1 National Blood Service, Division of Transfusion Medicine, East Anglia Blood Centre, Long Road, Cambridge CB2 2PT, UK
2 Department of Haematology, Division of Transfusion Medicine, East Anglia Blood Centre, Long Road, Cambridge CB2 2PT, UK
Correspondence
Jean-Pierre Allain
jpa1000{at}cam.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Hypervariable region 1 (HVR1), a sequence of 27 residues at the N terminus of the main envelope protein E2 of HCV, is a target for neutralizing antibodies (Farci et al., 1994, 1996
; Rosa et al., 1996
; van Doorn et al., 1995
) and a possible ligand of HCV binding to cells (Basu et al., 2004
; Hamaia et al., 2001
; Kurihara et al., 2004
; Penin et al., 2001
; Scarselli et al., 2002
). However, HVR1 is highly mutated, which allows HCV to escape the host's immunity (Farci et al., 1996
; Korenaga et al., 2001
; Kumar et al., 1994
; Ray et al., 1999
; Shimizu et al., 1994
). In recent years, a number of investigators have observed that antibodies from HCV-infected patients, or from mice and rabbits immunized with HVR1 peptides, cross-react with a wide range of HVR1 peptides and can be used to capture HCV variants and inhibit HCV binding to cells (Esumi et al., 1998
; Mondelli et al., 1999
; Puntoriero et al., 1998
; Shang et al., 1999
; Watanabe et al., 1999
; Zibert et al., 1995
). Monoclonal antibodies (mAbs) are a potent treatment against many infectious agents (Casadevall et al., 2004
). As HVR1 is highly heterogeneous in its primary sequence, to obtain a mAb broadly recognizing HCV would appear difficult. Currently, most mAbs to HVR1 are poorly cross-reactive with HVR1 variants and have limited ability to recognize multiple HCV strains (Allander et al., 2000
; Cerino et al., 2001
; Triyatni et al., 2002
; Zhou et al., 2000
).
The specific immunotherapy and prevention of HCV infection by using broadly cross-reactive antibodies to HVR1 of HCV might be effective in clinical applications. In a previous study (Li et al., 2001), we discovered that a conserved epitope based on the G--Q motif at positions 2326 within HVR1 was critical to induce cross-reactive antibodies to HVR1 variants. Two high-affinity murine mAbs to the G--Q epitope of HVR1, cross-reacting with 87 % of HVR1 peptides and highly effective in capturing HCV strains and blocking HCV binding to cells, were obtained. In order to avoid the antigenicity of murine mAbs in humans, chimeric monoclonal antibodies (cAbs) keeping only the murine variable regions were produced and characterized in comparison with the parental murine mAbs to HVR1 of HCV.
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METHODS |
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Cloning of mAb variable-region genes.
The variable-region genes of the heavy (VH) and light kappa (VK) chains of mAbs 2P24 and 15H4 (Li et al., 2001) were amplified by using the mouse primers VH1 (forward, 5'-GGAACCCTTTGGCCCAGCCGGCCATGGCCSAGGTYCAGCTBCAGCAGTC-3') and CH (reverse, 5'-TARCCYTTGACMAGGCATCC-3'), and VK1 (forward, 5'-TATTCGTCGACGGATATTGTGATGACBCAGDC-3') and CK (reverse, 5'-CGTTCACTGCCATCAATC-3'), obtained from Dr T. Grunwald (Medical Research Council, Cambridge, UK), and the primers CK and MKS11 (forward, 5'-GCCCAGTTCCTGTTTCTG-3') (for the 2P24 VK chain only) obtained from Dr I. Harmer (Division of Transfusion Medicine, University of Cambridge, UK). The PCR products were cloned and sequenced as described previously (Li et al., 2001
). The nucleotide and deduced amino acid sequences were analysed and defined by the Kabat numbering system (Kabat et al., 1991
).
Construction of genes for cAbs.
The Kabat-numbered variable-region sequences of the VH and VK chains of mAbs 2P24 and 15H4 were isolated and modified by overlapping-extension PCR with Pwo DNA polymerase (Roche). The VH and VK DNA fragments were cloned into pSVgpt-B2VH-hucIgG1 and pSVhygFog-1V-HuCK vectors with HindIII and BamHI sites by replacement of the V regions, respectively. These two vectors (Furtado et al., 2002
) were kindly provided by Dr K. Armour (Department of Pathology, University of Cambridge, UK).
