Establishment, validation and application of fluorescence focus assay for rapid titration of Japanese encephalitis virus / 中华微生物学和免疫学杂志

Objective:To establish and validate a fluorescence focus assay (FFA) for rapid titration of Japanese encephalitis virus (JEV) and to evaluate its application in the production of Japanese encephalitis vaccine.Methods:Recombinant JEV non-structural protein 1 (NS1) was expressed in a prokaryotic expression system. After purification, JEV-NS1 was used to immunize rabbits to induce polyclonal antibody. FFA was established with the polyclonal antibody to titer JEV. The accuracy of FFA was validated by comparing with plaque assay, and the specificity, precision, linearity, range and robustness of FFA were also validated. Twenty-eight batches of live-attenuated Japanese encephalitis vaccine were titrated with FFA and plaque assay to analyze the relationship between the two assays.Results:FFA established with polyclonal antibody against JEV-NS1 could be used to titrate JEV, and there was no cross reaction with other viruses (tick-borne encephalitis virus, yellow fever virus, coxsackievirus A2, coxsackievirus A4). Results of the validation tests showed that FFA met the requirement of quality control for live-attenuated Japanese encephalitis vaccine. FFA was more consistency than plaque assay.Conclusions:The established FFA could be used for virus titration in the production of live-attenuated Japanese encephalitis vaccine.

The Immunogenicity and Safety of the Live-attenuated SA 14-14-2 Japanese Encephalitis Vaccine Given with a Two-dose Primary Schedule in Children

Effective and tolerable vaccination is an essential strategy to prevent Japanese encephalitis (JE) in endemic areas. Although the live attenuated SA 14-14-2 JE vaccine (LAJEV) has been widely used since its introduction, the systemic data of LAJEV was very rarely available in Korea. We conducted the open-label, prospective cohort study to assess the immunogenicity and safety of this vaccine. Ninety subjects were enrolled, and LAJEV in a 2-dose primary series was given with a 12-month interval. Neutralizing antibody titers were measured before and after each vaccination, and active monitoring for adverse events was performed. After the first dose, 91.1% of subjects had seroprotection with a geometric mean titer (GMT) of 40.9. Seroprotection rate after the second dose was 97%, and GMT showed an increase of 6.5-fold. Most adverse events following immunization were self-limited, and no serious adverse events were reported until 42 days after each dose. The 2-dose administration of LAJEV in the primary immunization schedule appeared to be highly immunogenic and safe.

Chemical components analysis and morphology observation of virus in Japanese encephalitis vaccine produced from Beijing strain

The first 5 lots of Japanese encephalitis vaccines produced from Beijing-1 strain: JB011203, JB021203, JB030104, JB040204 and JB050304 were controlled of quality on chemical components and virus morphology in vaccine, using reference vaccine EJP034A (BIKEN-Japan). The results showed that: TCA-protein was 13.2- 14.5 µg/ml, the reference was 11.1 µg/ml (WHO standard 5-40 µg/ml). The thimerosal content was 82-84 µg/ml, the reference 76,3 µg/ml (WHO standard ≤120 µg/ml). The formaldehyde content was 0.061-0.063 µg/ml, the reference was 2.19 µg/ml (WHO standard ≤100 µg/ml). pH was 7.02-7.04, the reference was 7.10 (WHO stand. 6.8-7.4). The viruses morphology after negative stain were observed on electromicroscope JEM1010 with enlarge x 90.000 and x 150.000. The identical ball form of purified viruses with the intact surface antigen. 5/5 lots were passed the minimum requirements of biological products of WHO.

Evaluation of safety and potency of Japanese encephalitis vaccine produced by Beijing-1 strai

The pilot study was performed on the first 5 lots of the Japanese encephalitis vaccine produced by Beijing-1 strain: JB011203, JB021203, JB030104, JB040204 and JB050304 to evaluate their safety and potency. The quality control consists of abnormal toxicity, specific safety, pyrogen, sterility and potency test. All of 5 lots were passed the minimum requirements of biological products of Japan. The mice and guinea pigs were healthy, body weigh increased. Non of bacteria and fungi grown in both medium Thioglycholate and Soybeancasein. The pyrogen test on rabbits was passed in 5/5 lots. Summed temperature response of 3 rabbits per lot were 0°C-0.3°C (WHO requirement ≤1.4°C). Potency test was evaluated by PRNT compared with reference EJP034A of BIKEN. After the second immunization the neutralizing antibody response > reference from 0.04 to 0.2 log. 5/5 lots were passed the safety and potency test and met the minimum requirement of biological products of WHO.

Gianotti-Crosti Syndrome Following Japanese Encephalitis Vaccination

We report a three-year-old Korean boy who presented with itching symmetrical erythematous macules and papules on his face, trunk, and extremities for 1 week. Lymphadenopathies were detected on physical examination. He was vaccinated against Japanese B Encephalitis (JE) 1 day before developing skin rashes. The patient's serum JE antibody titer by hemagglutinin inhibition (HI) test was 1:40. Under the diagnosis of Gianotti-Crosti syndrome following JE vaccination, he was conservatively treated with an antihistamine agent, and his symptoms were all cleared 2 weeks after treatment.

Immune Response to SA14-14-2 Live Attenuated Japanese Encephalitis Vaccine

PURPOSE: SA14-14-2 live attenuated Japanese encephalitis (JE) vaccine has been administered safely and effectively to more than 100 million children in China since 1988, and recently licensure of the vaccine in Korea has been sought. Immune response to the vaccine was investigated. MEHTODS: In the first clinical evaluation of the vaccine outside of China, we monitored side effects in 93 children and evaluated plaque reduction neutralizing test (PRNT) antibody and IgM antibody responses to a single dose given as primary JE vaccination in 74 children, 1-3 years old (mean age 27 months). RESULTS: No significant adverse events were noted. PRNT antibodies (geometric mean titer [GMT] of 183) were produced in 96% of the 74 subjects. In 10 other children who previously had been immunized with two or three doses of inactivated JE vaccine, the booster administration of SA14-14-2 vaccine produced an anamnestic response in all, with a GMT of 3378. In a comparison group of 25 children previously immunized with two doses of inactivated vaccine, neutralizing antibody titers were detected in 16 (64%). Viral specific IgM was detected in nine primary vaccinees (13%) but in others, IgM may have declined to undetectable levels in the four week postimmunization sample. CONCLUSION: Live attenuated SA14-14-2 JE vaccine is a promising alternative to the only commercially available live attenuated JE vaccine for national childhood immunization programs in Asia.