Junko Namazue

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.

Comparison of antiviral assay methods using cell-free and cell-associated varicella-zoster virus

Assay methods for varicella-zoster virus (VZV) susceptibility to acyclovir (ACV) of VZV were compared by using cell-free (CF) and cell-associated (CA) virus of 6 x plaque-purified VZV. The 50% effective doses (ED50) of ACV, as required to reduce virus plaque formation by 50%, were about 8 times higher for CA virus than for CF virus. Also, the ED50 of 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU) for CA-VZV was higher than for CF-VZV, and fresh clinical isolates of VZV gave higher ACV ED50 values than CF virus. CA virus prepared at various times after CF virus infection showed a gradual increase of the ACV ED50 with time, ranging from the ED50 for CF virus to that for CA virus.

Induction of neutralizing antibody against varicella-zoster virus (VZV) by VZV gp3 and cross-reactivity between VZV gp3 and herpes simplex viruses gB

Glycoprotein gp3 (64K) is one of the major proteins specified by varicella-zoster virus (VZV). This glycoprotein was purified on an immunoadsorbent consisting of monoclonal antibody (clone 8) against gp3 linked to protein A-Sepharose. Rabbits were then immunized with the purified antigen to obtain monospecific antisera against gp3. The monospecific antisera and monoclonal antibody immunoprecipitated polypeptides with the same molecular weights of approximately 64,000 (64K), 106K, and 116K from a lysate of labeled cells infected with VZV. The monoclonal antibodies against gp3 did not have neutralizing activity against VZV, but anti-gp3 monospecific sera neutralized VZV infectivity. The antigenic relation of VZV to herpes simplex virus (HSV) was investigated by the immunofluorescent test, immunoprecipitation followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the neutralizing antibody test with monoclonal antibodies and monospecific antisera. In the indirect immunofluorescent test, the cytoplasm of cells infected with HSV-type 1 or HSV-type 2 was stained with anti-gp3 monospecific antiserum but not with anti-gp3 monoclonal antibodies. This serum also precipitated the polypeptides of HSV-type 1 and HSV-type 2 with molecular weight of approximately 120,000, possibly corresponding to gB of HSV-1 or HSV-2, and this immunoprecipitation was blocked by anti-gB monoclonal antibody. However, anti-gp3 monospecific antisera did not neutralize either HSV-type 1 or HSV-type 2 infectivity. These results suggest that gp3 induces neutralizing antibody against VZV and that it also has a cross-reacting antigenic determinant with gB of HSV.

Superior immunogenicity of a freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated).

Japanese encephalitis is an infectious disease caused by the Japanese encephalitis virus, which is widespread throughout Asia. The worldwide incidence is 50,000 cases per year. There is no specific treatment available, but inactivated mouse brain-derived vaccine was used from the 1950s to prevent infection. However, quality control of mouse brain-derived vaccines is difficult, and therefore a new freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated) (JEBIK V; development code: BK-VJE) was developed. In this paper, we report an analysis of neutralizing antibody titers in vaccinated subjects enrolled in clinical study of BK-VJE at various doses, and study of BK-VJE with the mouse brain-derived vaccine as a control. The results show that BK-VJE has superior immunogenicity compared to mouse brain-derived vaccine.

Immunosuppressive dose of azathioprine inhibits replication of human cytomegalovirus in vitro

SummaryAzathioprine (Aza) was found to have anti-human cytomegalovirus (HCMV) activity in vitro at concentrations used for immunosuppression therapy. The dose of Aza for 50% plaque reduction was 0.592µg/ml for HCMV in human embryonic lung (HEL) cells, but those of Aza for 50% plaque reduction for herpes simplex virus (HSV) and varicella-zoster virus were more than 20µg/ml. The dose of Aza for 50% reduction of the HCMV yield in infected cells was 0.25µg/ml, while that for 50% reduction of the HSV yield in infected cells was more than 50µg/ml. The dose of Aza for 50% growth inhibition of HEL cells was 30µg/ml, and 50.7 and 120 times greater than the doses for 50% reduction of the plaque formation and the yield of HCMV, respectively. Thus Aza was found to have a strong anti-HCMV activity at concentrations used for immunosuppression. When HCMV infected cells were treated with cyclosporine (CsA: 0.2µg/ml) and prednisolone (Pred: 0.3µg/ml) simultaneously with Aza, the doses of Aza for 50% reduction of plaque formation and the yield of HCMV were 0.73 and 0.32µg/ml, respectively. Thus an inhibitory effect of Aza was also observed in HCMV-infected cells treated with CsA and Pred at their concentrations used for immunosuppression. Maintenance of an anti-HCMV dose of Aza in combination with CsA and Pred might establish not only satisfactory immunosuppression but also suppression of HCMV infection in transplant recipients.

