| Japanese encephalitis (JE) is an acute viral infection of the central nervous system in humans, with an estimated 2-30 000 cases and of them 10% deaths annually, and about 15% of the survivors have severe neuropshychiatric sequelae in China. Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Asia. JEV remains a major public health threat. The formalin inactivated JE vaccine has several limitations: it is expensive to produce the vaccine, does not provide long-term immunity and multiple immunizations are needed to provide adequate protection, and may cause allergic reaction. For the risk of virulence reversion, the attenuated JE vaccine has not find wide acceptance outside the China. It is currently undergoing development for new types of JE vaccine.We evaluated the prophylactic efficacy of three genetically -engineered JE vaccine candidates in mice. These vaccines are HIV-1 gag virus-like participates carrying of JEV E protein (VLPs) expressed in baculovirus/insect cells, E protein particulates (EPs) expressed in drosophilia cells and DNA vaccine based on PrM/E gene of JEV. After immunized twice with the above neonatal vaccines, the cytotoxic T lymphocyte activities, anti-JEV antibodies and neutralizing antibodies in the immunized BALB/c mice were detected. The immunized mice were challenged with 10 LD50 JEV.The CTL activities were detected between 33% - 56% of mice immunized with 3 neonatal vaccines, compared to 22% in mice immunized with formalin inactivated vaccine, 17% and 18% for the vector and PBS control groups, respectively. Seroconversion was 90% (72/80) in vaccine groups after first immunization and 95% (76/80) after the second dose. Except for group pV/JE, mice immunized twice with the vaccines yield higher anti-JEV antibody than that of mice immunized once (P0.05). Mice immunized twice with VLPs emulsified with complete Freud's adjuvant (CFA) exhibited highest anti-JEV antibody detected by indirect immunofluorescence assay, and followed by group Eps+CFA. Both are significantly higher than that of pV/JE and FIV groups (PO.05). Neutralizing antibodies were also detectable in mice immunized with these neonatal vaccines. All immunized mice survived challenge with 10 LD50 JEV, compared to 37.5% mortality observed in the unimnumized groupDNA vaccine (pV/JE) in protecting immunized suckling mice against Japanese encephalitis virus was also evaluated. Sulking mice were immunized twice with the JEV DNA vaccine. The cytotoxic T lymphocyte activity andJEV-specific antibody were detected. And the immunized mice were challenged with 100 LD50 JEV. The results showed that the CTL activity in mice immunized with the DNA vaccine was 37.5 %, while 18% and 8.5% for vector and PBS control groups, respectively. DNA vaccine group revealed a seroconversion of 80% (8/10) with antibody 1:28.3 (geometrical mean titer). detected by immuno-flouresence assay. Neutralizing antibody of the mixed serum was also detectable (1:40) in mice immunized with the DNA vaccine. Six immunized mice survived from challenge with 100 LD50 JEV, compare with all mice dead in vector and PBS control groups. It revealed that the E protein epitopes of JEV have been functionally expressed in these engineered vaccine candidates. They induce stronger specific immune responses than a commercialized inactivated vaccine and demonstrated protective efficacy in mice against JEV infection. These contribute valuable data for investigation and production of JE neonatal vaccines.It is complicated in preparing VLPs and the risk remains for immunization of this kind of vaccine. There are different viewpoints on the safety of DNA vaccines. The EPs expressed in drosophilia cells is not sufficient for practical use. Pichia pastoris has been shown to express proteins glycosylated correctly in most of cases. It can be grown to high cell density and the regulation of expression by methanol is simple, easy to scale up and cost effective for industrial fermentation. It has been an excellent system...
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