Akihisa Takamizawa

Enhancement of immunity against VZV by giving live varicella vaccine to the elderly assessed by VZV skin test and IAHA, gpELISA antibody assay

The enhancement of immunity against varicella-zoster vaccine (VZV) by subcutaneous injection of a live varicella vaccine was assessed by the VZV skin test for cell-mediated immunity (CMI), and immunoadherence hemagglutination assay (IAHA) and gpELISA antibody assays in the elderly people of 50-79 years of age. A total of 127 subjects were examined: 79 aged 50-59, 25 aged 60-69, and 25 aged 70-79. All were seropositive by the gpELISA assay (one was seronegative in the IAHA antibody assay). In contrast, a notable decline was observed in the VZV skin test with increasing age. Negative reaction was observed in 16/79 (20.2%) of the subjects in their 50s, 12/25 (48.0%) in their 60s and 14/25 (56.0%) in the 70s. After the vaccination, the results of the VZV skin test changed from negative to positive in 15/16 (91.8%) of subjects in their 50s, 11/12 (91.7%) in their 60s and 12/14 (85.7%) in their 70s. The mean antibody titer in the IAHA and the gpELISA increased approximately two-fold after the vaccination in each group. Immunity to VZV in 35 elderly subjects who were vaccinated previously was followed up for 4 years. All were positive by the VZV skin test after the previous vaccination. After 4 years, 31 (88.6%) were positive by the skin test, 4 were negative and became positive after revaccination. Although this study was uncontrolled open study, the results suggest that administering live varicella vaccine to the elderly is effective for enhancing immunity, particularly CMI to VZV.

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.

Stable High-Producer Cell Clone Expressing Virus-Like Particles of the Japanese Encephalitis Virus E Protein for a Second-Generation Subunit Vaccine

ABSTRACT We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 μg per 104 cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 × 106 PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.

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.

Shortening of the window period in diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgG to p17 and reverse transcriptase in serum with ultrasensitive enzyme immunoassay

Following HIV infection, there is a window period of 6-8 weeks, during which HIV antibodies are not detectable and the infection cannot be diagnosed by methods for detecting HIV antibodies. However, HIV antigens are detectable in the latter part of the window period, although the level of HIV antigens declines as the level of HIV antibodies increases. We developed an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for the simultaneous detection of both p24 antigen of HIV-1 and antibody IgGs to p17 and reverse transcriptase of HIV-1 in a single assay tube and tested 11 HIV-1 seroconversion serum panels and serum samples randomly collected from 79 HIV-1 seropositive subjects and 100 HIV-1 seronegative subjects. The simultaneous detection was shown not only to shorten the window period significantly as compared with conventional methods for HIV-1 antibody detection but also to make possible a reliable diagnosis of HIV-1 infection from the time of seroconversion until late stages of the infection.

Expression of MBP-HCV NS1/E2 fusion protein in E. coli and detection of anti-NS1/E2 antibody in type c chronic liver disease

To characterize the putative NS1/E2 (non-structural protein 1/envelope 2) domain of HCV (hepatitis C virus), we expressed the hydrophilic three-quarters of this domain in a form of MBP (maltose binding protein) fusions in Escherichia coli. When we checked the positive frequency of antibody to this fusion protein, 17% of patients with type C chronic liver disease had this antibody. However, they were all positive for HCV-RNA in sera. These results suggest that the appearance of anti-NS1/E2 antibody does not serve as evidence of viral clearance.

Diagnosis of HIV-1 infection with whole saliva by detection of antibody IgG to HIV-1 with ultrasensitive enzyme immunoassay using recombinant reverse transcriptase as antigen.

Whole-saliva samples were collected from 45 asymptomatic carriers, 18 patients with AIDS-related complex (ARC) or AIDS, and 76 medical students by simple spitting with no stimulation and tested by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-HIV-1 IgG using recombinant reverse transcriptase as antigen and beta-D-galactosidase as label. With as little as 1 microliter of whole saliva, the lowest signals among the 45 asymptomatic carriers, 8 patients with ARC, and 10 patients with AIDS were 38-, 78-, and 3-fold, respectively, higher than the highest signal among the medical students. When the volume of whole saliva for test was increased up to 100 microliters, no significant effect was observed on signals for seropositive cases and signals for the medical students increased only very slightly. Therefore, whole-saliva samples containing extremely low levels of anti-HIV-1 IgG, even 2,000-fold lower than the lowest level among the 45 asymptomatic carriers tested, were considered to be discriminated from those of seronegative individuals. Thus, the sensitivity and specificity were expected to be both 100% with whole saliva even for a larger number of samples, although the number of samples tested was limited.

