Murine monoclonal antibody to a single protein neutralizes the infectivity of human cytomegalovirus.

Abstract

Murine monoclonal antibodies to human cytomegalovirus (CMV) strain AD169 were selected that neutralized virus infectivity. One monoclonal antibody-producing hybridoma, 1G6, was used to produce ascites fluid from which immunoglobulin was isolated. This antibody efficiently new tralized CMV AD169, other laboratory strains (Towne, Davis), and clinical isolates of CMV in early tissue culture passage (<10) in the absence of complement. The antibody immunoprecipitated a single 86,000-dalton protein from both laboratory and clinical strains. This viral protein was demonstrated by indirect immunofluorescence to be localized in the cytoplasm of CMV-infected cells.Human cytomegalovirus (CMV) infection initiates a cascade of protein synthesis in cell culture. There are approximately 30 proteins associated with the virions of CMV and more than 50 proteins specified by the viral genome that are synthesized in infected cells (1-5). The proteins of CMV from both virions and infected cells have been analyzed immunologically by using polyvalent hyperimmune sera (3, 6-8) and monoclonal antibodies (9, 10). These studies have shown that the major immunological determinants are probably glycoproteins and are present both in infected cells and on the viral envelope.The specific proteins that stimulate cell-mediated or humoral immunity have not yet been identified. In order to investigate the viral proteins that provide the target for antibody-mediated virus neutralization, we have selected murine monoclonal antibodies that neutralize CMV infectivity. A single viral protein was identified by immunoprecipitation that may be important for stimulation of neutralizing antibody in vivo. MATERIALS AND METHODSViruses. Human CMV strain AD169 was obtained from S. St. Jeor (University of Nevada). Laboratory strains (Towne, Davis) and clinical isolates from congenitally infected newborn infants were provided by J. Waner (University of Oklahoma). Viruses were grown in cultures of human embryonic lung (HEL) fibroblasts (11) with Dulbecco's minimal essential medium (DME medium) supplemented with 1% fetal calf serum. Cell-free infectious virus stocks that contained 106 plaque-forming units (pfu)/ml for laboratory strains and 102 to 104 pfu/ml for clinical isolates were prepared by sonication of infected cells (11).Assays for Antiviral Antibody. CMV binding antibody was detected by solid-phase RIA (11). Antigen was prepared by pelleting virus from the supernatant of roller bottles (490 cm2) of CMV-infected cells showing 90% cytopathic effect. Approximately 100 ml of supernatant with 106 pfu/ml was clarified at 800 x g for 10 min, then centrifuged for 1 hr at 200,000 x g. The sedimented virus was resuspended in 1 ml of Tris/saline/EDTA (0.05 M Tris/0.15 M saline/0.00i M EDTA, pH 7.4) and diluted 1:100 in the same buffer for binding to plates. Bound antibody was detected with iodinated goat anti-mouse IgG and IgM (Tago, Burlingame, CA).Virus-neutralizing antibody was detected by both microneutralization and plaque reduction. Microneutralization...

Characterization of neutralizing monoclonal antibodies to murine cytomegalovirus

“…In contrast, hyperimmune immunoglobulin was able to neutralize infectivity after virus adsorption and, whereas neutralization was enhanced in the presence of complement, the virus was neutralized at low antibody concentrations even in the absence of complement. These results are consistent with previous reports of the requirement of complement for neutralization of MCMV and HCMV by some sera (Kim & Carp, 1973;Rasmussen et al, 1985;Masuho et al, 1987;, but not by others (Rasmussen et al, 1984).…”

Human Monoclonal Antibodies Neutralizing Human Cytomegalovirus

“…It is known that CMV strains possess different antigenicities (Rasmussen et al, 1984). Thus, the neutralization by C23 and C41 was examined using five clinical CMV isolates (Table 1).…”

Biochemical and Immunological Analysis of Discontinuous Epitopes in the Family of Human Cytomegalovirus Glycoprotein Complexes Designated gC-I

“…Both gp52 (Lussenhop et al, 1988) and gp93-130 (Karl et al, 1990) contain epitopes recognized by neutralizing murine monoclonal antibodies (MAbs). However, most MAbs made using whole virus as antigen have recognized gp52 (Banks et al, 1989;Karl et al, 1986;Britt, 1984;Rasmussen et al, 1984). Many of these MAbs recognize epitopes that map between amino acids 400 and 700 on gp52 (Banks et al, 1989;Utz et al, 1989).…”