1 Affitech AS, Oslo Research Park, Gaustadalleen 21, 0349 Oslo, Norway
2 Agricultural University of Norway, Institute for Biotechnology, Ås, Norway
3 The Norwegian Institute of Public Health, Division for Infectious Disease Control, Oslo, Norway
Correspondence
O. H. Brekke
ole.henrik.brekke{at}dynalbiotech.com
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ABSTRACT |
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Present address: Dynal Biotech ASA, Ullernchausseen 52, 0379 Oslo, Norway.
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INTRODUCTION |
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Only a few human monoclonal antibodies against VZV have been generated to date (Foung et al., 1985; Sugano et al. 1987
; Lloyd-Evans & Gilmour, 2000
). The generation of human hybridomas is often associated with instability of the cell line. Thus, alternative methods for isolating human antibodies are in great demand. With the introduction of phage display technology the generation of antibody libraries is now being performed routinely, and they have been used successfully for the isolation of antibodies to virtually any antigen (Griffiths et al., 1994
; Vaughan et al., 1996
; Sheets et al., 1998
; de Haard et al., 1999
; Little et al., 1999
; Knappik et al., 2000
; Sblattero & Bradbury, 2000
; Soderlind et al., 2000
). However, whereas the isolation of specific antibodies against a pathogen is routine, it is more difficult to isolate functional antibodies (the neutralizing antibodies). As long as the antibody can be detected in the serum of a patient, it can also be isolated from the corresponding library from this donor (Williamson et al., 1993
). Such patient-derived antibodies have developed to fight infections and thus are probably the best source for functional anti-infective antibodies.
VZV is an enveloped virus expressing seven glycoproteins in the membrane, of which glycoprotein E (gE) is highly immunogenic. However, the VZV-specific antibody with the highest documented neutralization titre is directed against glycoprotein H (gH) (Grose, 1990). One antibody, a murine monoclonal antibody against gH, mAb 206, is shown to neutralize VZV in the absence of human complement (Montalvo & Grose, 1986
). Recently, it was reported that humanized fragments of this antibody neutralized VZV, although monomer fragments did not (Drew et al., 2001
).
We have utilized recombinant antibody technology to isolate and select VZV-neutralizing human antibodies from an antibody phage display library derived from a varicella patient. The isolated clones show a high neutralization effect both as monomer single-chain variable fragments (scFv) and as intact immunoglobulins. This is, to our knowledge, the first report of VZV neutralization of human monomeric scFv fragments. These antibodies may also serve as agents for the prophylaxis and post-infection prophylaxis of VZV infection.
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METHODS |
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The PCR product includes restriction-enzyme cleavage sites for PvuII and NotI (underlined) in oligonucleotides 1 and 2, respectively. The PCR product was digested with PvuII and NotI enzymes and cloned into a version of pHOG dummy (Stacy et al., 2003) in which the C-terminal myc and his polypeptide tags are removed and a FLAG tag is inserted N-terminal to the gE protein. The construct was sequenced and verified as the extracellular part of gE. Recombinant FLAGgE was expressed in E. coli. The denaturation and renaturation of gE inclusion bodies produced soluble recombinant gE. The recombinant gE was verified as structurally functional by positive binding of gE-specific monoclonal antibodies (data not shown).
Generation of VZV cell lysate and control antigen.
Early passage laboratory strain RG was used for infection of human embryonic fibroblasts (HE cells). HE cells did not undergo more than 17 passages before use. Then 0·51·5 ml virus incubated/absorbed the cells at 11·5 h at room temperature. Eagle's minimal essential medium supplemented with penicillin and streptomycin, 10 % fetal calf serum and 1 ml 5 % NaHCO3 per 100 ml medium was added and further incubated at 37 °C overnight. The medium was changed after 24 h, and cells were harvested after 52 h. Cells were sonicated for 15 s and centrifuged at 1000 r.p.m. Non-infected cells were treated in an identical way for the generation of control antigen.
Isolation of lymphocytes and serum from the patient.
Blood was drawn on days 5 and 11 after appearance of the first vesicular lesion on an adult male with primary varicella infection. Blood (10 ml) was spun at 4000 r.p.m. (2969 g) for 15 min and the serum was frozen at 20 °C. Peripheral blood lymphocytes were isolated by lymphoprep separation (Boyum, 1968). The mononuclear cells were washed twice in PBS. Cells were counted and frozen in batches of 5x107 cells.
