Hitoshi Ohnuma

Lengths of hepatitis B viremia and antigenemia in blood donors: preliminary evidence of occult (hepatitis B surface antigen-negative) infection in the acute stage.

BACKGROUND: The Japanese Red Cross (JRC) implemented a fully automated pooling and nucleic acid amplification test (NAT) system for testing seronegative donations. The JRC sample repository and repeat blood donations allowed for lookback and follow‐up studies of hepatitis B virus (HBV) DNA–positive donors, who tested negative for hepatitis B surface antigen (HBsAg) and anti–hepatitis B core antigen in the JRC screening system.

A solid-phase enzyme immunoassay for the determination of IgM and IgG antibodies against translation products of pre-S1 and pre-S2 regions of hepatitis B virus

The envelope of hepatitis B virus is coded for by pre-S1, pre-S2 regions and the S gene. A method was developed to determine antibody to the product of pre-S1 region (anti-pre-S1) and antibody to the product of pre-S2 region (anti-pre-S2), either of IgM or IgG class, by a solid-phase enzyme immunoassay. For the determination of anti-pre-S1, tubular particles containing translation products of pre-S1, pre-S2 regions and the S gene were broken into constituent envelope polypeptides and immobilized on a solid support. Serums were absorbed with spherical particles containing translation products of pre-S2 region and the S gene, obtained from plasma positive for hepatitis B e antigen (HBeAg) and deprived of particles carrying pre-S1 product by an affinity column. They were then tested for the binding with tubular polypeptides fixed on a solid support, and the bound antibody representing anti-pre-S1 was detected by monoclonal antibody to human IgM/mu or IgG/gamma labeled with horseradish peroxidase. For the determination of anti-pre-S2, test serums were absorbed with spherical particles containing the product of the S gene, obtained from plasma positive for antibody to HBeAg and deprived of particles bearing pre-S2 product by an affinity column. They were then tested for the binding with polypeptides, fixed on a solid support, composed of products of pre-S2 region and the S gene. The assay was applied to the determination of anti-pre-S1 and anti-pre-S2 of IgM or IgG class in asymptomatic carriers and in persons who had recovered from infection with hepatitis B virus.

The first large-scale nucleic acid amplification testing (NAT) of donated blood using multiplex reagent for simultaneous detection of HBV, HCV, and HIV-1 and significance of NAT for HBV.

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV‐1) of voluntarily donated blood after serological pre‐screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV‐1 reagent for blood transfusion including short shelf‐life platelets. Among them, 112 HBV DNA‐positives, 25 HCV RNA positives and 4 HIV‐1 RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adw/B, adw/C, ayr/C and ayw/D were found and adr/C was predominant. A total of 61.6% of them (69/112) were negative by overnight EIA. Sixth three of HBV NAT‐positive samples carried virus loads less than 104 copies/mL and 92.1% of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follow‐up study showed exponential straight lines in the early stage of serological window periods and the log times of HBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.

Epidemiology of blood donors in Japan, positive for hepatitis B virus and hepatitis C virus by nucleic acid amplification testing

Background and Objectives  The Japanese Red Cross screens seronegative blood donors by nucleic acid amplification testing (NAT) for hepatitis B, hepatitis C and human immunodeficiency virus‐1 markers. NAT‐positive donors thus identified seemed to have a different infectious background from serologically positive donors. The purpose of our study was to characterize this background in the hepatitis B virus (HBV) and hepatitis C virus (HCV) NAT‐positive donors.

Production and product quality assessment of human hepatitis B virus pre-S2 antigen in submerged and solid-state cultures of Aspergillus oryzae

Pre-S2 is a diagnostically important antigen of human hepatitis B virus (HBV). In order to produce pre-S2 antigen in Aspergillus oryzae, the gene [pre-S2]3, which encodes a tandemly triplicated repeat of pre-S2 polypeptides was fused with the partial glaA gene encoding glucoamylase lacking the starch-binding domain. In submerged culture, A. oryzae transformants carrying glaA-[pre-S2]3 secreted a heterogeneously glycosylated form of the fusion protein that was partially degraded. Contrarily, utilization of a wheat brain solid-state culture system resulted in the secretion of a homogeneous glycosylated form of the whole fusion protein. This is the first report of a dissimilarity in glycosylated modification between submerged and solid-state culture conditions in heterologous protein production in A. oryzae.

A monoclonal antibody against a hepatitis B e antigen epitope borne by six amino acids encoded by the precore region

Hepatitis B e antigen (HBeAg) polypeptide in the circulation (p17e) is composed of ten amino acids (aa) coded for by the precore region and 149 aa by the core gene of hepatitis B virus. A monoclonal antibody (Y0583A) was raised against the N-terminal ten amino acids (SKLCLGWLWG) encoded by the precore region. The binding of Y0583A with a panel of 203 decapeptides on multipins, which covered the precursor of HBeAg polypeptide made of 212 aa shifting by one aa, recognized an epitope sequenced LGWLWG representing the C-terminal six aa coded for by the precore region. This HBeAg epitope was not readily accessible on HBeAg in serum, but it became exposed and bound with Y0583A by treatment with 0.2 N NaOH. Using Y0583A, an enzyme-linked immunosorbent assay was developed for specific determination of HBeAg. The test sample was incubated with the monoclonal antibody to an HBeAg determinant encoded by the core gene (904) that had been immobilized on a solid support. Captured HBeAg was treated with 0.2 N NaOH, neutralized and released into the fluid phase. The reactant was then tested for a sandwich between monoclonal antibody (C33) to the C-terminus of the HBeAg polypeptide immobilized on a solid support and Y0583A labeled with horseradish peroxidase.