]. The study compared the safety and immunogenicity of a single dose of IMOJEV with that of three doses of JE-VAX. The study was designed to administer either two doses of placebo (saline) and one dose of IMOJEV, or all three doses of JE-VAX. The serum samples collected from the vaccinees 30 days p.i. were then assayed for the neutralizing immune responses against the vaccine viruses. The study found that the vaccine met the pri-mary immunization end points (lot-to-lot variation) and the neu-tralizing immunity requirements of the study. Moreover, the study reported a seroconversion rate of 99.1% for IMOJEV compared with 95.1% seroconversion after JE-VAX administration, demon-strating statistical noninferiority of one dose of IMOJEV compared with three doses of JE-VAX. In addition, the chimeric vaccine induced rapid neutralizing immunity in approximately 93% of the vaccinees within 14 days p.i. Consistent with the previous Phase III data, the occurrence of systemic adverse events were similar in the IMOJEV and JE-VAX groups, whereas injection-site reactions were less frequently reported in the IMOJEV-administered population compared with JE-VAX. Thus, these data clearly support the ben-efits of a live-attenuated vaccine approach – single-dose adminis-tration, rapid immunity and high immunogenicity. Accordingly, Sanofi Pasteur has filed the application for the vaccine approval in Thailand and Australia and is awaiting registration.

Use of the ChimeriVax platform for expression of other vaccine antigensSimilar to IMOJEV, recombinant vaccines have been developed for other flaviviruses, such as tetravalent dengue vaccine and West Nile vaccine (ChimeriVax-WN or WN-CV) for use in humans [80,82]. The tetravalent dengue vaccine consists of four chimeric dengue viruses representing the prM and E cDNAs from all the four den-gue serotypes circulating in nature. These recombinant viruses were highly attenuated in human beings as well as in the Aedes mosquito species, confirming the clinical safety of the tetravalent dengue vaccine and the inability to be transmitted from vaccinated hosts to healthy individuals [83–85]. Although no mutations were introduced into the dengue 1–4 serotype sequences during the con-struction of chimeras, an amino acid substitution in the envelope

(K204R) was observed during the passage on Vero cells, which was later found to be responsible for the complete attenuation of the chimeric viruses in the brain as well as in liver and mosquito cells [86]. In addition, artificially constructed recombinants, pro-duced with the dengue 1–4 prM and E sequences and the virulent YFV Asibi backbone sequences (instead of the YFV17D back-bone), were found to be highly attenuated in mosquitoes, human liver cells and in nonhuman primates [51,79,82,84]. Moreover, the tetravalent dengue vaccine was found to be highly immunogenic in preclinical and clinical studies and the vaccine induced strong responses against all four serotypes [82,87–89]. However, immuniza-tion of nonhuman primates with the tetravalent dengue vaccine induced weak immune responses to less immunogenic serotype antigens compared with those against the immunodominant serotypes. These interfering factors were identified and alternate methods used to overcome these interfering effects were success-ful in inducing protective immunity against all four serotypes [90]. Similarly, two recombinant, chimeric West Nile vaccine viruses were made, one carrying the native prM and E sequence of the West Nile virus (ChimeriVax-WN01), which was used to develop a vaccine for horses, and the other with three mutations in the E sequence to produce a more attenuated virus for the development of ChimeriVax-WN02 for human use [80,91,92]. ChimeriVax-WN01, developed by Intervet (Boxmeer, The Netherlands), has been commercially available in the USA since 2006 for use in horses. ChimeriVax-WN02, on the other hand, has passed all the safety and immunogenicity tests in preclinical as well as in initial clini-cal trials [80,93,94]. A Phase II trial is warranted to test the vaccine efficacy and safety in a large number of volunteers.

Another chimeric flavivirus expressing the M2e peptide of influ-enza A virus has also been developed as a model for the insertion of for-eign immunodominant epitopes into flavivirus chimeric viruses [94]. The virus was constructed by randomly inserting the sequence encoding the M2e peptide (SLLTEVETPIRNEWGSRSNDSSD; mutated residues are underlined) into the NS1 sequence of JE-CV and selecting viable recombinants by plaque purification. Two amino acid mutations (C→S) were inserted into the M2e sequence to avoid the formation of disulfide bridges. All viable clones had the M2e sequence inserted after position 236. Upon serial passages, the selected virus clone became more cytopathic and showed larger plaque sizes in Vero cells. These changes were attributed to two amino acid changes in the NS1–M2e protein. The insertion of M2e into NS1 inhibited the in vivo dimerization of NS1, without affecting the virus replication and immunogenicity, but dimeriza-tion was restored by the two adaptative mutations selected by serial passages. When tested in mice models, the vaccine virus induced a strong IgG2a-type immune response, indicative of Th1 immu-nity, whereas the hepatitis B core protein–M2e subunit vaccine used for comparison induced Th2-type immunity. The immune responses elicited by the chimeric YFV-based influenza virus were, however, insufficient to protect mice against a virulent challenge with mouse-adapted influenza A/H1N1 virus, while five of the seven mice immunized with the subunit vaccine survived. Such a double chimera is likely to provide an immune response to two different antigens, although this was not investigated [94].



