Hideo Asada

Analysis of a glycoprotein of human herpesvirus 6 (HHV-6) using monoclonal antibodies

The virus-specific polypeptides of human herpesvirus 6 (HHV-6) were studied. Six hybridoma clones secreting monoclonal antibodies were established. Localized cytoplasmic antigen and surface antigens were found in and on the infected cells, respectively, by using the immunofluorescent test with those antibodies. In the neutralization test, two clones (OHV3 and OHV9) neutralized HHV-6, but the other five monoclonal antibodies did not. When infected cells were labeled with [35S]methionine and the antigens were immunoprecipitated by OHV3 and OHV9 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, two polypeptides with molecular weights of 98,000 (98K) and 92K were detected. Furthermore, it was found that these polypeptides were glycosylated, because they were labeled with [14C]mannose. Pulse-chase studies revealed that precursor molecules were cotranslationaly glycosylated to produce 98K glycoprotein and they were replaced by 92K glycoprotein. Endo-beta-N-acetylglucosaminidases H and F reduced those glycoproteins to putative precursor molecules of 75 kDa. This indicates that N-linked high-mannose sugars associate to 98K and 92K glycoproteins.

Characterization of glycoproteins of viruses causing hemorrhagic fever with renal syndrome (HFRS) using monoclonal antibodies

Viruses causing hemorrhagic fever with renal syndrome (HFRS) encode two glycoproteins, G1 and G2. For determination of the biological functions of these glycoproteins, we isolated 15 hybridomas secreting monoclonal antibodies directed against the glycoproteins of the B-1 and Hantaan viruses (HV). From results of neutralizing and hemagglutination inhibition (HI) tests, and studies on the antigenic reactivities of the antibodies with other HV-related viruses by immunofluorescence, we classified these hybridoma clones into two groups producing antibodies to the G1 proteins of the B-1 virus, six groups producing antibodies to G2 proteins of the B-1 virus, and four groups producing antibodies to the G2 protein of HV. Of the antibodies to G2 produced by 12 clones, groups A and B had high HI activity with HV-related virus cross-reactivity and moderate neutralizing activity, group C had moderate HI activity with virus specificity but low neutralizing activity, group G had high neutralizing activity and low HI activity, and five other groups had little or no HI or neutralizing activity. Group A reacting with G1 protein had low level of both neutralizing and HI activity, while group B had no HI activity. One clone of monoclonal antibody had high neutralizing activity and no HI activity, but it did not react with either polypeptide by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by the immunoblotting method. These data suggest that both glycoproteins are the targets of neutralizing antibodies. Furthermore, the results indicate that the antigenic determinants with hemagglutination activity are mainly on the G2 protein, and that the domains related to neutralizing activity and to HI activity are separate.

Enhancement of immunity against VZV by giving live varicella vaccine to the elderly assessed by VZV skin test and IAHA, gpELISA antibody assay

The enhancement of immunity against varicella-zoster vaccine (VZV) by subcutaneous injection of a live varicella vaccine was assessed by the VZV skin test for cell-mediated immunity (CMI), and immunoadherence hemagglutination assay (IAHA) and gpELISA antibody assays in the elderly people of 50-79 years of age. A total of 127 subjects were examined: 79 aged 50-59, 25 aged 60-69, and 25 aged 70-79. All were seropositive by the gpELISA assay (one was seronegative in the IAHA antibody assay). In contrast, a notable decline was observed in the VZV skin test with increasing age. Negative reaction was observed in 16/79 (20.2%) of the subjects in their 50s, 12/25 (48.0%) in their 60s and 14/25 (56.0%) in the 70s. After the vaccination, the results of the VZV skin test changed from negative to positive in 15/16 (91.8%) of subjects in their 50s, 11/12 (91.7%) in their 60s and 12/14 (85.7%) in their 70s. The mean antibody titer in the IAHA and the gpELISA increased approximately two-fold after the vaccination in each group. Immunity to VZV in 35 elderly subjects who were vaccinated previously was followed up for 4 years. All were positive by the VZV skin test after the previous vaccination. After 4 years, 31 (88.6%) were positive by the skin test, 4 were negative and became positive after revaccination. Although this study was uncontrolled open study, the results suggest that administering live varicella vaccine to the elderly is effective for enhancing immunity, particularly CMI to VZV.

