Yoshinobu Miyatsu

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.

Retrospective survey to evaluate the safety and efficacy of Japanese botulinum antitoxin therapy in Japan.

Japanese botulinum antitoxins have been used for more than 50 years; however, their safety and therapeutic efficacy are not clear. In order to analyze the available data on botulinum antitoxin therapy in Japan, we surveyed published reports about botulism cases in which botulinum antitoxins were used, and retrospectively analyzed the safety and efficacy of the therapy. A total of 134 patients administered botulinum antitoxins were identified from published reports. Two cases of side effects (1.5%) were detected after antitoxin administration, both not fatal. The fatality rate was 9.4%, and more than 70% of the patients showed improvement in their symptoms and better clinical conditions than those not treated with antitoxins. These data suggest that the therapy with Japanese antitoxins is safe and highly effective.

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.

Freeze-dried equine-derived redback spider antivenom: a local irritation study by intramuscular injection in rabbits and a repeated-dose toxicity study in rats

The redback spider (Latrodectus hasseltii) is nonindigenous to Japan but has now spread throughout the country. Bites to humans are rare but can be fatal. We prepared freeze-dried redback spider antivenom for therapeutic use against bites in Japan by immunization of horse plasma. This study included two nonclinical tests of the antivenom: a local irritation study involving a single intramuscular administration to rabbits (with injections of physiological saline and an existing freeze-dried diphtheria antitoxin as control and comparison substances, respectively) and a 2-week repeated intermittent intravenous-dose toxicity study in rats. The irritation study showed the antivenom’s irritancy to be comparable with that of the saline and the existing antitoxin preparations under the test conditions. In a repeated-dose toxicity study, no toxicity change was found in male or female rats, and the no-observed-adverse-effect level (NOAEL) was judged to be a dose volume of 20 mL/kg (1082 units/kg antivenom activity) in both male and female rats. In addition, there was no toxicological difference between proteinaceous diphtheria antitoxin and redback spider antivenom prepared to have the same protein content and the same additive composition. Based on these findings, we will further advance our research towards clinical application of the redback spider antivenom. This research was supported by the Research Program on Emerging and Re-emerging Infectious Disease of the Japan Agency for Medical Research and Development.

Venom and Antivenom of the Redback Spider (Latrodectus hasseltii) in Japan. Part II. Experimental Production of Equine Antivenom against the Redback Spider

This is the first report on large-scale experimental production of an equine antivenom against the redback spider (Latrodectus hasseltii) lived in Japan. We captured 10,000 redback spiders in Japan and prepared the toxoids of crude venom extract, mixed the toxoids with a mineral oil adjuvant, and immunized healthy horses repeatedly over a period of several weeks. Thereafter, we separated the horse plasma, purified the γ-globulin fraction, and stocked it as a purified antivenom concentrate. Consequently, we manufactured approximately 6,500 vials of a single-dose freeze-dried test lot from a portion of the purified γ-globulin fraction, equivalent to the extract derived from 520 spiders. This test lot had an antitoxin titer comparable to that of a similar drug commercially available overseas (a liquid preparation), and the other quality met all quality reference specifications based on the Minimum Requirements for Biological Products and other guidelines relevant to existing antivenom drug products in Japan.

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.