Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration

Abstract

Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process.

Mechanisms of transmissible gastroenteritis coronavirus neutralization

“…These results strongly suggest that both infectious and noninfectious virions have a functional spike protein mediating the binding of the virus to the cells . The limitations derived from the inhability to follow isolated infectious virions have been accepted in all the studies analyzing the interaction between viruses and cells, by electron microscopy or using radiolabeled virions (Fuller and Spear, 1985 ;FulleretaL, 1989 ;Highlander et al, 1987 ;Miller and Hutt-Fletcher, 1988) . In all cases, the observed virions, or most of the radioactivity, corresponds to noninfectious particles .…”

“…Under these conditions (4°C), about 10% of the virus has been protected from cell dissociation at pH 3 ( Fig . 5B) (Highlander at al ., 1987), and was not accessible to MAb, which prevented the extensive neutralization observed when the virus interacted first with the antibody (Table 4) .…”

Geldanamycin, a Ligand of Heat Shock Protein 90, Inhibits the Replication of Herpes Simplex Virus Type 1 In Vitro

“…Measurement of viral penetration. The method for measurement of viral penetration was described before (7). Vero cell monolayers were placed on ice for 2 h followed by infection with HSV-1 (1 PFU/cell).…”

Critical role of cholesterol in bovine herpesvirus type 1 infection of MDBK cells

“…To determine if the cholesterol depletion at the virus entry stage affects the virus replication, the monolayer cells seeded in 24-well plates were mock treated or treated with MbCD at the concentrations of 2.5, 5, 10, 20 mM for 30 min at 37 8C before incubation for 1 h with BoHV-1 of 50TCID 50 . After treatment with citrate buffer (40 mM citric acid, 10 mM KCl, 135 mM NaCl, pH 3.0) for exactly 1 min to inactivate cell membrane bound but unpenetrated virions as described elsewhere (Chung et al, 2005;Highlander et al, 1987), the cells were further incubated to determine the virus yield with plaque formation assay.…”