Transfection and antibody production.
DNA constructs containing the VH or VK chain of cAbs 2P24 and 15H4 were prepared by using an EndoFree Plasmid Maxi kit (Qiagen). The PvuII-linearized VH chain construct DNA (10 µg) was mixed with 20 µg PvuII-linearized VK chain construct DNA and co-transfected into 1x107 SP2/0 myeloma cells by electroporation (0·4 cm gap Bio-Rad cuvette, 180 V and 960 µF). After electroporation, the cells were added immediately to 20 ml RPMI 1640 medium containing 10 % FCS in a 75 cm2 flask and incubated for 4872 h at 37 °C. The cells were resuspended in 40 ml RPMI 1640 medium with 10 % FCS containing 0·8 µg mycophenolic acid ml1 and 250 µg xanthine ml1 and distributed over two or three 96-well plates at 200 µl per well. After 3 weeks, the wells containing cell colonies were screened for the presence of cAb in the culture supernatant by ELISA.
Transfectomas producing cAb 2P24 or 15H4 were cloned repeatedly in RPMI 1640 medium containing 15 % FCS and 5 % BM Condimed H1 supplement (Roche). Clones were selected according to antibody-expression levels and stability. Several clones for each cAb were adapted to hybridoma serum-free medium (SFM; Gibco), serum-free and protein-free medium (SPF; Sigma) or a mixture of both SFM and SPF. Cells were passaged in the media for 35 days and supernatants were collected for antibody-level tests.
cAbs were purified from the supernatant of SFM or SFP cultures in flasks by using Protein G columns. Some antibodies were produced by MiniPerm (Vivascience) and purified by Protein G columns.
Antibody biotinylation.
cAbs and mAbs were biotinylated by using a Micro-Biotinylation kit (Sigma). Biotinylated antibodies were used to assess the ability to cross-react with HVR1 peptides or to capture HCV.
Enzyme immunoassay (EIA).
Indirect ELISA was used for the measurement of cAb-expression levels in the supernatants of cell cultures. Goat anti-human IgG Fc fragment-specific antibody (Sigma) was coated on the plates. Goat anti-human IgGalkaline phosphatase conjugate was used as a secondary antibody for detecting cAbs bound to the coated anti-human IgG Fc fragment. Human IgG1
(Sigma) was used as a standard.
The reactivity of biotinylated cAbs and mAbs with various HCV peptides was measured by peptide EIA in Nunc-Immuno plates (Maxisorp; Nalge Nunc) as described previously (Li et al., 2001). Levels of antibody reactivity to HVR1 peptides by EIA were presented as sample/cut-off (S/CO) ratios. The cut-off was calculated as the mean of the A405 values of non-HVR1 peptides+6SD.
Affinity measurements.
The affinity of the cAbs and mAbs was determined against seven selected keyhole limpet haemocyanin (KLH)/BSA-conjugated HVR1 peptides (EH, MH2, MH5, S67, S85 S90 and L1.1) (Jackson et al., 1997) by using an IAsys optical biosensor (Affinity Sensor) as described previously (Li et al., 2001
; Zhai et al., 1999
). Affinity constants (Kd) were calculated from these measurements as Kdiss/Kass by using the FASTFIT program.
Real-time quantitative RT-PCR analysis of HCV RNA.
HCV RNA was measured by real-time quantitative RT-PCR using the Mx4000 Multiplex Quantitative PCR system (Stratagene) as described previously (Candotti et al., 2003). For each run, duplicates of a tenfold serial dilution of WHO International Standard for HCV RNA for NAT assays 96 and 790 (NIBSC) containing 4x1024x105 IU HCV genome ml1 were used as a standard curve for quantification of HCV RNA. Each sample was analysed in duplicate and the results were averaged. The sensitivity of quantitative RT-PCR for detecting HCV in plasma was 100 IU ml1.
HCV capture.
