Hiroshi Mizokami

Non-clinical and phase I clinical trials of a Vero cell-derived inactivated Japanese encephalitis vaccine

The safety and effectiveness of a Vero cell-derived inactivated Japanese encephalitis (JE) vaccine were compared with those of a current JE vaccine in non-clinical studies and a phase I clinical trial. The single-dose toxicity study showed no toxicity of either the current JE vaccine or the investigational Vero cell-derived JE vaccine. In a local irritation study, the degree of irritation caused by both vaccines was determined to be the same as that induced by normal saline. To investigate genotoxicity, a chromosomal aberration test was conducted and the results were negative. Both JE vaccines were administered to a group of 30 subjects who were seronegative (neutralizing antibody titer <10(1)) for JEV virus (Beijing-1 Strain). Each subject was subcutaneously inoculated twice at an interval of 1-4 weeks, followed by an additional booster inoculation 4-8 weeks later, and clinical reactions and serological responses were subsequently investigated. Adverse drug reactions of local reaction, headache and malaise were mild, occurring at a rate of 6.7 and 20.0% after administration of the Vero cell-derived JE vaccine and the current JE vaccine, respectively. The seroconversion rate after three doses of both JE vaccines was 100%, while the geometric mean titer for the Vero cell-derived and current JE vaccines was 10(2.35) and 10(2.03), respectively. These results suggest that the safety and effectiveness of the Vero cell-derived inactivated JE vaccine are equal to those of the currently available conventional vaccine in humans, and that the Vero cell-derived vaccine could be a useful second-generation JE vaccine.

Purification and characterization of the hepatitis B virus core antigen produced in the yeast Saccharomyces cerevisiae

Hepatitis B virus core antigen gene was expressed in Saccharomyces cerevisiae and the product (yHBcAg) was purified from a crude lysate of the yeast by three steps: sucrose step-gradient ultracentrifugation, hydroxyapatite chromatography and CsCl-isopycnic ultracentrifugation. yHBcAg was synthesized in yeast cells as a particle consisting of polypeptides which have a molecular weight of 21.5 kDa (p21.5). In the CsCl-density gradient, yHBcAg particles synthesized with the expression vector pYG701c (the GAP promoter) had two peaks, at 1.35 g cm−3 (HP; high-density particle) and 1.31 g cm−3 (LP; low-density particle). On the other hand, the particles synthesized with expression vector pAC701 (the PHO5 promoter) had only one peak at 1.32 g cm−3. The isoelectric points of HP and LP were estimated to be 4.05 and 4.07, respectively. Absorption spectrum analysis showed that the HP contains nucleic acids. yHBcAg particles possessed the immunogenicity of HBcAg and its component polypeptide (p21.5) possessed that of HBeAg in addition to HBcAg. Moreover, Western blotting analysis of p21.5 using a monoclonal antibody against yHBcAg or yHBeAg indicated that the hepatitis B virus C-gene-coded protein shares the antigenic sites responsible for both antibodies.

A convenient method for preparation of the calcium ion-binding protein annexin V

This study presents a novel method for the production and purification of annexin V using cation exchange chromatography in the presence of calcium ions at neutral or alkaline pH. This method enables the handling of a large quantity of sample at one time without deterioration of the adsorption capacity of the cation exchange carrier by contaminating proteins, as well as the production of the calcium ion-binding protein annexin V with high purity on an industrial scale from both recombinant products and native products.

Development of a recombinant vaccine against infectious coryza in chickens.

Infectious coryza is an acute respiratory disease of chickens caused by Avibacterium paragallinarum, and this infection is associated with growth retardation and reduced egg production. Previous studies have shown that HMTp210, a 210-kDa outer-membrane protein, is the major protective antigen of Av. paragallinarum both serovars A and C. Region 2 is a serovar-specific domain in the HMTp210 protein. Although the serovar C region 2 has been reported to be an effective vaccine antigen for infectious coryza, there have been no reports on the efficacy of region 2 from serovar A. In the current study, region 2 from serovars A and C was expressed as a fusion peptide. Chickens inoculated with vaccine consisting of 0.6 μg of the fusion peptide showed no clinical signs of disease after challenge with either serovar A or C, and there were no side effects such as swelling at the injection site. These results demonstrate that the recombinant fusion peptide derived from HMTp210 could be useful for producing effective and safe vaccines against infectious coryza in chickens.

