The safety and immune efficacy of SA14-14-2 live attenuated Japanese encephalitis (JE) vaccine have been proved not only by studies in China［1,2］ but also confirmed by researchers of other countries.［3-6］ In recent decades, JE has gradually spread to many Asian countries that has alert WHO's great concern against JE. In WHO position paper, JE vaccination is concerned as one of the most important control. Currently an inactivated mouse brain-derived JE vaccine has been used in Asian countries. However a combination of adverse side effects, relative high expense, and a problem of insufficient supply has limited its wide distribution. Therefore an effective and saved live attenuated JE vaccine, developed in China, has drawn much concern of WHO .［7］ In order to study the immunogenicity of this live JE vaccine against JE wild virus strains isolated from different Asian countries, we report here the results of immune protection tests on mice.
SA14-14-2 live attenuated JE vaccine was produced by Chengdu Institute of Biological Products, China. The vaccine lot, 980107, had an infectious titer of 6.53 LogPFU/ml.
Nine strains from Asian countries orher than China were provided by American Center of Disease Control, others were collected and identified by NICPBP.
Children aged 12-14 years, from non-epidemic area ,were immunized with SA14-14-2 JE vaccine, 0.5 ml subcutaneously. Bleed after 14 days, serum were separated and pooled together.
Virus titration and neutralization test
Virus titer was measured by PFU method and neutralizing antibody was assayed by plaque reduction neutralization tests (PRNT).
Vaccination-challenge protection test on mice
SA14-14-2 JE vaccine was reconstituted with 2.5 ml diluent ,and then diluted by 10-fold to 10-3, 10-4, 10-5. 10 mice (Kunming strain) weighing 8-10 g were used for each dilution. Each mouse was inoculated with 0.1 ml vaccine of one of the diluted vaccine subcutaneously. Two weeks after immunization, each mouse was challenged intraperitoneally with 0.3 ml JE virus (about 1000-10000 LD50) and intracerebral inoculated with diluent simultaneously. Mice were observed for 14 days.
As shown in Tables 1 and 2 , even the immune virus dose was as low as 34 pfu, most of the protection rates against 22 virus strains were over 80%, except the P3 strain. Mice were protected. The protection rates to all the 22 challenge virus were 90%-100% when 340 pfu/0.1 ml virus doses were administered. This results showed good and broad-spectrum immunogenicity of SA14-14-2 live JE vaccine.
As shown in Table 3 , the sera from children immunized with SA14-14-2 live JE vaccine can neutralize all the 11 domestic and foreign virus strain.
As demonstrated previously in mice experiment, SA14-14-2 live JE vaccine induced a higher and broader protection against different Chinese JE virus isolates than the PHK-derived inactivated JE vaccine,［9］ Wills et al［10］ reported that mice immune serum, derived from immunization of SA14-14-2 live JE vaccine, could neutralize the JEV strains isolated from Japan, Malaysia, India, Vietnam, Thailand, Nepal and Indonesia, and Taiwan province, and demonstrated that live JE vaccine has broad immunogenicity.
In this study, the immunogenicity of SA14-14-2 live JE vaccine was studied in mice by vaccination-challenge protection test and by plaque reduction neutralization test (PRNT), indicating that SA14-14-2 live JE vaccine could protect 10 Chinese JE strains, one strain from Taiwan province of China, 3 strains from Japan and 8 strains from other five Asian countries, and the protection rates reached 90%-100%. Human immune serum could also neutralize 11 strains from China and other Asian countries. All the above-mentioned results strongly support the broad immunogenicity of SA14-14-2 live JE vaccine against JE viruses in many Asian countries. Although the neutralizing antibody titer is not high, the protective effect is strong, and it is consistent with previous animal tests, showing that mice immunized with SA14-14-2 live vaccine or mouse brain/PHK killed vaccines induced similar neutralizing antibody levels in the both groups of mice, but much higher protection effects was demonstrated in live vaccine immunized mice than that in the latter.［11］ Our previous studies［12］ also showed that guinea-pigs immunized with one single dose of SA14-14-2 vaccine induced low neutralizing antibody levels but viremia formation was total prevented after challenge with virulent JE virus. Besides, Lee et al［13］ reported that mice immunized with SA14-14-2 live vaccine induced much higher specific CTL response (79.2%) than did by the PHK killed vaccine (29%), although neutralizing antibody titers appeared similarly. Jia et al［14］ reported that the rates of protection in mice receiving transferred spleen cells from mice immunized either with live or killed vaccine showed significant difference, 50% protection by live vaccine & only 10% by killed vaccine. Therefore the protection effects of the live vaccine is considered to be induced not only by the neutralizing antibody but also by the cell-mediated immunity,which differs from the killed JE vaccines.
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