Sadao Manabe

Application of the Arming System for the Expression of the 380R Antigen from Red Sea Bream Iridovirus (RSIV) on the Surface of Yeast Cells: A First Step for the Development of an Oral Vaccine

The cell surface is a functional interface between the inside and the outside of the cell. Moreover, cells have systems for anchoring surface specific proteins and for confining surface proteins to particular domains on the cell surface. For use in bioindustrial processes applied to oral vaccination, we consider that cell‐surface display systems must be useful and that the yeast Saccharomyces cerevisiae, the most suitable microorganism for practical purposes, is available as a host for genetic engineering because it can be subjected to many genetic manipulations. In particular, the rigid structure of the cell makes the yeast suitable for several of the applications. In this study, we describe the expression of one of the target antigens, 380R, from the red sea bream iridovirus (RSIV), which is one of the most common viral diseases in the cultured marine fish Pagrus major in Japan, using the arming yeast system and aiming at its application for oral vaccination. We first performed the molecular cloning and expression of the 380R antigen from RSIV in Escherichia coli. The nucleotide sequence of the 380R antigen was composed of an open reading frame (ORF) of 1360 bp encoding a protein of 453 residues. To prepare a specific antibody against the 380R antigen, the recombinant protein was overexpressed and purified in E. coli. As a result of indirect immunofluorescence with the specific antibody, we could observe the expression of the 380R antigen on the surface of the yeast cells. Thus, we have successfully prepared the source of an oral vaccine using cell‐surface display technology in yeast.

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.

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.

Characterization of neutralizing antibodies in adults after intranasal vaccination with an inactivated influenza vaccine

The levels and properties of neutralizing antibodies in nasal wash and serum collected from five healthy adults were examined after intranasal administration of an A/Uruguay/716/2007 (H3N2) split vaccine (45 µg hemagglutinin (HA) per dose; five doses, with an interval of 3 weeks between each dose). Prior to the assays, nasal wash samples were concentrated so that the total amount of antibodies was equivalent to about 1/10 of that found in the natural nasal mucus. Vaccination induced virus‐specific neutralizing antibody responses, which increased with the number of vaccine doses given. Neutralizing antibodies were produced more efficiently in the nasal passages than in the serum: A ≥4‐fold increase in nasal neutralization titres was observed after the second vaccination in four out of five subjects, whereas a rise in serum neutralization titres was observed only after the fifth vaccination. Nasal and serum neutralizing antibodies were mainly found in the polymeric IgA and monomeric IgG fractions, respectively, after gel filtration. Taken together, these results suggest that intranasal administration of an inactivated split vaccine induces high levels of nasal neutralizing antibodies (primarily polymeric IgA) and low levels of serum neutralizing antibodies (primarily monomeric IgG). J. Med. Virol. 84:336–344, 2012.

Development and evaluation of a formalin-inactivated West Nile Virus vaccine (WN-VAX) for a human vaccine candidate.

A formalin-inactivated West Nile Virus (WNV) vaccine (WN-VAX) derived from the WNV-NY99 strain was tested for its safety, efficacy, dilution limit for complete protection, and cross-neutralization. Safety tests performed with experimental animals, bacteria, or cultured cell lines showed no evidence of short- or long-term adverse effects. WN-VAX also protected 100% of 4-week-old mice against a lethal challenge from the WNV-NY99 strain after two doses of intraperitoneal inoculation-even when the vaccine was diluted to 3.2ng/dose. Moreover, very limited cross-neutralization activity against Japanese encephalitis virus, Dengue virus, Murray Valley encephalitis virus, Yellow fever virus or St. Louis encephalitis virus was observed. Therefore, the WN-VAX satisfies the requirements for human trials planned to be done in Japan.

