Yahiro Uemura

Urokinase Inhibitor in Human Placenta

FIBRINOLYTIC activity in the blood markedly decreases in the later months of pregnancy and in labour, but rapidly returns to normal after delivery1–3. Brakman and Astrup4 observed a significant increase in the selective inhibition of fibrinolysis induced by urokinase in plasma and serum of pregnant women, and their findings suggested that an inhibitory mechanism was involved in depressing fibrinolysis during pregnancy. The same characteristics of fibrinolytic inhibition in saline extract of human placenta have been reported by one of us5, that is placental extract inhibited only urokinase but not streptokinase or glycerol-activated plasmin. Because the placental extract contained blood components, however, it was uncertain whether the inhibition was derived from pregnancy blood or from placenta itself.

Inactivation and Elimination of Viruses during Preparation of Human Intravenous Immunoglobulin

We report here the results of our evaluation of virus inactivation during the manufacturing steps of two intravenous immunoglobulin (IGIV) preparations. Virus inactivation and/or removal by processing steps, such as ethanol fractionation and polyethylene glycol precipitation, and deliberate virucidal steps, such as solvent/detergent treatment and pasteurization, were tested on a variety of human pathogenic and experimental model viruses, including human immunodeficiency, Hepatitis C, Mumps, Vaccinia, Chikungunya, Vesicular Stomatitis, Sindbis, and ECHO viruses. All viruses were successfully inactivated and/or eliminated by the processing steps studied. In some cases, however, multiple steps were required. We conclude that the incorporation of steps deliberately designed to inactivate or remove viruses during the production of IGIV provides an extra measure of viral safety.

INACTIVATION AND ELIMINATION OF VIRUSES DURING THE FRACTIONATION OF AN INTRAVENOUS IMMUNOGLOBULIN PREPARATION: LIQUID HEAT TREATMENT AND POLYETHYLENE GLYCOL FRACTIONATION

Abstract. A method for the heat treatment of human IgG solution at 60 °C for 10 h was established. Human immunodeficiency, mumps, vaccinia and 4 other viruses were added to the IgG solution in 33% sorbitol and heated at 60 °C. Those viruses were inactivated within 1 h. Heat‐treated intravenous IgG (IVIG‐H) was prepared by heat treatment and polyethylene glycol (PEG) fractionation. Conventional nonheated intravenous IgG (IVIG‐C) was prepared from the same source paste by the fractionation method. No physicochemical or biological difference was observed between the heated and control IVIG preparations.

Elimination of Viruses (Human Immunodeficiency, Hepatitis B, Vesicular Stomatitis and Sindbis Viruses) from an Intravenous Immunoglobulin Preparation

Abstract. More than 104 plaque‐forming units (pfu)/ml of HIV are inactivated during the alcohol fractionation step from plasma to fraction (Fr)‐II+III, >104 pfu/ml is inactivated from Fr‐II+III to Fr‐II and >104 pfu/ml is inactivated during the polyethylene glycol (PEG) fractionation process from Fr‐II+III to intravenous IgG (IVIG). The total inactivation rate from plasma to IVIG via Fr‐II+III or Fr‐II was calculated to be greater than 108 or 1012, respectively. The PEG fractionation method produces an intact and unmodified IVIG. In addition, the PEG fractionation method at a low ionic strength was found to be effective for the elimination of greater than 105 units of other viruses, including hepatitis B, vesicular stomatitis and Sindbis viruses.

Down regulation of a harmful variant protein by replacement of its normal protein.

To compensate for the hypoprotein and hypoalbuminemia of familial amyloidotic polyneuropathy (FAP) patients, 800 ml of fresh frozen plasma (FFP) was intravenously administered and change in total and variant transthyretin (TTR) levels were measured in the plasma. After injection of FFP, total plasma TTR levels were elevated and variant TTR levels decreased from 24 to 48 h, accompanied by an elevation of plasma total protein, albumin levels and TTR levels. To elucidate the mechanism of this phenomenon, a large amount of purified normal TTR from normal human plasma was intravenously injected in mice and FAP patients. By intravenous injection of 3 mg of the purified TTR to C57Black6, the expression of TTR mRNA decreased from 6 to 24 h post injection, and gradually increased up to 48 h post injection. After injecting 400 mg of normal TTR in each of 3 FAP patients, total plasma TTR levels were elevated and variant TTR levels decreased significantly from 24 to 48 h. These results suggested that down regulation of the harmful protein by replacement of its normal form of the protein occurred by this method. This phenomenon should be applied as the basis for one of the useful methods for decreasing the harmful proteins in the circulation.

