Vector-Borne Viral Infections
Japanese encephalitis (JE) is the most common cause of viral encephalitis in the Asian Pacific region. The virus exists in a transmission cycle between mosquitoes and pigs and/or water birds such as herons and egrets which are the main host reservoir. JE is therefore a mosquito-borne zoonotic viral infection, the reservoir of which is water birds and in which pigs play the role of amplifying host in rural areas. JE used to be mostly prevalent in countries with a temperate climate, including Japan, but data from tropical countries (Thailand, Cambodia, Indonesia) show that these zones also are favorable for JE transmission . Indeed, JE can now be found from the extreme south-eastern part of Russia to the North of Australia and Papua New Guinea, and from Japan to the west of India. Some 50,000 cases of JE occur annually, with 25%-35% case fatality rates, and more than 30 % severe long-term disabilities in survivors
Recent epidemics have led to increased demand for more effective, safe and affordable JE vaccines, leading to an extended registration and use of a live-attenuated vaccine that was previously largely confined to China. To meet increasing demand and to comply with GMP standards, a new plant for the vaccine is being built in China, which should become operational by 2010 and produce vaccines at GMP standards  . Together with the licensing of second generation JE vaccines, the global need for this vaccine should thus be met. It is anticipated that the mouse brain inactivated vaccine will be replaced by the less reactogenic and better controlled Vero cell-derived inactivated vaccine, which, together with the live attenuated SA14-2-14 vaccine and the live YF-JE chimeric vaccine hold great promise for programmatic use in developing countries. Their accelerated clinical development and evaluation has been facilitated by the WHO.
JE is endemic in parts of China and in Eastern, Southern and Southeastern Asia, and Papua New Guinea . In the 1990s, JEV spread westward into Pakistan and eastwards into the western Indonesian archipelago, New Guinea and northern Australia
. The mechanism by which the virus reached the Torres straight in 2000, perhaps through a migratory bird, remains unknown
. JE is principally a disease of rural areas in which vector mosquitoes proliferate in close association with birds and pigs. The spread of the disease into non-endemic regions has been correlated with agricultural development and intensive rice cultivation and the breeding of pigs supported by irrigation programs. The disease is currently considered hyperendemic in northern India and southern Nepal, as well as in parts of central and southern India.
JE is the most important cause of acute encephalitis in eastern and southern Asia and carries with it a heavy burden of permanent neuropsychiatric sequelae. The figure of 50 000 cases of illness a year probably is an underestimate, because of inadequate surveillance and reporting and because most infections are asymptomatic, with a ratio of symptomatic to asymptomatic infections that can range from 1 in 25 to probably 1 in 250 infections  . In rural villages, exposure and infection occur at a very early age with half of all cases occurring in children less than 4 years of age. Typical incidence rates in those younger than 19 years range from 10 to 100 per 100 000 population per year  . Seroprevalence studies indicate nearly universal infection by early adulthood in those areas  . Transmission of JE is mostly seasonal in temperate areas, but year-round transmission is seen in Indonesia  . Large outbreaks of JE with clear summer seasonality  also are periodically reported on the Indian subcontinent, as illustrated in Uttar Pradash, where 6097 suspected cases, including 1398 deaths, were reported between July 1 and November 10, 2005.
Needless to say, JE also poses risks to travelers and expatriates and to military personnel deployed overseas.
The JE virus, JEV, belongs in the Flaviviridae family, where it forms a common serogroup with West Nile, Kunjin (a subtype of WNV), Murray Valley encephalitis and Saint Louis encephalitis (SLE) viruses. All members in the serogroup have avian vertebrate hosts and are vectored by Culex spp mosquitoes. JEV has also occasionally been recovered from Aedes spp mosquitoes. The virus is a 40-50 nm enveloped, positive single-stranded RNA virus, with an isometric 30 nm nucleocapsid core. The envelope is spiked with a mature membrane (M) protein and a glycosylated envelope (E) protein which is stabilized by disulfide bonds and comprises three domains (I, II and III) involved in antigenic properties, cell receptor binding and penetration of the virion into the host-cell. The 10 976 bases long single-stranded viral RNA encodes an uninterrupted open reading frame that is translated into a polyprotein precursor eventually processed into capsid (C) pre-M and E structural proteins and into seven non-structural proteins (NS1-NS5). All known JEV isolates, although comprising five genotypes, belong to a single serotype
The control of JE is based essentially on three interventions: mosquito control, avoiding human exposure to mosquitoes and immunization. Mosquito control has been very difficult to achieve in rural settings and avoidance of exposure is difficult as Culex mosquitoes bite during day time. Immunization is the only effective method for sustainable control. Routine immunization of school-age children is currently in use in Korea, Japan, China, Thailand and Taiwan. The introduction of the JE vaccine into the Expanded Program of Immunization has helped curb the disease in countries like Thailand, Vietnam, Sri Lanka and China
Among the currently available vaccines is a formalin-inactivated vaccine derived from mouse brain-grown JEV strain Nakayama   , which still is produced by manufacturers in Korea, Thailand and Vietnam. The vaccine is relatively expensive, requires three doses on days 0, 7 and 30, followed by a booster at 1 year and thereafter at intervals of 3 years. The vaccine can often generate neurological adverse reactions. In addition to local and systemic side effects, individual cases of generalized urticaria and angioedema were reported in about 1 case per 1000 vaccinees after vaccination of travelers from western countries.