Contribution of transcription factor, SP1, to the promotion of HB‐EGF expression in defense mechanism against the treatment of irinotecan in ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is a worst histological subtype than other ovarian malignant tumor. Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is a promising target for ovarian cancer therapy. The aims of this study were to validate the efficacy of HB‐EGF–targeted therapy for OCCC and to identify the transcription factor that contributed to the induction of HB‐EGF by SN38 treatment in OCCC cells. HB‐EGF was highly expressed in OCCC cells, and an increase of HB‐EGF was induced by SN38 which had only antitumor effect among conventional anticancer agents on OCCC. A specific inhibitor of HB‐EGF, a cross‐reacting material 197 (CRM197), led to a synergistic increase in the number of apoptotic OCCC cells with the treatment of SN38. The luciferase assay with 5′‐deletion promoter constructs identified a GC‐rich element between −125 and −178 (the distal transcription start site was denoted +1) as a cis‐regulatory region, and the treatment of SN38 induced luciferase activity in this region. An in silico and chromatin immunoprecipitation analysis estimated that SP1 bound to the cis‐regulatory region of HB‐EGF in OCCC cells. Real‐time PCR and cell viability assays showed that the transfection of a small interfering RNA targeting SP1 suppressed the expression of HB‐EGF induced by SN38, resulting in the enhanced sensitivity of SN38. Taken together, these results indicate that induction of HB‐EGF expression contributed to defense mechanism against treatment of SN38 through the transcriptional activity of SP1 in OCCC cells.

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.

Evaluation of varicella zoster virus-specific cell-mediated immunity by using an interferon-γ enzyme-linked immunosorbent assay.

BACKGROUND Administration of the varicella vaccine induces both varicella-zoster virus (VZV)-specific humoral and cell-mediated immunity (CMI). OBJECTIVE To assess VZV-CMI, we developed an interferon γ enzyme-linked immunosorbent assay (IFN-γ ELISA) that measures the quantity of total IFN-γ in culture supernatants of human peripheral blood mononuclear cells. STUDY DESIGN We evaluated this method by comparing the pre- and post-vaccination immune response in peripheral blood mononuclear cells of 30 healthy children who were administered an initial varicella vaccination at Konan Kosei hospital. RESULTS IFN-γ ELISA showed well-validated results; CMI was not detectable pre-immunization but became detectable post-immunization. Seroconversion was detected in 92.6% of subjects by the immune adherence hemagglutination test; however, half of the subjects did not display an increase in CMI levels. We also compared the incidence of breakthrough varicella and herpes zoster development between CMI post-positive and post-negative vaccinees at 1-2years after the last VZV vaccination. Eight subjects had a history of varicella or herpes zoster exposure post-VZV vaccination. Two of them with post-negative CMI contracted breakthrough varicella 15-16months after the last vaccination, even though they had sufficient VZV-specific antibody levels to be considered seropositive and seroprotected. Conversely, the others with post-positive CMI did not contract breakthrough varicella, despite experiencing extensive VZV exposure through casual contact with playmates and family. CONCLUSIONS The CMI data generated by this IFN-γ ELISA may accurately reflect real-world immune status, and CMI may be closely related to immunoprotection against breakthrough varicella development.

Characterization of antibody response to four different hepatitis C virus antigens.

We studied antibody responses to four different hepatitic C virus (HCV) antigens using sera collected from hemophilia patients. Three HCV antigens were expressed in E. coli which contained a fragment of the putative core, nonstructural (NS)3 and NS5 region of the genome, respectively. Antibody to each of these three antigens (core, NS3, NS5) was determined by enzyme-linked immunosorbent assay (ELISA). The remaing one antigen used was C100 developed by Chiron Corporation and antibody to it was also detected by ELISA. In 182 sera collected from 71 patients, seropositive rates for C100, core, NS3 and NS5 antigens were 79%, 950, 95% and 91%, respectively. Sequencial serum samples were obtained twice or three times at intervals of 1 to 4 years from 69 patients and were tested for antibody to four antigens. Forty seven patients were repeatedly seropositive and 4 were repreatedly seronegative for all of four antigens. One was seronegative for all of four antigens on first testing and became seropositive for core and NS3 after 1 year. At that point, antibody to C100 and NS5 was still negative. Eight were seropositive for three antigens except C100 on first testing and remained seropositive for core and NS3. The remaing 9 patients were seropositive for all of four antigens on first testing but lost antibody to C100 and/or NS5 on repeat testing. These results show that the antibody response to core and NS3 appears earlier and persists for a longer period than that to C100 and NS5, and suggest that detection of the antibody to core and NS3 is more useful method for serological diagnosis of HCV infection.