Antibody Fc Functional Activity of Intravenous Immunoglobulin Preparations Treated with Solvent‐Detergent for Virus Inactivation

We report here results of in vitro comparisons of the Fc functional activity of a second‐generation intravenous immunoglobulin (IGIV) preparation (Venoglobulin®‐I) and a third‐generation IGIV product that includes a deliberate virus‐inactivation step (Venoglobulin®‐S). Both formulations showed equivalent Fc‐mediated function against viral antigens (rubella, influenza A, and influenza B) by single‐radial hemolysis test, and against group B Streptococcus, Staphylococcus aureus and Escherichia coli by opsonophagocytosis assay. In addition, we showed by three different immunochemical reactions and by HPLC analysis that both preparations consisted of mostly monomeric IgG and contained very low levels of complement‐fixing IgG aggregates. However, IgG aggregation induced by heating at 63°C markedly enhanced fixation of C1q and C3 and binding to Raji cells, indicating that the IgG molecules retained their complement‐fixing capacity. Thus, the incorporation of a virus inactivation step in the manufacture of our third‐generation IGIV did not alter the Fc functional activities of the IgG, as measured by these in vitro assay systems.

Properties of recombinant hepatitis B vaccine

A large-scale purification method for hepatitis B surface antigen produced in a recombinant yeast (Saccharomyces cerevisiae) was established. The resulting HBsAg was greater than 99% pure and suitable for vaccine use. The yeast-derived HBsAg was structurally and biochemically similar to plasma-derived HBsAg. The anti-HBs antibody producing potency of the yeast-derived vaccine in mice was significantly higher than that of the plasma-derived vaccine. The yeast-derived vaccine induced protective antibody against hepatitis B virus of either adr or ayw subtype in a chimpanzee efficacy study. These observations demonstrate the usefulness of the yeast-derived vaccine as a second-generation hepatitis B vaccine.

Immunoglobulin Preparation: Safe from Virus Transmission?

Abstract. The incidence of non‐A, non‐B hepatitis associated with the administration of immunoglobulin preparations, especially intravenous preparations, which had been considered to be free from virus transmission, is reported. Research efforts to improve the safety of intravenous immunoglobulin preparations which could be administered in a large volume must be continued. Adding sorbitol under weakly acidic conditions, heat treatment of IgG at 60°C for 10 h is possible. Intravenous immunoglobulin preparation manufactured by polyethylene glycol fractionation followed by the heat treatment is not only intact, but also much closer to the ideal intravenous immunoglobulin preparation in the safety and stability.

Efficacy of an Immunoglobulin Preparation from HIV Carriers in Preventing HIV Replication in vitro

Abstract. Immunoglobulin samples (HIV‐Ig) were prepared by cold ethanol fractionation of human plasma containing antibody against human immunodeficiency virus (HIV). The ability to prevent viral spreading was studied using either human T‐cell leukemia virus type I (HTLV‐I)‐carrying MT‐4 cells or in a coculture system using MOLT‐4 cells and virus‐producing MOLT‐4/HIVHTLV‐IIIB cells. Treatment of HIV‐infected MT‐4 cells with HIV‐Ig effectively blocked the appearance of antigens of HIV and the virus‐induced cytopathic effect. HIV‐Ig blocked multinucleated giant cell formation in the MOLT‐4 and MOLT‐4/HIVHTLV‐IIB coculture system.

HEPATITIS C VIRUS MORE RESISTANT TO INACTIVATION THAN HUMAN IMMUNODEFICIENCY VIRUS

The prevalence of hepatitis C virus (HCV) seropositivity is reportedly over 80% in patients with moderate to severe hemophilia B [l, 21. In contrast to the human immunodeficiency virus (HIV), HCV appears to be relatively more resistant to inactivation during the purification processes for factors VIII and IX [l]. The development of a highly sensitive, two-stage, nested reverse transcription-polymerase chain reaction (NRT-PCR) has made it possible to detect at least 10chimpanzee-infectious doses of HCV per milliliter, making it approximately 10 times more sensitive than the one stage PCR [3]. The twostage PCR was also found to be sensitive and specific in analyzing human sera, detecting HCV in 11 of 12 (92%) serum samples from individuals with non-A, non-B hepatitis who were seronegative for HCV antibodies 131. Thus, the NRT-PCR assay provides a means to directly evaluate the HCV content of purified coagulation factors. Three typical lots of AlphaNine@ coagulation factor IX (human) were selected for NRT-PCR analysis. The purified AlphaNine was prepared from lots of plasma that had not been screened for anti-HCV antibody. Total mouse RNA was added to each sample of AlphaNine to provide an internal control. RNA obtained from these samples was used as a template €or cDNA synthesis; the twostage PCR was then performed using sequence primers from three regions of the HCV genome: 5’-noncoding, core, and C-100. By this procedure, the three lots of factor IX concentrate were found to be nega-. tive for HCV by the NRT-PCR (table 1). Hepatitis C Virus More Resistant to Inactivation than Human Immunodeficiency Virus