Department of Immunotechnology, Lund University, PO Box 7031, S-220 07 Lund, Sweden1
Department of Immunology, Royal Free and University College Medical School, Royal Free Campus, London NW3 2PF, UK2
Author for correspondence: Mats Ohlin. Fax +46 46 2224200. e-mail mats.ohlin{at}immun.lth.se
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The gene encoding the AE11 single chain variable fragment (scFv) corresponding to the human antibody ITC88 (Ohlin et al., 1993 ), which recognizes AD-2 on HCMV gB, has been described previously (Ohlin et al., 1996
). This gene was recloned into the pPICZ
vector (Invitrogen) for large-scale production of soluble protein carrying the FLAG epitope (Hopp et al., 1988
) (named AE11F) in Pichia pastoris. Following induction of protein expression with methanol, the scFv was purified by a combination of ultrafiltration (Filtron Ultrasette with Omega membrane, MWCO: 10 kDa; Pall Gelman Sciences), ion exchange chromatography (SP Sepharose Fast Flow; Amersham Pharmacia) and size exclusion chromatography on Superdex 75 (Amersham Pharmacia). Both the monomeric and dimeric forms of the scFv were obtained in this way. Microsequencing of the N terminus of the product confirmed that the yeast-derived leader sequence was properly removed.
The antibody neutralization assays were performed with the AD169 strain of HCMV (European Collection of Animal Cell Cultures, Salisbury, Wiltshire, UK) in 48-well plates, essentially as described by Roy & Grundy (1992) . In addition to purified monomers and dimers of the AE11F scFv, dimerized monomeric scFv were also analysed. These were obtained by incubation of monomeric scFv in minimal essential medium containing 2% fetal calf serum for 30 min at 37 °C with anti-FLAG antibody (anti-FLAG M2; Sigma Chemical Company) at a molar ratio of 2:1. Serial dilutions of the various scFv preparations were incubated for 60 min at 37 °C with the concentration of virus required to produce 50100 viral plaques per well. The various dilutions were then inoculated in triplicate for 60 min on to fibroblast monolayers. The cell monolayers were washed twice and a 2% methylcellulose overlay was added. After incubation for 12 days at 37 °C in 5% CO2, the cells were fixed in 10% (v/v) formalin and stained with 0·03% methylene blue. The numbers of viral plaques per well were counted, and the mean number of p.f.u./ml was calculated for each dilution of a given antibody format. The plaque reduction and the effective concentration giving 50% plaque reduction (EC50) were calculated using the ReedMuench method (Thorpe et al., 1987
). In order to investigate the antigen-specificity of the interaction, neutralization assays were also performed in the presence of an AD-2-mimicking peptide (ANETIYNTTLKYGDV). A peptide incorporating residues known to be important for recognition by the scFv (Ohlin et al., 1996
) in scrambled order (YTLETYINKT) was used similarly as a negative control. The assays were performed with dimeric scFv at a concentration approximately equal to their EC50 value (1 µg/ml).
The reaction rate kinetics of the scFv with the peptide antigen and recombinant gB produced in eukaryotic cells (Spaete, 1991 ) were studied using the BIAcore technology (BIAcore AB). The antigens were bound to a CM5 sensor chip either through direct coupling using the Amine Coupling Kit (BIAcore AB) in the case of gB (approximately 15000 RU) or through binding to similarly immobilized streptavidin (approximately 800 RU) in the case of the biotinylated peptide antigen.
Immunofluorescence studies showed that both monomers and dimers of the AE11F scFv bound to virus-infected cells and displayed similar titration endpoints (data not shown). When assessing the neutralizing capacity of the various preparations, it became apparent that the dimeric but not the monomeric scFv format could mediate virus neutralization in vitro as assessed by their effect on the ability of the virus to infect human embryonic lung fibroblasts (Fig. 1). The EC50 value of the dimeric scFv was calculated at 0·6 µg/ml, while virus neutralization could not be detected with the monomeric form, even at a concentration of 10 µg/ml (data not shown). The neutralizing activity of the monomeric protein could, however, be restored on addition of a cross-linking antibody, which recognizes the C-terminal FLAG epitope on each scFv (Fig. 1
). The EC50 value of this format was found to be 0·5 µg/ml, i.e. more or less the same as for the dimeric scFv. We thus concluded that neutralization of HCMV with AD-2-specific antibodies or fragments thereof requires a bulkier, divalent construct to be effective. The antigen specificity of the interaction was demonstrated by the fact that the scFv-mediated neutralization could be inhibited by the addition of a soluble 15-mer peptide recognized by the scFv (Fig. 2
).
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References |
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Received 17 December 2001;
accepted 26 April 2002.