ELISA of patient serum.
Microtitre plates were coated with 4 µg VZV cell lysate and control antigen ml1. All sera were diluted 1 : 50 in water and 100 µl of each diluted serum was added to both the VZV cell lysate and control antigen-coated microtitre wells.
The negative serum was from a person with no visible signs of ever having had a VZV infection. The positive control is serum from a person who had a varicella infection many years ago (low-positive). mAb7.88, a mouse anti-VZV monoclonal antibody (Norwegian Institute of Public Health) was diluted 1 : 1000 before use.
RNA isolation and cDNA synthesis.
Cells (2x107) were washed once in ice-cold PBS, and the cell pellet was lysed. Between 5 and 25 µg total RNA was isolated by Strataprep Total RNA miniprep kit, as described in the manufacturer's protocol (Stratagene). Donor RNA was used for first strand cDNA synthesis by way of random hexamer oligonucleotides according to the protocol of SuperScript RNase H reverse transcriptase (Gibco-BRL). Briefly, 5 µg RNA, 0·5 mM dNTPs (Fermentas) and 500 ng random hexamers (Promega) were incubated at 65 °C for 5 min, followed by rapid cooling to 4 °C. Then, 5x reverse transcriptase reaction buffer (Gibco-BRL), 10 mM DTT and 2 U RNasin µl1 (Promega) were added and incubated at 25 °C for 2 min. During incubation, 200 U SuperScript II reverse transcriptase (Gibco-BRL) was added. After 10 min, the reaction was incubated at 42 °C for 1 h. The reverse transcriptase was inactivated at 70 °C.
Variable (V) gene PCR amplification.
Oligonucleotides used for the primary amplification of V genes are described by Sblattero & Bradbury (1998). A secondary set of oligonucleotides was designed, including restriction enzyme sites in the 5' ends corresponding to the VH and VL cloning sites (see below). The PCR reactions were thus performed in two steps. First, 125 ng cDNA was used as template for the amplification of V genes. A total of nine IgG variable heavy-chain (VH), 20 variable light-chain kappa (V
) and nine variable light-chain lambda (V
) reactions were set up with V-gene-specific oligonucleotide sets. The reactions were run at annealing temperatures of 55, 58 or 61 °C (depending on the primer pair) for 30 cycles with 2 U Vent DNA polymerase (New England Biolabs) and 20 pmol of each primer pair per reaction.
One microlitre of each primary PCR reaction (100 ng) was used as template for the secondary PCR reactions. A total of 45 VH, nine V
and 20 V
PCR reactions were run for 30 cycles at an annealing temperature of 58 °C. All the oligonucleotides used in the secondary PCR introduce restriction enzyme sites: NcoI and HindIII for all VH genes, and MluI and NotI for all V
and V
genes.
Cloning of V genes into phagemid pSEX81.
A modified pSEX phagemid (G. Å. Løset, Affitech AS, personal communication) was digested with MluI and NotI and ligated with V and V
pools and electroporated into XL-1 Blue cells. The two light-chain libraries (V
and V
) showed between 3x106 and 5x106 clones. The bacterial colonies were scraped from agar plates and a total plasmid preparation from each library was isolated by DNA miniprep (Qiagen).
The light-chain plasmid libraries (pSEX VL) were digested with NcoI overnight at 37 °C. Linearized plasmid was isolated from agarose gels and purified by gel extraction kit (Qiagen) followed by secondary digestion with HindIII overnight. The digestion reaction was incubated with calf intestinal phosphate (New England Biolabs) before the double NcoI/HindIII-digested plasmid was isolated from agarose gel and purified by gel-extraction kit (Qiagen).
The NcoI and HindIII-digested pSEX VL plasmids were ligated with seven variable heavy chain pools and electroporated into XL-1 Blue cells. Colonies were grown on agar plates with ampicillin (100 µg ml1) and 100 mM glucose.
Packaging of phage libraries.