Another formalin-inactivated JE vaccine is prepared in China using the JEV P3 strain propagated in primary hamster kidney-cell cultures. The vaccine appears to be more immunogenic than that based on the Nakayama strain and can be integrated into the routine childhood immunization schedule but is not distributed outside of China. It is now largely being replaced by the live attenuated vaccine.
Several attempts are in progress to prepare inactivated JEV vaccines starting from virus grown in controlled cell line cultures. Several manufacturers are developing Vero cell-derived purified inactivated JE vaccines, either using the virulent Nakayama strain, as done by Japanese manufacturers, or starting from the attenuated SA14-14-2 JEV strain, as done by the Austrian biotech company Intercell. Phase I and Phase II clinical trials have shown that the vaccine was safe and immunogenic  and a Phase III trial was recently completed  . The Japanese vaccine candidates have been recently licensed in Japan, while the Intercell vaccine, Ixiaro, was licensed by the US FDA for adults. A two-dose rapid immunization schedule has been worked up for administration to travelers. Most people immunized with the Intercell vaccine developed protective neutralizing antibody levels that lasted for at least one year   and the vaccine was well tolerated   . The company pursues a separate clinical development for pediatric indication for endemic countries in a joint venture with Biological E, an Indian manufacturer. A large pediatric Phase IIb trial is currently taking place in endemic settings in India. Similarly, a Vero cell inactivated vaccine is now being produced in China by the Beijing Institute of Biological Products.
Live attenuated vaccines
The live attenuated JE vaccine strain, SA14-14-2, which was obtained after 11 passages in weaning mice followed by 100 passages in primary hamster kidney cells, has been developed and used in China since 1988. The vaccine, which is produced by the Chengdu Institute of Biological Products in China, was licensed in recent years in several Asian countries and was extensively used from 2006 to 2008 in mass immunization campaigns in India. Although the product is not WHO prequalified at this time, much investment and efforts have been made to bring the production and quality control to international standards. The vaccine is produced on primary hamster kidney cells, lyophilized, and administered to children at one year of age and again at two years, in annual spring campaigns  . Initial observational studies in southern China involving more than 200 000 children had demonstrated the vaccine safety, immunogenicity (99-100% seroconversion rate in nonimmune subjects) and protective efficacy over 5 years . The short-term effectiveness of a single dose of SA14-2-14 was demonstrated in 2001 in a case control study on Nepalese children where an efficacy of 99.3% was reported  . A five year follow-up study found the single-dose efficacy was maintained at 96.2%  . Another five-year follow up study showed that neutralizing antibody persistence was close to 90% at 4 years and 64% at 5 years after a single-dose of the vaccine in adult volunteers  . Recent studies in the Philippines have demonstrated the safety and efficacy of the vaccine even when co-administered with measles vaccine at 9 months of age. Similar studies in Sri Lanka and Indonesia will help confirm these findings in other Asian settings .
Currently, more than 30 million doses of the live SA14-2-14 vaccine are distributed annually in southern and western China and exported to Nepal, India and Korea. Starting in May 2006, the SA14-2-14 live attenuated vaccine was used in India to vaccinate 9.3 million children in 11 districts scattered among 4 states where JE was considered as highly endemic. More than 500 adverse events were reported during the campaign, including 66 severe AE, of which 22 were fatal. These cases were reviewed by an expert committee which concluded that none of the deaths were attributable to the vaccine. The severe adverse events and critical press coverage nevertheless had a deep negative impact on vaccine acceptance in the rest of the country, highlighting the need for proper safety monitoring and case investigations.
A promising approach for a future JE vaccine has been the construction of a YF-JE chimera based on the attenuated 17D YF virus genome, in which the YFV sequences encoding viral structural proteins prM and E were replaced by the corresponding prM and E sequences from JEV strain SA14-2-14. The resulting YF-JE chimeric virus, ChimeriVax-JETM, developed by Acambis and now licensed to Sanofi Pasteur, was grown on Vero cells and shown to elicit JEV neutralizing antibodies as well as protection against nasal and intracerebral virus challenge in rhesus monkeys    . The vaccine was tested in human adult volunteers in the USA, showing good safety and immunogenicity, with 94% of the vaccinees in the Phase II trial developing protective neutralizing antibody levels after a single dose  . The chimeric virus was shown not to replicate in mosquitoes which were fed the Chimerivax-JE vaccine  , a further proof of attenuation. The vaccine has been undergoing Phase III clinical trials in the USA and Australia for adult indication, whereas a parallel pediatric development program has been launched in Thailand by SanofiPasteur.
Live recombinant JEV vaccines
Replication-defective canarypox (ALVAC) and the highly attenuated vaccinia virus strain NYVAC were used as vectors to express the pr-M, E, NS1 and NS2a gene from JEV. The vaccine candidates were found to be well tolerated but their immunogenicity was too weak, especially in non-vaccinia immune volunteers, to warrant further development  .