Processing of virus-specific glycoproteins of varicella zoster virus

Abstract Monoclonal antibodies to varicella zoster virus (VZV) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83K–94K (gp 2), 64K (gp 3), and 55K (gp 5). Immunoprecipitation experiments performed with VZV-infected cells, pulse labeled with [3H]glucosamine in the presence of tunicamycin, suggest that O-linked oligosaccharide is present on the glycoprotein of gp 2. Use of the enzyme endo-β-N-acetylglucosaminidase H revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of N-linked oligosaccharides. Experiments with monensin suggest that the precursor form (116K) of gp 3 is cleaved during the processing from Golgi apparatus to cell surface membrane. The extension of O-linked oligosaccharide chain and the complex type of N-linked oligosaccharide chains also occurs during this processing.

Safety and immunogenicity of a freeze-dried, cell culture-derived Japanese encephalitis vaccine (Inactivated) (JEBIK(®)V) in children.

Freeze dried, cell culture-derived Japanese encephalitis vaccine (Inactivated) (JEBIK(®)V) is approved for a three-dose primary immunization followed by a one-dose booster immunization in Japan. We conducted a multicenter, double-blinded, randomized controlled trial of the safety and immunogenicity of the vaccine in 370 healthy children who received three doses of 5, 2.5 or 1.25 μg of virus protein per 0.5 mL formulation subcutaneously. Children received two doses of test vaccine 7-28 days apart followed by a dose 6-12 months after the second vaccination. The three-dose regimen showed a good safety profile with no serious vaccine-related adverse events. Fever and reactions at the injection site were common adverse reactions at each dose of vaccine. The seroconversion rates were 100%, 99.2% and 95.0% after two doses in the 5, 2.5 and 1.25 μg groups, respectively, and 100.0% after three doses in all groups. The geometric mean titers were high for all three formulations after the second and third doses, with a very clear dose-response relationship. These results indicate that JEBIK(®)V is likely to be a useful vaccine.

Varicella infection complicated with meningitis after immunization

A healthy 45 month old boy who had received varicella vaccine 21 months previously developed aseptic meningitis along with an episode of varicella. The presence of viral DNA in cerebrospinal fluid (CSF) detected by polymerase chain reaction (PCR) with Southern blot hybridization confirmed the relationship between the symptoms and the CSF pleocytosis. This is the first reported case of this complication of varicella meningitis occurring in a child with documented immunization and seroconversion.

Evidence for attachment of fatty acid to varicella-zoster virus glycoproteins and effect of cerulenin on the maturation of varicella-zoster virus glycoproteins.

Varicella-zoster virus (VZV) contains four major glycoproteins designated gpI, gpII, gpIII, and gpIV. In the present study the presence and role of fatty acids in these glycoproteins were examined. The incorporation of fatty acid into the glycoproteins that span the membranes of VZV was studied. For this, virus-infected human embryo fibroblast cells were labeled with [3H]-palmitic acid, an extract of the cells was treated with monoclonal antibodies to VZV glycoproteins, and the precipitate was analyzed by SDS-PAGE. The electrophoretic pattern showed that gpI was heavily labeled and that gpII and gpIV were lightly labeled. Cerulenin, an antibiotic that inhibits de novo fatty acid biosynthesis, was used to examine the effects of fatty acids on replication of VZV and posttranslation processing of glycoproteins. Treatment of the cells with cerulenin significantly inhibited viral growth, but did not affect protein synthesis in the cells. Examination of processing of viral glycoproteins in these cerulenin-treated cells revealed the accumulation of precursor proteins and a decrease in the amount of mature glycoproteins. These data suggest that fatty acid acylation of VZV glycoproteins is necessary for formation of complete infectious virions.

Isolation of varicella-zoster virus from vesicles in children with varicella.

Varicella‐zoster virus (VZV) was isolated from 29 samples of the vesicular fluid in 13 otherwise healthy children with varicella who were aged from 7 months to 7 years. Human embryonic lung cells were used for viral isolation, and VZV was identified by a characteristic cytopathic effect and an indirect immunofluorescence assay. VZV was found in 17 samples; in two (12%) of which it was also detected after filtration (0.45 μm). The rate of isolation was 100% in the first two days after the onset of the disease. It declined gradually with time to 1 of 6 in the samples 6 days after the clinical onset. Specific IgG antibody to VZV was investigated in the same materials. The positive rate was 0% (0/13) in the first 3 days and increased to 7 of 16 in the following 3 days after the onset. VZV was not isolated from samples with specific antibody. In conclusion, VZV can be isolated easily from vesicles within the first 3 days of onset, but the filtration of samples affects its isolation. Infective VZV disappears gradually in vesicles after the first 3 days, and this may be related to the establishment of immune reactions including specific antibody.