Large-scale production of major house dust mite allergen Der f 2 mutant (C8/119S) in Escherichia coli

Hyposensitization, in which causative antigens of allergic diseases are injected, is the sole means of a radical cure for allergic diseases. Since the therapeutic allergens currently used are naturally extracted, producing preparations with a stable titer from such extracts is extremely difficult. There are several reports on the expression of recombinant mite allergens in Escherichia coli using inducers. The use of an inducer for industrial production will lead to high costs and, for therapeutic use, it must be removed in the purification process. C8/119S is a mutant of Der f 2, a major house dust mite allergen. The C8/119S gene was integrated downstream of the trp promoter to produce the expression plasmid (pWU11-C8/119S). Then, this expression plasmid was used to transform E. coli strain HB101 (pWU11-C8/119S/HB101). A recombinant E. coli pWU11-C8/119S/HB101 did not express C8/119S in a low-temperature culture (32 degrees C), but C8/119S was induced to a high level of expression in a high-temperature culture (37 degrees C). pWU11-C8/119S/HB101 proliferated when expression was induced by high temperature and an approximately 3-fold greater proliferation was obtained compared with the use of an inducer in a large-scale culture. The C8/119S protein was expressed as inclusion bodies and obtained by refolding and chromatography purifications. The immunological properties of C8/119S were assessed by western blotting. Western blotting demonstrated that purified C8/119S reacted with a monoclonal anti-Der f 2 antibody (18G8). pWU11-C8/119S/HB101 can be used as an easy, low cost expression system on a large scale. It is also advantageous for industrial production in that the addition of an inducer is not required to achieve expression of the mite allergen.

A yeast system for stable expression of hepatitis B surface antigen

We have constructed a yeast strain that has integrated into its chromosomal ribosomal RNA gene site two copies of Hepatitis B virus surface (HBS) antigen gene under the control of the yeast (Saccharomyces cerevisiae) glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and terminator. The level of expression of HBS gene was low in the strain, but upon chemical and physical mutageneses, in combination with an immunological screening procedure, a mutant clone which expressed HBS protein at a high level was obtained. This mutant strain produces HBS antigen stably under non-selective conditions.

Production-scale purification of the recombinant major house dust mite allergen Der f 2 mutant C8/119S.

A WHO position paper states that allergen immunotherapy is an effective treatment for allergic diseases, and well characterized allergens should be used in immunotherapy. The house dust mite is a major cause of allergic disease. However, the biological activity of the mite extracts currently used cannot be clearly determined, since these extracts contain various impurities. The use of recombinant allergens can avoid this problem. However, there remains a risk of contamination by other impurities, such as host cell-derived proteins (HCPs). Advanced purification techniques are thus required to remove these contaminants. C8/119S is a mutant of the major house dust mite allergen Der f 2, and is expressed and accumulated as an inclusion body in Escherichia coli. The C8/119S was refolded and purified through three column chromatography steps. Using this method, we could obtain about 2g of the purified C8/119S in one purification batch. This amount is equivalent to 100,000 of the maintenance doses required for immunotherapy based on the WHO position paper. The purity of the C8/119S was 99% or more. The antigenicity of HCPs in the C8/119S was examined by passive cutaneous anaphylaxis assays. When the C8/119S was administered at 40 μg/kg, no local anaphylaxis was observed. C8/119S was thus highly purified with an extremely low level of impurities, and our procedure was shown to be an effective advanced production-scale purification process for this Der f 2 mutant. In this study, we established an advanced purification processes for C8/119S, then characterized the purified C8/119S and evaluated its purity.

Characterization of the Recombinant Der F 2 Mutant C8/119S and Evaluation of C8/119S in a Rder F 2-Sensitized Rhinitis Mice Model

Immunotherapy is the only curative approach to treat allergy, but carries the risk of anaphylaxis. C8/119S is a mutant of Der f 2, which is one of the causative allergens of perennial allergic diseases, and has been selected to decrease the risk of anaphylaxis in immunotherapy. In this study, the physical properties of C8/119S were determined, and the efficacy of C8/119S was evaluated in a NC/Nga mouse rhinitis model. C8/119S and recombinant Der f 2 (rDer f 2) were expressed in Escherichia coli . Purified allergens were analyzed by physicochemical and immunological techniques. In addition, rhinitis was provoked in rDer f 2-sensitized NC/Nga mice by nasal administration of rDer f 2, and C8/119S was administered. After provocation tests with rDer f 2, the number of eosinophils infiltrating the nasal mucosa was determined. C8/119S had a disordered structure, and the binding activity of allergic patients’ IgE to C8/119S was decreased compared with rDer f 2. In the NC/Nga mouse rhinitis model, eosinophil infiltration provoked by rDer f 2 was significantly controlled by the administration of C8/119S. Although similar therapeutic effects were also observed with rDer f 2 administration, 11 of 20 animals died during the rDer f 2 treatment period. On the other hand, no deaths occurred during C8/119S treatment. C8/119S appears to be an effective allergen vaccine for immunotherapy in patients with mite allergy and also appears to be safer than wild-type allergen vaccines.