The plates with pSEX library-transformed E. coli were scraped. 2x YT (50 ml; Difco) with 100 µg ampicillin ml1, 30 µg tetracycline ml1, 100 mM glucose (2x YT-ATG) was inoculated to OD600 value of 0·025. The culture was incubated at 37 °C for 2 h. At OD600 value of 0·1, helper phage M13KO7 was added at an m.o.i. of 8. The infection continued at 37 °C for 30 min at 80 r.p.m. and for 30 min at 260 r.p.m. The culture was centrifuged at 4000 r.p.m. (2969 g) and the medium was changed to 40 ml 2x YT with 100 µg ampicillin ml1 and 50 µg kanamycin ml1 (2x YT-AK). The culture was further incubated for 6 h at 30 °C. The culture was centrifuged and the supernatant was added to 0·25 vol PEG 20 % solution. The phage library was allowed to precipitate at 4 °C overnight before it was centrifuged at 8500 r.p.m. (8885 g) for 30 min. The phage pellet was dissolved in 1 ml TE buffer (pH 7·4) and transferred to microcentrifuge tubes for the last centrifugation at 14 000 r.p.m. for 10 min to remove insoluble material.
Panning conditions.
In both rounds of selection the library was pre-incubated with control antigen. Unbound phages were subjected to further incubation with VZV cell lysate antigen. Antigen was immobilized in immunotubes (NUNC) in bicarbonate buffer pH 9·6 at concentration of 300 µg ml1 in the first round and 30 µg ml1 in the second round. After 10 times washes with PBS/0·05 % Tween 20 followed by 10 times washes with PBS, which was increased to 20 times washes in the second selection round, the bound phages were eluted with 500 µl triethylamine followed by neutralization with 500 µl Tris/HCl pH 5·5. The eluted phages were allowed to infect XL-1 Blue at OD600 value of 0·4. The infected cells were plated on LB-ATG plates and incubated at 30 °C overnight. After the first round of selection, colonies were scraped and handled as described in the previous section. From both rounds, single colonies were picked and monoclonal phages isolated and assayed by ELISA.
V-gene analysis.
Sequencing was performed at GATC GmbH, Constance, Germany. All sequenced V genes were analysed by DNAPLOT at the V-base (www.mrc-cpe.cam.ac.uk). All amino acid sequence alignments were performed using CLUSTAL W 1.8 on the BCM-search launcher (http://searchlauncher.bcm.tmc.edu).
Expression and purification of soluble scFv.
The scFv genes from round two were collectively cloned into the pHOG expression vector as NcoI/NotI-digested inserts (Stacy et al., 2003) and transformed into XL-1 Blue. One hundred clones were picked and expressed in 96 deep-well plates. The plates were centrifuged and supernatants subjected to ELISA as described below. ELISA-positive colonies were picked from the master-stock plates and grown overnight at 37 °C in LB-ATG. The overnight culture from each positive clone was further inoculated into LB-ATG medium to OD600 value of 0·1. The cultures were grown to OD600 value of 0·8. Induction was performed by changing medium to LB with 100 µg ampicillin ml1 and 100 µM IPTG and incubated overnight at 30 °C. scFv in the supernatant and periplasmic fractions were subjected to purification by metal-chelating chromatography by Ni2+ chelating sepharose and eluted by 250 mM imidazole. The purified scFv were quantified by SDS-PAGE, ELISA and OD280 measurements.
Cloning and expression of intact IgG.
The V genes from selected clones were amplified by oligonucleotides specific for V genes with extensions including restriction-enzyme cleavage sites for cloning into the eukaryotic expression vectors pLNOH2 and pLNO, as described (Norderhaug et al., 1997
). The plasmids were co-transfected into NS0 cells by electroporation. For the selection of resistant clones, 600 µg G418 ml1 was added to transfected cells. Emerging G418-resistant colonies were analysed for production of soluble IgG by ELISA. Positive clones were subjected to limiting dilution followed by ELISA, the highest-producing clones were expanded, and the supernatant harvested.
ELISA.
Ninety-six-well microtitre plates (Maxisorp; NUNC) were coated with either 4 µg VZV cell lysate and control antigen ml1, or with 20 µg recombinant gE and BSA ml1 as control overnight at 4 °C. The wells were blocked with 4 % skimmed milk before dilutions of scFv or IgG were added and incubated for 1 h at room temperature. Plates were washed with PBS/Tween followed by incubation of anti-myc antibody (1 : 5000) for scFv detection or anti-human Fc antibody (1 : 10 000) for IgG detection for 1 h at room temperature. After washing in PBS/Tween, HRP-labelled anti-mouse IgG (1 : 2000) was added and incubated for 1 h at room temperature. The plates were washed in PBS/Tween and signal developed by adding ABTS (Calbiochem). The plates were read at OD405 after 20 min.
