Laboratory assays and field dengue vaccine evaluation at Ratchaburi province, Thailand: a preliminary result.

Laboratory-based epidemiological studies have been initiated in a cohort of school-aged children in Ratchaburi province, Thailand in preparation for imminent dengue vaccine field trials. In these studies, levels of neutralising antibodies were determined by the plaque reduction neutralisation test (PRNT) while virological confirmation was performed using RT-PCR or mosquito inoculation. Serological confirmation was performed using IgM and IgG ELISA. The incidence rate of dengue in the Ratchaburi cohort was 1635 per 100,000 during 2005-2006. Among 3547 patients in this cohort, 331 were classified with febrile disease and 58 with dengue. All four dengue serotypes were observed in Ratchaburi province. Efforts to identify the infecting serotype in symptomatic patients, based solely upon the neutralising antibody response, were complicated by cross- neutralising antibodies. In addition, 20 symptomatic and 49 asymptomatic cases were identified among 897 subjects investigated during 2005-2006, a ratio of 1:2.5. Serological analysis of asymptomatic dengue infections demonstrated boosting of immune responses due to subclinical infections with dengue or Japanese encephalitis. These results demonstrate that laboratory-confirmed dengue disease in areas of high transmission can be established. Epidemiological data of this kind are critical to dengue vaccine efficacy trials.

Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo.

Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys(179) (domain I), that for Fab B2 was Ile(126) (domain II), and that for Fab E3 was Gly(302) (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED(50)) of 0.84 microg, followed by MAb A3 (ED(50) of 5.8 microg) and then MAb E3 (ED(50) of 24.7 microg) for a 4-week-old mouse. Administration of 200 microg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.