HCV RNA and antibody to HCV core protein in Japanese patients with chronic liver disease

We evaluated the prevalence of hepatitis C virus (HCV) RNA and antibody (anti-HCVcore) to the putative HCV core protein in Japanese patients with chronic liver disease. Sera were screened by solid-phase enzyme immunoassay with a recombinant HCV core protein and by the reverse transcription-polymerase chain reaction (RT-PCR) test which directly detects the HCV genome. Anti-HCVcore was detected with high titers in 95% (69/73) of chronic non-A, non-B hepatitis, and 94% (65/69) of anti-HCVcore-positive patients had the genome. Anti-HCVcore was also found with lower titers in 24% (10/41) of chronic hepatitis B virus carriers, and three of them had the genome. Only one (3%) of the 35 patients negative for anti-HCVcore tested positive to RT-PCR. These findings indicate the overwhelming prevalence of HCV infection in Japanese patients with chronic non-A, non-B hepatitis and a close relationship between the presence of anti-HCVcore and chronic hepatitis C in this population.

A lambda gt11-cDNA clone specific for chronic hepatitis C generated from pooled serum presumably infected by hepatitis C virus

SummaryA lambda gt11-random-primed-cDNA clone specific for chronic hepatitis C was isolated from pooled serum preumably infected by hepatitis C virus. The translation product of the clone detect 50% of patients with chronic hepatitis C in 4 test panels but none of patients with acute hepatitis C, other liver diseases or normal controls was positive for the peptide. The nucleotide sequence of the cDNA clone, the size of which is 66 bp, has no homology to the complete sequences of known human viruses such as adenovirus, coxsackievirus, rhinovirus, immunodeficiency virus type 1, Epstein-Barr virus, polioma virus, poliovirus, papilloma virus, parvovirus, papovavirus, varicella-zoster virus, yellow fever virus, endogenous retrovirus, T-cell lymphotropic virus types I, II, and III Japanese encephalitis virus, and hepatitis A, B, and D viruses. Probably only one or two epitopes are present on the molecule encoded by the clone as the peptide consists of only 22 amino acid residues.

Ultrasensitive and more specific enzyme immunoassay (immune complex transfer enzyme immunoassay) for p24 antigen of HIV‐1 in serum using affinity‐purified rabbit anti‐p24 Fab′ and monoclonal mouse anti‐p24 Fab′

Previously, an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for p24 antigen of HIV‐1 was developed. The immune complex comprising 2,4‐dinitrophenyl‐biotinyl‐bovine serum albumin‐rabbit anti‐p24 Fab′ conjugate, p24 antigen, and rabbit anti‐p24 Fab′‐β‐D‐galactosidase conjugate was trapped onto polystyrene beads coated with affinity‐purified (anti‐2,4‐dinitrophenyl group) IgG, was eluted with ϵN‐2,4‐dinitrophenyl‐L‐lysine, and was transferred to polystyrene beads coated with streptavidin. β‐D‐Galactosidase activity bound to the streptavidin‐coated polystyrene beads was assayed by fluorometry. This assay was highly sensitive. However, bound β‐D‐galactosidase activity had to be assayed for a long time (20 h), and the nonspecific signal was observed in 5% serum samples from subjects with low risk of HIV infection. In the present study, the assay time for bound β‐D‐galactosidase activity was shortened to 2.5 h by using 2,4‐dinitrophenyl‐biotinyl‐bovine serum albumin‐affinity‐purified rabbit anti‐p24 Fab′ conjugate and affinity‐purified rabbit anti‐p24 Fab′‐β‐D‐galactosidase conjugate. Furthermore, the nonspecific signal was found to increase with increasing periods of time for storage of serum samples at ‐20°C, and this increase was prevented without prolongation of the assay time for bound β‐D‐galactosidase activity and without loss of the sensitivity by substituting monoclonal mouse anti‐p24 Fab′‐β‐D‐galactosidase conjugate for affinity‐purified rabbit anti‐p24 Fab′β‐D‐galactosidase conjugate.