Surface plasmon resonance (SPR) analysis of anti-gE scFv.
The CM5 dextran sensor chip was activated with a 30 µl (6 min) injection of 0·2 M N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide + 0·05 M N-hydroxysuccinimide (EDC/NHS) followed by a 30 µl injection of 1 M ethanolamine hydrochloride to deactivate excess NHS esters. The recombinant gE protein was amine-coupled at a concentration of 0·75 mg ml1. The scFv-binding analyses were performed on a BiacoreX (Biacore AB) at 25 °C with a flow rate of 50 µl min1.
VZV-neutralization assay.
A sensitive VZV micro-immunoplaque was used to analyse isolated scFv fragments and IgG molecules for the ability to neutralize VZV. Briefly, VZV cell-free stock (25x103 p.f.u. ml1) in optimal dilution were mixed with dilutions of scFv or IgG of the various anti-VZV clones, and incubated at room temperature for 1 h. The mixtures were then added to confluent monolayers of HE cells and incubated for another hour at room temperature before further incubation in a 5 % CO2 cabinet at 34·5 °C. After incubation, the cell monolayers were fixed using a mixture of ice-cold (20 °C) acetonemethanol (70 : 30) containing 0·5 % Triton X-100. The VZV plaques were visualized using a VZV-specific mouse monoclonal antibody (mAb gp7.88) and a peroxidase-conjugated rabbit anti-mouse IgG polyclonal serum (P0260; Dako). In this study, a human serum selected from a VZV- and HSV-negative blood donor was used as a source of complement (80 IU ml1). Controls used in various assays were an scFv clone of irrelevant specificity (anti-flunitrazepam), the RN donor serum (convalescent varicella patient), a VZV- and HSV-negative human serum, and an IgG1 human monoclonal antibody of irrelevant specificity (anti-6-mono-acetylmorphine). As a complement control the same serum as above was used, but had been inactivated by heating at 56 °C for 30 min.
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RESULTS AND DISCUSSION |
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Single colonies were randomly picked and expressed as soluble scFv. One hundred expressed clones were screened for binding to VZV cell lysate, control lysate, recombinant gE or BSA (data not shown). From this screening we found 20 positive anti-VZV antibodies, which were identified as eight unique clones by DNA sequencing. The deduced amino acid sequences are shown in Fig. 2.
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The functional concentration of antibodies is composed of a variety of elements such as affinity, specificity, valency, kinetics and external factors (Brekke & Sandlie, 2003). In this study, we observed an 180-fold increase in IC50 from the scFv format to IgG format for VZV5, and over 2000-fold for VZV39 (Table 1
), indicating a beneficial effect of bivalency and thus avidity. As the affinity of VZV39 is in the low micromolar area, we assume that the IC50 value can be reduced considerably if we are able to increase the antibody affinity. Methods for in vitro enhancement of antibody affinities are based on the introduction of mutations in the V genes, or introducing new variable light-chain repertoire together with the original variable heavy chain thus creating a small library repertoire of slightly altered antibodies. The subsequent small library is subjected to high-stringency selection. Thus, only antibodies with higher affinity than the mother clone will be selected (Brekke & Sandlie, 2003
). By the induction of complement, VZV39-IgG increased potency 16-fold from 5 to 0·3 nM (Table 1
). Compared with VZV4-IgG and VZV5-IgG, VZV39-IgG was about 100 and three times more potent, respectively. By genetic engineering of the IgG Fc-region, antibodies with higher complement-activation potential can be generated (Michaelsen et al.1990
; Brekke et al., 1993
). In this study, we have developed potent antibodies that can gain even higher functionality by the employment of genetic engineering of both the antibody affinity and effector functions.
Concluding remarks
The use of antibodies derived from human antibody libraries may prove to be a very successful method to develop neutralizing antibodies against certain human viruses. The isolation of one in vitro neutralizing human antibody against the S1 protein of SARS coronavirus derived from a phage display library has been described recently (Sui et al., 2004). It may also prove that human antibodies derived from human donors, as described here, will help the effort to produce better medicines for passive immunity, an important aspect of the increasing resistance to both antibiotics and anti-viral drugs.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 June 2004;
accepted 16 August 2004.
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