Toru Sugano

A human monoclonal antibody against varicella-zoster virus glycoprotein III

Hybridomas producing human monoclonal antibodies (HMAbs) against varicella-zoster virus (VZV) were generated by fusing murine myeloma cells with human lymphocytes immunized in vitro. An assay system was developed to select anti-glycoprotein (gp)III HMAbs from the pool of anti-VZV HMAbs. A murine anti-gpIII MAb, 4B7, did not react with a VZV-infected cell homogenate, but did react with a VZV-infected cell monolayer, whereas anti-gpI and anti-gpII MAbs reacted with both antigens. Hybridomas were screened to obtain HMAbs having a reaction profile similar to that of 4B7 and one such clone, V3, stably produces human IgG1 (kappa). HMAb V3 immunoprecipitated a VZV antigen of 115K to 120K, which was not immunoabsorbed by an anti-gpII HMAb, implying that V3 recognizes gpIII. V3 neutralized VZV independently of complement, unlike anti-gpI and anti-gpII HMAbs. All five strains of VZV tested were completely neutralized by V3, and the dose of V3 required to reduce the number of virus plaques by 50% ranged from 0.027 to 0.15 micrograms/ml. V3 was also able to inhibit the spread of virus infection from infected to uninfected cells, whereas anti-gpI and anti-gpII HMAbs could not. In addition, V3 mediated antibody-dependent cellular cytotoxicity but not complement-dependent cytotoxicity of VZV-infected cells. The results suggest that an anti-gpIII HMAb may provide a new means of passive immunoprophylaxis and also help to identify an antigenic epitope appropriate for a subunit vaccine.

Human monoclonal antibody against glycoproteins of human immunodeficiency virus

We have established a program to make human monoclonal antibodies to the human immunodeficiency virus (HIV). Lymphocytes of lymph nodes from patients with the acquired immunodeficiency syndrome (AIDS) related complex (ARC) spontaneously produced antibodies to HIV in vitro and their antibody production was suppressed by culturing them in the presence of HIV antigens. Therefore, in vitro stimulation with HIV antigens was not done but rather, donor lymph node or spleen lymphocytes were directly fused with mouse myeloma cells. One of the hybridomas thus generated has been stably producing human monoclonal antibody (MAb) of the IgG1 isotype with a kappa chain. This antibody, MAb86, bound to the surface membrane of HIV-infected cells but not to that of uninfected cells at all. MAb86 reacted in Western blot with both viral glycoproteins of 120,000 daltons (gp120) and 41,000 daltons (gp41). While not neutralizing alone, a combination of MAb86 with another human IgG1 MAb against gp120 showed viral neutralization. Based on these data it seems likely that this approach will result in human MAbs capable of viral neutralization and antibody-dependent cytotoxicity. These may have value for the prevention and/or treatment of AIDS.

A microneutralization enzyme immunoassay for antibody to human cytomegalovirus

We have developed a relatively rapid, sensitive and quantitative microneutralization assay for antibody to human cytomegalovirus (HCMV). Cell monolayers in 96-well microtiter plates inoculated with pre-incubated virus-antibody mixtures were fixed after 3 days. Infectious foci were stained with peroxidase-labeled human monoclonal antibody to a 64 kDa immediate early antigen of HCMV, and the plates were read at OD450. The 50% neutralization titer of the antibody was calculated. A study with 20 human sera and a human monoclonal antibody which neutralizes virus showed that this microneutralization enzyme immunoassay is more sensitive than, and as quantitative as, the conventional plaque reduction assay for antibody to HCMV. The neutralizing antibody titers of each sample measured by these two methods showed good correlation (n = 19, r = 0.884). Thus, this new assay is a useful and valid alternative to the conventional method for mass screening of sera and hybridoma fluids, and considerably more rapid.

A hybridoma producing human monoclonal antibody specific for glycoprotein 120kDa of human immunodeficiency virus (HIV-1)

A stable hybridoma producing anti-HIV human monoclonal antibody (HMCA) was generated by fusing CD3-depleted human splenic lymphocytes from an HIV sero-positive donor with the mouse myeloma cell line P3x63AgU1. The resultant hybridoma has been secreting IgG1, lambda chain for over nine months at a rate of 2.5 micrograms/10(6)cells/day. The HMCA shows specific reactivity in ELISA using HIV-infected cell lysates. Immunofluorescence tests have indicated that this HMCA binds specifically to the surface of H9 and C3 HIV/HTLVIIIb infected cells, HIV/N1T infected CEM cells and to MoT cells infected with an HIV clinical isolate. Western blotting revealed recognition of glycoproteins 120 and 160 kDa of HIV by the HMCA. Although this HMCA demonstrated no neutralizing activity, the production of an anti-HIV HMCA specific for glycoprotein 120 kDa indicates the possibility that a neutralizing HMCA can be developed as further fusions with lymph nodes and spleens from HIV positive donors are performed.