Analysis of human herpesvirus 6 glycoproteins recognized by monoclonal antibody OHV1

A virus-specific glycoprotein (gp) from human herpes-virus 6 (HHV-6) was studied using the anti-HHV-6 monoclonal antibody OHV1. Immunoprecipitation with extracts from infected cells revealed that the antibody recognized four glycosylated proteins (gps) with Mrs of 106K, 102K, 65K and 63K under reducing conditions. However, only two gps, of 106K (gp106) and 102K, were detected under non-reducing conditions. Pulse-chase experiments revealed that gp65 and gp63 were cleavage products of gp106 and gp102. When infected cells were treated with tunicamycin, none of these gps was detected. With endo-beta-N-acetylglucosaminidase H (endo H) and endo-beta-N-acetylglucosaminidase F (endo F) treatment, gp106 and gp102 disappeared. Moreover, gp65 and gp63 were not affected by endo H treatment but were sensitive to endo F treatment. These data suggest that sugar residues of gp106 and gp102 are high-mannose type N-linked oligosaccharides, whereas those of gp65 and gp63 are complex type N-linked oligosaccharides.

Effects of human and murine interferons against hemorrhagic fever with renal syndrome (HFRS) virus (Hantaan virus)

The effects of human interferons (IFN-alpha, IFN-beta, IFN-gamma) on the replication of Hantaan virus (HV) in Vero E6 cells were examined. Pretreatment of cells with human IFNs resulted in dose-dependent inhibition of HV plaque formation. Of the 3 human IFNs, IFN-beta inhibited virus replication most effectively. Pretreatment of murine macrophage cells with mouse IFN-beta also resulted in an inhibition of viral growth and then the effect of murine IFN-beta in newborn ICR mice infected with HV was also examined. When newborn mice were inoculated intraperitoneally with HV, their survival rate was approximately 20%. When they were treated with interferon 6 h before infection with virus, their survival rate was 85-90%. When IFN and virus were injected simultaneously into the intraperitoneal cavity, the survival rate of the mice was also higher than that of untreated mice. When the mice were treated with IFN for 2 or 7 consecutive days after infection, their survival rate was 70%. These results suggest that IFN may be effective for both prophylactic and therapeutic purposes in Hantaan virus infection.

Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells

We isolated a novel gene, termed MLZE, from a B16‐BL6 cDNA library after subtraction of B16‐F10 mRNA. Expression levels of mouse MLZE (mMLZE) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that hMLZE was expressed primarily in trachea and spleen. We mapped the hMLZE gene (by fluorescence in situ hybridization) to 8q24.1‐2, which contains the c‐myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze‐positive cases was significantly larger in Clark levels III, IV and V melanomas (6/11=55%) than in Clark levels I and II melanomas (2/15=13%). In two cases of hMlze‐positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.

Increased Expression of a Nucleolar Nop5/Sik Family Member in Metastatic Melanoma Cells : Evidence for Its Role in Nucleolar Sizing and Function

F10 and BL6 cells of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells can metastasize to lungs after subcutaneous injection. Differences in gene expression between the two cell lines were examined, and a greater expression of the Sik-similar protein (Sik-SP) gene was found in BL6 cells. Structurally, Sik-SP belongs to the nucleolar Nop5/Sik family whose members play central roles in ribosome biogenesis; however, the function of Sik-SP has not been examined. Cytology with green fluorescent protein-fused proteins showed that Sik-SP was localized to the nucleolus. To examine whether Sik-SP is involved in ribosome biogenesis, two parameters were measured: magnitude of ribosomal RNA synthesis per nucleus and magnitude of protein production from the same amount of mRNA of an exogenous luciferase gene. Both values and, in addition, nucleolar size were larger in COS-7 monkey kidney cells overexpressing Sik-SP and BL6 cells than in mock-transfected COS-7 and F10 cells, respectively. Sik-SP seemed to promote ribosome biogenesis in the nucleolus. Furthermore, the expression of Sik-SP seemed to confer a greater cell growth response to serum, because such a response was greater in BL6 cells and F10 cells overexpressing Sik-SP than in untreated and mock-transfected F10 cells. Sik-SP may render melanoma cells more competent to survive through augmenting the activity of nucleolus.