Expert commentaryJapanese encephalitis is a highly endemic disease in most Asian countries and is currently a major health concern in these settings. Although the availability of effective MBDVs reduced the disease burden in some of the Asian countries, these have limitations in terms of safety and availability. Moreover, the MBDVs requires multiple doses to induce long-term protective immunity, an issue which is practically not feasible. Furthermore, owing to the occur-rence of meningo–encephalomyelitis in some of the vaccinated individuals, the global JE vaccine manufacturer, BIKEN, has recently stopped production of JE-VAX, leading to a shortage in the MBDV supply. Recent advances in JE vaccine research resulted in the development of cell culture-based killed vaccine (IXIARO), live-attenuated SA14-14-2 vaccine and IMOJEV. IXIARO is in use in Europe and the USA, whereas the SA14-14-2 vaccine is licensed in some Asian countries. With respect to IMOJEV, its parent virus (YFV17D) has been in use as a vaccine for a long time with a proven record of safety and strong immunogenicity. IMOJEV is the first chimera to be developed using the YFV17D backbone by replacing the cDNA coding for the prM and E proteins of YFV with that of JEV. This vaccine preparation was at least as immunogenic and safe as the currently used MBDVs in both preclinical and clinical studies. Moreover, the vaccine is administered as a single dose in the adult population and this generates strong immunity in more than 95% of the vaccinees, which is equal to or better than the immunogenicity of MBDVs, depending on the study parameters. In addition, construction of artificial recombinants, in vitro testing for attenuation in different cell types and establishing the genetic stability to rule out the possibility of reversion to virulent strains have been carried out to answer some of the safety concerns related to live-attenuated IMOJEV and its genetically modified nature. As the etiology of rare cases of viscero tropic disease among YFV17D vaccinees was found to be host-related, more studies investigat-ing the possible risk of viscerotropism due to IMOJEV need to be carried out. Furthermore, the risk of recombination between the wild-type flaviviruses and the chimeric viruses, administered as vaccine antigens, need to be thoroughly investigated to rule out the possibility of evolution of pathogenic viruses, such as the YF/JE Nakayama or MOD/YFV17D strains that were neuroin-vasive in the host [23,53]. As YFV17D is contra indicated for use in immunodeficient individuals, it is unlikely that IMOJEV would be acceptable in this population.

With respect to the immunogenicity of IMOJEV, the vaccine was initially tested in adults to establish its safety and immuno-genicity and, recently, studies were initiated in pediatric popula-tions living in JE-endemic areas. Although these results are not yet published, information from various sources suggests successful completion of Phase II and Phase III trials in these regions. The safety and immunogenicity data obtained in these studies are highly promising, indicating that IMOJEV may be the correct replacement for MBDV. The most exciting attributes of IMOJEV are the requirement for a single dose to induce durable immunity and the mild adverse events, which are similar to that seen in placebo or control vaccine-treated subjects. Although proven to be effective in adult populations in several phases of clinical trials,

establishing the same in pediatric populations below the age of 15 years is necessary since JE is mainly a disease of children. In this direction, the outcomes of clinical trials being carried out in different parts of Asia are very important. With more such studies in the JE-endemic regions and with the cooperation of various health departments across Asia, the introduction of this vaccine into clinics seems to be a possibility in the next few years. Overall, accelerated efforts to introduce IMOJEV into clinics may save thousands of children living in JE-endemic regions. Since most of the JE-endemic regions are developing economies, attention should be given towards the affordability of IMOJEV in order to introduce it into the routine vaccination schedules in these regions.

Five-year viewWith the levels of success exhibited in clinical trials, the road ahead for IMOJEV in the next 5 years seems to be highly prom-ising. With proven safety and efficacy in adult populations over the last decade and due to the shortage in supply of the currently licensed MBDV, IMOJEV may be licensed for use in travelers visiting JE-endemic areas. Trials conducted in children in India, Thailand and the Philippines have also established the safety and immunogenicity of IMOJEV in the pediatric population. However, more such studies need to be conducted in children living in JE-endemic countries to convincingly establish the safety and efficacy of IMOJEV. Accelerated efforts from the industry to establish the safety and immunogenicity of IMOJEV in children less than 1 year of age, together with the cooperation from various health departments across the world, should help in the introduc-tion of this vaccine into the national immunization program of all the JE-endemic countries to effectively control the spread and incidence of JE.

As JE-CV was shown to protect immunized mice against MVE virus challenge, IMOJEV may find use in regions that are affected by other members of the JEV serocomplex, such as MVE in Australia. IMOJEV has worked very well in YFV- or JEV-exposed individuals perhaps due to the cross-reactive anti-bodies produced. As dengue is endemic in several parts of India and Southeast Asia that are experiencing frequent JE epidemics, it would be interesting to see how dengue antibodies affect the take of IMOJEV vaccine. It may also be of interest to study the effect of anti-JEV antibodies on the outcome of dengue virus infec-tion in the IMOJEV vaccinees. Work related to influenza vaccine development using the ChimeriVax platform has opened up the exciting possibility of using this system to develop nonflavivirus vaccines. This area is likely to recieve increasing attention as the ChimeriVax platform promises to be a versatile vaccine vector.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.


