Iwao Yoshida

Development of immunogenic recombinant Oka varicella vaccine expressing hepatitis B virus surface antigen

Recombinant Oka varicella vaccine expressing hepatitis B virus (HBV) surface antigen (HBs) was constructed by inserting the HBs gene into the viral thymidine kinase (TK) gene and was examined for its immunogenicity in guinea-pigs. The HBs gene encoding 25 amino acids of preS2 and the whole of the S region was inserted into the TK gene of the cloned plasmid. The chimeric plasmid DNA and Oka varicella vaccine DNA were cotransfected and recombinant virus was isolated after immunofluorescence screening using a monoclonal antibody to HBs and a fluorescein-conjugated anti-mouse antibody. Expression of viral HBs was detected in the cytoplasm of infected cells and was stable over several repeated passages in vitro. The recombinant virus expressed 26K and 30K HBs molecules in infected cells and the culture supernatant contained 30K and 35K HBs molecules. HBs was purified at a density of 1.20 g/ml from the culture supernatants. The recombinant virus induced an antibody response to HBs as well as to varicella-zoster virus (VZV) in guinea-pigs, and the antibody titre to HBs was comparable to that induced by a recombinant HBs subunit vaccine produced in yeast. Thus a single dose of live recombinant Oka varicella vaccine could induce good immunity to VZV and HBs. The recombinant Oka varicella vaccine expressing HBs may be a good candidate for a combined HBV and VZV vaccine.

Development of inactivated vaccine against virus causing haemorrhagic fever with renal syndrome

B-1 virus belonging to the hantavirus group was serially passaged in the brains of newborn mice. Inactivated vaccine was prepared from the brains after inactivation with formalin and then purification by ultracentrifugation. The antigenic potency of this vaccine in vitro was determined by antibody-bound enzyme-linked immunosorbent assay (ELISA) and serial diluted vaccine bound to an aluminium hydroxide gel was inoculated into Balb/c mice to test immunogenicity. After two injections of this vaccine preparation, antibodies were detected in the mice by immunofluorescent, neutralizing and haemagglutination inhibition antibody tests. When mice immunized with this vaccine were challenged with B-1 virus and Hantaan virus (KHF-83-61BL strain), the virus titres in their lungs and spleens were significantly less than those in non-immunized mice. These results suggest that inactivated B-1 virus vaccine is effective against virus challenge by homotypic (B-1 virus) and heterotypic (Hantaan virus) viruses.

A Unique Mutation of Glycoprotein Gene of the Attenuated RC-HL Strain of Rabies Virus, a Seed Virus Used for Production of Animal Vaccine in Japan

Although the RC‐HL strain of rabies virus is avirulent in adult mice, the amino acid at position 333 of its G protein is arginine, which is thought to be necessary for virulence in adult mice upon intracerebral inoculation of the virus. This result suggests that besides arginine at position 333, some other positions of G protein might also be involved in determining the virulence of rabies virus.

The purification and characterization of an acellular pertussis vaccine

An acellular pertussis vaccine manufactured by Biken was investigated for purity, potency and toxicity. The vaccine was composed of almost equal proportions of pertussis toxin (PT) and filamentous hemagglutinin (FHA). The purity of the vaccine was 97-99%. The protective effects of component vaccines containing various ratios of PT and FHA were tested and it was found that the ratio of 1:1 provided the most effective vaccine.

Expression of the hepatitis B surface antigen gene containing the preS2 region in Saccharomyces cerevisiae.

We constructed a plasmid, pBH103-ME5, in which the region encoding the 10 preS2 amino acid residues and the S domain of the hepatitis B surface antigen (HBsAg) were regulated by the promoter of the yeast repressible acid phosphatase gene. Saccharomyces cerevisiae carrying pBH103-ME5 produced the HBs antigen (yHBsAg), when it was cultured in a medium containing a low concentration of phosphate. The antigen was purified to homogeneity. Its molecular weight was determined by Western blotting to be 24,000, and its amino acid composition agreed well with that deduced from the nucleotide sequence. The C-terminal amino acid sequence of yHBsAg was exactly the same as that predicted from the nucleotide sequence, while the N-terminal amino acid acetylserine, which was followed by 8 amino acid residues coded by the preS2 region. These results indicate that the recombinant yeast produced a single polypeptide consisting of the preS2 region and the subsequent S domain after being processed at the N-terminus.