1 Institut für Virologie, Philipps-Universität Marburg, Robert-Koch-Strae 17, 35037 Marburg, Germany
2 Department of Immunotechnology, Lund University, PO Box 7031, S-220 07 Lund, Sweden
Correspondence
Klaus Radsak
radsak{at}mailer.uni-marburg.de
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ABSTRACT |
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MAIN TEXT |
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HCMV infection requires viral envelope glycoproteins and the respective cellular receptors to engage in a series of interactions, ultimately resulting in fusion of the viral envelope with the plasma membrane. The mechanisms by which gB-specific antibodies interfere with the complex processes involved leading to loss of viral infectivity have not been precisely defined so far, although a previous report suggested that antibodies may neutralize infection before or after adsorption to the cell surface (Ohizumi et al., 1992). In order to analyse further antibody-mediated HCMV neutralization an activity with potential therapeutic and protective function in vivo the effects of human monoclonal antibodies (mAbs) against AD-1 and AD-2 of HCMV gB on virus attachment as well as on virus-induced cellcell fusion were investigated. Virus induced cellcell fusion was measured by a new reporter gene activation assay (Gicklhorn et al., 1999
) based on two stably transfected astrocytoma cell lines, U373-CAT and U373-VP16, in which induction of the chloramphenicol acetyltransferase (CAT) gene is triggered on experimentally induced cellcell fusion by the transactivator GAL4-VP16. For the fusion assay, U373-CAT and U373-VP16 cells were seeded at a ratio of 1 : 1 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % foetal calf serum (FCS) into 15 mm dishes to obtain a subconfluent monolayer the next day (2x105 cells). To induce cellcell fusion, cell-free HCMV strain AD 169, grown and titrated on human foreskin fibroblasts (HF) as previously described (Eggers et al., 1992
), was routinely used at an m.o.i. of 0·5. Higher m.o.i.s were previously found to result in reduced CAT activity, most likely as a result of pronounced cytopathogenicity (Gicklhorn et al., 1999
).
The neutralizing gB AD-1-specific mAbs ITC48, ITC52, ITC63B, or the AD-2-specific mAb ITC88 (recognizing site II of AD-2) and the non-neutralizing AD-1-specific mAb ITC39, as control antibody, were used (Ohlin et al., 1993) after standard ammonium sulfate precipitation and dialysis against PBS. Human IgG was quantified with a human IgGFc ELISA Quantification kit (NatuTec) according to the manufacturer's instructions. For routine fusion-inhibition experiments, the virus inoculum was incubated with the respective mAb (6·2 µg IgG ml-1 each, diluted in DMEM) for 60 min at 37 °C. This concentration was selected as it is within the range of concentrations of AD-1-specific antibodies seen in vivo (Ohlin et al., 1997
). As negative and positive controls, a human anti-pp65 mAb (MO58) (Ohlin et al., 1991
) and an HCMV-positive human serum, respectively, were used. To obtain a baseline for the fusion process, virus was preincubated without antibody. Co-cultivated U373-CAT/VP16 cells were incubated with 200 µl of virus/antibody mixture for 60 min at room temperature to allow virus adsorption. The inoculum was removed and culture medium (DMEM supplemented with 2 % heat-inactivated FCS) was added. At 48 h post-infection, cell monolayers were solubilized in 50 µl 1x lysis buffer (Promega) and analysed for CAT activity by thin-layer chromatography according to a standard protocol (Gorman et al., 1982
). Since total cell-free virus preparations were used, the possibility that dense bodies and non-infectious enveloped particles contributed to the observed fusogenic activity (Schmolke et al., 1995
) in addition to virions cannot be ruled out.
Efficient fusion inhibition by the standard IgG concentration (6·2 µg IgG ml-1) was obtained for the human control serum as well as for the neutralizing AD-2-specific mAb ITC88, whereas with AD-1-specific antibodies (ITC52, ITC48, ITC63B) only 2060 % fusion inhibition was found (Fig. 1). Since all of these antibodies exhibited neutralizing activities (Ohlin et al., 1993
), this result underlines the relevance of AD-2 for the fusion process. HCMV-mediated cellcell fusion was unaffected by the control mAb MO58 as well as by non-neutralizing mAb ITC39.
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Subsequent experiments were thus designed to examine whether the observed inhibitory effect of gB-specific mAbs occurred at the level of virus attachment or at a later stage. To allow accurate comparisons, identical numbers of cells, inoculum volume and proportion of virus and antibody were used. Radioactively labelled [35S]AD 169 was prepared and titrated in HF cultures after purification by sucrose cushion ultracentrifugation as previously described (Eggers et al., 1992; Compton et al., 1993
). HCMV inoculums and mAbs were preincubated for 60 min at 37 °C prior to inoculation of duplicates of co-cultivated U373-CAT/VP16 cells and incubation for 90 min at 4 °C. As negative controls, heparin-sodium (Serva) at 100 IU ml-1 in DMEM was added for 60 min at 4 °C before addition of virus or during virus adsorption. The virus inoculum was removed and the cells were washed three times with PBS followed by lysis in lysis buffer (1 % Triton X-100, 1 % SDS in PBS). Cell extracts were prepared for scintillation counting (LS1801; Beckmann) as previously described (Radsak et al., 1985
).
Attachment of HCMV in the presence of heparin was strongly inhibited, whereas for mAbs ITC48, ITC52 and ITC39 and control mAb MO58, an increased attachment was observed compared with the positive control (virus without mAb) (Table 1). This enhancement of infectivity most likely reflects a phenomenon described mainly for neutralizing mAbs called antibody enhancement of infectivity (Dimmock, 1993
). Attachment in the presence of mAb ITC88 and ITC63B was slightly reduced by 48 %. This minor effect, in particular for mAb ITC88, did not correlate with the strong inhibition observed in the fusion assay. Loss of infectivity of HCMV in the presence of AD-1-specific mAbs and AD-2-specific mAb ITC88 at concentrations of 6·2 µg IgG ml-1 appeared therefore to result primarily from inhibition of viruscell fusion mechanisms and not from interference with HCMV attachment to HSPGs. This finding agrees with a previous report suggesting that another AD-2-specific antibody blocks virus penetration but not virus attachment to the cell surface (Ohizumi et al., 1992
), and also extends such findings to AD-1-specific human antibodies. Inhibition of attachment at higher IgG concentrations, however, cannot be excluded but was not considered relevant in this context.
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In the presence of the control mAb MO58 or the AD-1-specific mAbs ITC52 or ITC63B, heparin-resistant receptor binding of HCMV did occur and was enhanced by up to 20 %, whereas the AD-2-specific mAb ITC88 reduced heparin-resistant receptor binding by 30 % (Fig. 2). These results suggested that the neutralizing activity of AD-1- and AD-2-specific mAbs may involve different stages of the virus adsorption process to cells.
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ACKNOWLEDGEMENTS |
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Received 28 November 2002;
accepted 3 February 2003.