Development of the second generation Japanese encephalitis vaccine by recombinant DNA technology

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Studies were made to develop the second generation Japanese encephalitis (JE) vaccine by recombinant DNA technology. In order to obtain basic information for this objective, complete nucleotide sequence of JE virus was analyzed. The virus genome consisted of 10,976 nucleotides, of which 95 bases existed in 5' noncoding region, 585 bases at 3' noncoding region, and 10,296 bases formed a long open reading frame (ORT) between both noncoding region. The N-terminal amino acid sequences of the structural proteins of JE virus showed that the structural protein gene existed in the 5' 1/4 of the ORF in the order of C, PreM (precursor of M), and E. Sicne E protein is known to produce neutralizing antibodies which play most important roles in the protection of je, e protein gene was inserted into various recombinant plasmids or viruses in order to express the E protein in the cells transformed or infected with these plasmids or viruses. The expression of E protein was detected in recombinant yeast, mammalian cells, vaccinia virus and bombix mori nuclear polyhedrosis virus, respectively, however, the immunogenicity of these expressed.E protein was much lower than that of the current vaccine in terms of eliciting neutralizing antibodies. The reason of the low immunogenicity could be (1) amount of the expressed E protein, (2) stability of the E protein in the expressed cells, (3) dependence of immunogenicity on the highly conformational structure of the E protein, (4) purity of the e protein in the expressed cells used for immunization. These problems should be overcome by further studies to improve the levels of expression, purification of the expressed E protein, and trying to express the neutralizing epitope which is resistant to protein denaturation and exists near C-terminal of the E protein.