Unselected plasmas from chronically HCV-infected patients were centrifuged for 2 min at room temperature. The plasma supernatant was collected for two sample preparations: (i) plasma or its dilution (1 : 10 in PBS) (native sample) was untreated and used directly for the HCV capture test; (ii) plasma or its dilution (1 : 5 in PBS) was chromatographed through a 1 ml Protein G column (Amersham Biosciences) to obtain IgG-depleted HCV plasma for use in the HCV capture test. Native or IgG-depleted HCV plasma (100 µl) was added to a 50 µl mixture of biotinylated cAbs 2P24 and 15H4 or mAbs 2P24 and 15H4 in PBS (20 µg ml1) containing 0·1 % Tween 20 and 4 % BSA and pre-incubated at 37 °C for 1·5 h and then overnight at 4 °C. Fifty microlitres of 2 mg streptavidin-coated magnetic particles ml1 (Promega) was added to the biotinylated antibodyHCV mixture and incubated for 1 h at room temperature. Biotinylated mouse myeloma IgG1 (Sigma) was used as a negative control in each assay. After four washes with PBS containing 0·1 % Tween 20, HCV RNA was extracted from the magnetic particles by using a High Pure Viral RNA kit (Roche) and detected by real-time quantitative RT-PCR (Candotti et al., 2003).
Inhibition of HCV binding to target cells.
One hundred microlitres of various dilutions of cAbs and normal mouse myeloma IgG1 was pre-incubated with 50 µl IgG-depleted HCV-containing plasma or native HCV-containing plasma for 2 h at 37 °C and then at 4 °C overnight. The mixture was added to 2x105 Molt-4 cells in a 250 µl final volume and incubated at room temperature for 1 h. The cells were washed four times, and viral and cellular RNA was extracted by using an RNeasy Mini kit (Qiagen) and tested for the presence of HCV RNA by real-time quantitative RT-PCR as described above. Under identical conditions, 50 µl HCV-containing plasma without cAb pre-incubation and normal plasma were added to cells as positive and negative controls, respectively.
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RESULTS |
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Production of cAbs
Two human IgG1 versions of mAbs 2P24 and 15H4 were produced. Chimeras of VK and VH were made, retaining the original murine complementarity-determining regions and framework regions, but with the human CK and CH1 (IgG1) constant regions, respectively. In the cAb-expressing vectors (Furtado et al., 2002), the complete DNA sequence of cAb 2P24 or 15H4 contained, at its 5' end, the Ig promoter, the eukaryotic exon and intron leader sequences, the last 4 aa of the leader region (GVHS, which form part of the V region exon) and, at its 3' end, the 5' end of the first half of the VC intron.
After co-transfection of SP2/0 with the VH and VK chain construct DNAs containing murine mAb variable regions and human antibody (IgG1) constant regions, the full-length cAb VH and VK chains were expressed and whole molecules of cAb were assembled and secreted in the culture medium. The cAb 2P24-producing cell line was cloned seven times and adapted to a mixture of 25 % SFM and 75 % SPF. The cAb 15H4 cell line was cloned three times and adapted to SFM. The final clones of the cAb 2P24 cell line in SFM/SPF medium and the cAb 15H4 cell line in SFM were passaged for more than 6 months and remained stable. Levels of cAbs 2P24 and 15H4 in the supernatants ranged from 2 to 4 µg ml1 and from 3 to 5 µg ml1, respectively, after 35 days in culture. The concentrations of cAbs 2P24 and 15H4 reached 10 µg ml1 in the saturated cell-culture flasks and 36 µg ml1 in MiniPerm (Vivascience) culture.