Natural killer cell-derived large granular lymphocyte lymphoma of lung developed in a patient with hypersensitivity to mosquito bites and reactivated Epstein-Barr virus infection

A 17‐year‐old female developed natural killer (NK) cell‐derived large granular lymphocyte (LGL) lymphoma of the lung. She had a past history of hypersensitivity to mosquito bites (HMB). After an eight‐year chronic, active Epstein‐Barr virus (EBV) infection, she developed multiple lung lesions and pleural effusion. In the effusion, 60% of the cells were LGL. They were CD2+, 3−, 16+, 56+, 57+, 45RO+/RA + weak, and possessed strong NK activity. No rearrangement of T‐cell–receptor genes was detected. From all these results, a diagnosis of NK‐LGL lymphoma of the lung was made. EB virus DNA was detected in cells infiltrating the pleural effusion. The clonality of the LGLs was determined by Southern blot hybridization with the terminal repeat sequence of EB virus as a probe, and by chromosomal abnormalities. The patient died from respiratory failure. Necropsy of the lung revealed diffuse lymphoma composed of polymorphic cells with typical angiocentric lesions. Reportedly, lymphomas of NK lineage show predominantly extranodal involvement, and primary lung lesions are rare. In the pleural effusion of the present case, abnormally high levels of soluble Fas ligand, interleukin‐10 and interferon γ were detected. This hypercytokinemia, reflecting the microenvironment of lymphoma cells, may play a role in the progression of the lymphoma and organ injury in the lung. Am. J. Hematol. 59:309–315, 1998.

Development of inactivated vaccine against virus causing haemorrhagic fever with renal syndrome

B-1 virus belonging to the hantavirus group was serially passaged in the brains of newborn mice. Inactivated vaccine was prepared from the brains after inactivation with formalin and then purification by ultracentrifugation. The antigenic potency of this vaccine in vitro was determined by antibody-bound enzyme-linked immunosorbent assay (ELISA) and serial diluted vaccine bound to an aluminium hydroxide gel was inoculated into Balb/c mice to test immunogenicity. After two injections of this vaccine preparation, antibodies were detected in the mice by immunofluorescent, neutralizing and haemagglutination inhibition antibody tests. When mice immunized with this vaccine were challenged with B-1 virus and Hantaan virus (KHF-83-61BL strain), the virus titres in their lungs and spleens were significantly less than those in non-immunized mice. These results suggest that inactivated B-1 virus vaccine is effective against virus challenge by homotypic (B-1 virus) and heterotypic (Hantaan virus) viruses.

Annexin VII as a novel marker for invasive phenotype of malignant melanoma.

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. While examining the genetic difference between the two sublines, we found a marked reduction of annexin VII expression in BL6 cells. In addition, fusion cell clones of both sublines, were as poorly metastatic as F10 cells after subcutaneous injection, and contained the annexin VII message as abundantly as F10 cells. Hence, we examined whether the annexin VII expression was correlated with the less malignant phenotype of clinical cases by immunohistochemistry. Immunoreactivities to anti‐annexin VII antibody in melanoma cells were evaluated quantitatively by using skin mast cells as an internal positive control. Eighteen patients with malignant melanoma were divided into two groups: lymph node metastasis‐negative and positive groups. The ratio of numbers of patients positive versus negative to the antibody was significantly larger in the former than in the latter group. These results not only indicated that annexin VII serves as a marker for less invasive phenotype of malignant melanoma, but also suggested a possible role of annexin VII in tumor suppression.