Vaccine Profile Appaiahgari & VratiAppaiahgari & Vrati

ReferencesPapers of special note have been highlighted as:• of interest•• of considerable interest

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Key issues

• Japanese encephalitis (JE) is a serious disease of the CNS caused by Japanese encephalitis virus (JEV), a flavivirus.

• Although JE can occur in all age groups, it is primarily a disease of children that can lead to serious neurological problems leading to encephalitis and death.

• JEV infections are reported frequently in the form of epidemics of encephalitis in most south and southeast Asian countries and the virus has become endemic in many of these regions.

• Mouse brain-derived vaccines are produced using the mouse brain-derived, formaldehyde-inactivated virus. The vaccine has limitations in terms of safety, availability and the multiplicity of doses required to induce protective immunity.

• Yellow fever vaccine based on the 17D strain of Yellow fever virus (YFV17D) has a proven record of safety and immunogenicity in humans.

• IMOJEV® is the first chimeric vaccine based on YFV17D by replacing the cDNA encoding premembrane and envelope proteins of YFV with that of JEV.

• Preclinical studies performed in mice and monkey models have established the safety and immunogenicity of ChimeriVax™-JE.

• Several clinical trials have established the safety and immunogenicity of IMOJEV in adult human populations.

• Phase II studies in JE-endemic regions have been carried out in children from India, Thailand and the Philippines, where IMOJEV was found to be safe and immunogenic.

• A single dose of IMOJEV is sufficient to provide a protective immune response that is similar to that generated by three doses of a mouse brain-derived vaccines.

• More studies need to be carried out in other JE-endemic settings in people of all ages to extend the recommendation of IMOJEV to other age groups.



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• DescribesthestrategyusedintheconstructionofJE-CV,thechimericYellowfevervirusexpressingJEVenvelopeproteins.ThestudyalsoreportedthebiologicalpropertiesofJE-CVincellcultureandinamousemodel.

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• EstablishedtheinabilityofmosquitoestotransmitJE-CVvirusfromtheimmunizedsubjectstothereservoir/amplifyinghostsand,thus,ruledoutthepossibilityofspreadofchimericvirusesintheenvironment.

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81 Monath TP, Levenbook I, Soike K et al. Chimeric yellow fever virus 17D-Japanese encephalitis virus vaccine: dose-response effectiveness and extended safety testing in rhesus monkeys. J. Virol. 74, 1742–1751 (2000).

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83 Higgs S, Vanlandingham DL, Klingler KA et al. Growth characteristics of ChimeriVax-Den vaccine viruses in Aedes aegypti and Aedes albopictus from Thailand. Am. J. Trop. Med. Hyg. 75, 986–993 (2006).

84 Johnson BW, Chambers TV, Crabtree MB et al. Growth characteristics of ChimeriVax-DEN2 vaccine virus in Aedes aegypti and Aedes albopictus mosquitoes. Am. J. Trop. Med. Hyg. 67, 260–265 (2002).

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89 Guirakhoo F, Kitchener S, Morrison D et al. 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. Hum. Vaccin. 2, 60–67 (2006).

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94 Rumyantsev AA, Zhang ZX, Gao QS et al. Direct random insertion of an influenza virus immunologic determinant into the NS1 glycoprotein of a vaccine flavivirus. Virology 396, 329–338 (2010).

• FirstattempttoconstructanonflaviviruschimeraexpressinganinfluenzaAviruspeptideusingtheChimeriVaxplatformvaccinevector.



Websites

101 Acambis commences paediatric clinical trial of ChimeriVax-JE in India. APM news www.apmhealtheurope.com/story.php?mots=JAPAN&searchScope=1&searchType=0&depsPage=4&numero=5823&ctx=6ce45e47ae9323371d1dc39b12537f2b

• ElectronicreportontheresultspertainingtotwoPhaseIIItrialsusingIMOJEV®.

102 New medical therapies – Vaccines. Center Watch www.centerwatch.com/clinical-trials/results/new-therapies/nmt-details.aspx?CatID=372

• ElectronicreportbriefingtheresultsobtainedinthesafetyandimmunogenicityPhaseIIItrialusingIMOJEV.

103 Vaccines for Emerging Infectious Diseases. The accomplishments of Acambis. Emerging Technologies 2006 http://adisonline.com/innovation/Fulltext/2006/04120/Vaccines_for_Emerging_Infectious_Diseases__The.4.aspx

• ThisreportontheaccomplishmentsofAcambisdiscussestheresultsobtainedinthePhaseIIIsafetystudyusingIMOJEV.

imojev ® : a yellow fever virus-based novel japanese encephalitis...

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