Hiroyoshi Ohba

Serological response and detection of viraemia in acute hepatitis C virus infection

The serological response during acute hepatitis C virus (HCV) infection was examined by enzyme-linked immunosorbent assay (ELISA) in sequential serum samples from 13 haemophiliacs following their first exposure to factor VIII concentrates contaminated with HCV. The commercially available C100-3 peptide and a new 22 kDa recombinant protein (p22) encoded by the nucleocapsid region of the viral genome were used for antibody detection, whilst a nested polymerase chain reaction (PCR) method was used for the detection of viraemia. In addition, eight sporadic cases of acute HCV infection were studied. The results in haemophiliacs demonstrated that seroconversion to the C100-3 antigen occurred in only one-third of the patients within 12 weeks of disease onset, but all of the patients had a diagnostic serological response to p22 during this phase of the disease. The new test was positive in all the sporadic cases at a time when the commercially available test was negative. Although PCR offers a sensitive method for the detection of recent HCV infection, the complex methodology makes it unsuitable for diagnostic laboratories. The new ELISA test with p22 may therefore have a useful diagnostic role in acute disease.

Generation of neutralizing antibody to the reverse transcriptase of human immunodeficiency virus type 1 by immunizing of mice with an infectious vaccinia virus recombinant

Antibodies inhibiting the reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) were found to be generated in the serum of mice repeatedly infected with a vaccinia virus recombinant, WRRT, expressing the enzyme. A monoclonal antibody (mAb), 7C4, which specifically and almost completely inhibits the RNA-dependent DNA polymerase activity of HIV-1 RT was produced from a mouse repeatedly immunized with WRRT. 7C4 seems to be specific for HIV-1 among retroviruses: 7C4 inhibited RT activity of three strains of HIV-1 (IIIB, Bru, and IMS-1) but not of two strains of HIV-2 (GH-1 and LAV-2) or two strains of SIV (MAC and MND). The immunoglobulin isotype of three out of four mAbs produced from spleen cells of the immunized mouse were IgG2a. This immunization method that avoids protein denaturation may preferentially induce a T helper type-1 immune response and increase the chances of producing the only occasionally obtainable mAb capable of recognizing a conformational epitope and completely inhibiting enzyme activity.

Common Cell‐Surface Antigens Functioning in Self‐Recognition Reactions by Both Somatic Cells and Gametes in the Solitary Ascidian Halocynthia roretzi

The “contact reaction” is an extremely rapid allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the solitary ascidian Halocynthia roretzi. It has been proposed that regulation of the alloreactivity of hemocytes may be involved in preference for fertilization or self‐sterility in this species. To identify the receptors and target ligands involved both in self‐recognition by somatic cells and self‐discrimination by gametes, we produced monoclonal antibodies (mAbs) that inhibit the ACR mediated by hemocytes and tested their effects on fertilization. Six different mAbs that inhibit the ACR were prepared and categorized into three groups. Although all three mAbs seemed to have the same ability to inhibit the ACR, almost constant and statistically significant inhibition (CRB1.1) and infrequent but significant inhibition (CRB2.1, and CRB3.1) of the ACR were observed in the same pairs of animals. Pretreatment of the unfertilized eggs with CRB1.1, CRB2.1, and CRB3.1, resulted in the constant and statistically significant inhibition, infrequent but significant inhibition, and no inhibition, respectively, of fertilization. Antigens recognized by CRB1.1 (CRB1.1 antigens) were detected on the cell surface of all types of hemocytes and on the vitelline coat and follicle cells of unfertilized eggs. CRB2.1 and CRB3.1 antigens were detected on the surface of certain types of hemocytes and follicle cells, but not on the vitelline coat. CRB mAbs were directed against different epitopes in the N‐linked glycan on glycoproteins. These common carbohydrate antigens on somatic cells and gametes may function in some recognition processes in ACR and fertilization in H. roretzi.

Mutual and directional allogeneic cytotoxic reaction of hemocytes in the solitary ascidian Halocynthia roretzi revealed by one-step quantitative fluorimetric assay.

Abstract A novel one-step microplate cytotoxicity assay using the cytoplasmic fluorescent viability dye calcein AM was established for simple, rapid, sensitive, and quantitative measurements of the allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the ascidian Halocynthia roretzi. The mutual and directional ACR was distinguishable by the assay using the hemocytes from pairs of animals with different alloreactivities. The ACR assay may allow more precise genetic analysis of the gene that controls allore-activity of hemocytes, since the mutual and directional ACR may be related to levels of expression or numbers of the gene product or products on the target cells. The directional ACR will be useful in elucidating the cellular and molecular mechanisms of self-recognition in H. roretzi, since it allowed us to equate hemocytes from one animal with “effector cells” and those from the other animal of the pair with “target cells”. In addition, the quantitative ACR assay in a large number of samples is possible and it will allow production of monoclonal antibodies that may recognize receptors or ligands functioning in self-recognition processes by the H. roretzi hemocytes.

Follow‐up study of anti‐hepatitis C virus antibodies in blood donors implicated in post‐transfusion non‐A, non‐B hepatitis

Summary. We retrospectively examined the antibodies to p22, a hepatitis C virus (HCV) nucleocapsid protein, and to c100‐3, a HCV nonstructural protein, in donors whose blood was transfused to patients who later developed post‐transfusion non‐A, non‐B hepatitis. Of 13 such blood donors, three seroconverted and three seroreverted with the anti‐c100‐3 test. In contrast, 12 of the 13 blood donors showed the same results at transfusion and follow‐up, and one donor showed seroconversion with the anti‐p22 assay. The follow‐up study shows that the anti‐p22 antibody test provides consistent results and is far more suitable for screening blood than the anti‐c100‐3 test.

Common Cell Surface Ligands Functioning in Allogeneic Cytotoxic Reaction and Fertilization in Halocynthia roretzi

Two distinct oligosaccharide ligands that function in both the allogeneic contact/cytotoxic reaction (ACR) by hemocytes and gamete fertilization were identified by means of the monoclonal antibodies (Mabs), CRB1 and CRB2, which inhibit both the ACR and fertilization in Halocynthia roretzi. The CRB1 epitope, determined to be the “core” α1–6 fucosylated oligosaccharide epitope (CFOE) in hybrid-type N-glycans, was detected on the cell surfaces of all types of hemocytes as well as on those of follicle cells and the vitelline coat of eggs. The CRB2 epitope, which was indicated as an outer-side sulfated and phosphorylated ojigosaccharide epitope (OSPOE) in complex-type N-glycans, was demonstrated on the cell surfaces of some types of hemocytes and egg follicle cells. Both epitopes, OSPOES and CFOE, may function as ligands in the initial attachment and binding, respectively, in the sperm-egg interaction. These epitopes may also function in the same way in the self-recognition of the ACR. We propose that the fucose residue in CFOE is among the key molecules, and perhaps the most important, involved in self-recognition in the ACR and fertilization.