Fine mapping of the conserved HVR1 epitope recognized by the cAbs
Our previous data suggested that mAbs 2P24 and 15H4 recognize a G--Q-based motif of HCV HVR1, as HVR1 peptide substituted at G and Q (positions 23 and 26 of the HVR1 sequence) with V and L, respectively, no longer reacts with the mAbs (Li et al., 2001). To confirm that the basic structure of G--Q was a conserved epitope, four 9mer HVR1 peptides, MH2-C, MH5-C, G1245-C and EH-C (covering positions 1927), and a 15mer, MH2, containing the G--Q motif with substitutions at positions other than positions 23 and 26, were tested in a competitive EIA. Fig. 1
(a) shows that the binding of cAb 2P24 to the 15mer HVR1 peptide MH2 was inhibited competitively by 9mer HVR1 peptides MH2-C, MH5-C, G1245-C, EH-C and MH2, but not by a control core peptide, S5. In contrast, the binding of cAb 15H4 to MH2 was not affected by any of the 9mer HVR1 peptides or by S5, but was inhibited competitively by the 15mer HVR1 peptide, MH2 (Fig. 1b
). The results further confirmed that the recognition of cAb 2P24 was based entirely on the conserved G--Q motif and that the recognition of cAb 15H4 was not limited to that motif. The recognition of the conserved HVR1 epitopes by cAbs 2P24 and 15H4 was thus maintained from the original murine mAbs 2P24 and 15H4.
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HCV capture by cAbs and mAbs
Eighteen non-selected HCV-containing plasmas were used for HCV capture by the cAb or mAb mixture. HCV was captured from native plasmas that contained antibodyHCV complexes and from IgG-depleted plasmas that contained mostly uncomplexed virus. The captured HCV was detected by real-time RT-PCR. Eighty-nine per cent of HCV strains were captured (Table 3). Results suggested that the antibody capacity for HCV capture was not dependent on the genotype of HCV and that mostly free HCV was captured. The first round of HCV capture from IgG-depleted plasmas ranged from 1 to 60 % of total HCV, but complexed virus in native plasma was captured poorly. The ability to capture more HCV in a second round of capture of the unretained (free) virus fraction suggested that the majority of free virus was susceptible to capture (data not shown).
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DISCUSSION |
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Two cloned transfectomas harbouring cAb 2P24- or 15H4-expressing construct DNA were stabilized in SFM or SPF, facilitating antibody purification for clinical use.
The epitope recognized by mAbs 2P24 and 15H4 to HCV HVR1 is located at the C terminus of HVR1 and based on the G--Q motif (positions 2326). This was confirmed by the fact that mAb recognition of HVR1 no longer occurred after substitution of G at position 23 or Q at position 26 with V or L, respectively (Li et al., 2001). Four 9mer HVR1 peptides spanning positions 1927 competitively inhibited the 15mer HVR1 peptide MH2 binding to cAb 2P24, but not to cAb 15H4 (Fig. 1
). The results suggested that cAb 2P24 essentially reacts with the conserved G--Q epitope of HVR1. In contrast, cAb 15H4 interacts with a broader region of the C terminus of HVR1, as determined previously with the parental mAbs.
The affinity of cAbs and mAbs was generally similar, except that cAb 15H4 did not react with S85 HVR1 peptide, whereas mAb 15H4 did. This feature was retained in the cAb recognition of HVR1. A mixture of the two biotinylated cAbs or mouse mAbs reacted almost equally with a panel of 37 HVR1 peptides. The slight discrepancy in S/CO values in some HVR1 peptide EIAs between cAbs and mAbs was seen in both directions and could be related to slightly different affinities between cAbs and mAbs.
The capacity of cAbs to cross-react broadly with wild-type HCV variants was demonstrated by HCV capture. Eighty-nine per cent of HCV strains were captured by cAbs, which was consistent with the 86 % of HVR1 peptides recognized by cAbs, suggesting that the recognition of cAbs for HCV is based on the conserved HVR1 epitope exposed on HCV. However, some HCV strains containing PGAKQN in the HVR1 sequence, such as UKEH, G4720 and US (Li et al., 2001; Yanagi et al., 1997
), were captured at low levels (ranging from 0·9 to 4·8 % in Table 3
) by cAbs, but their corresponding HVR1 peptides containing the same sequence reacted strongly with cAbs, indicating that some particular HVR1 peptides might not fully represent the partially conformational epitopes of HVR1 on wild-type HCV particles. The percentages of captured HCV from IgG-depleted HCV plasmas were much higher than from native plasmas, suggesting that mostly uncomplexed (free) HCV was captured. Free HCV is considered the infectious portion of HCV. HCV genotypes 1, 2 and 3 were used in the capture assay and the results showed no difference among genotypes, suggesting that the capture was independent of HCV genotype.
The lack of HCV replication in cell culture limits the measurement of protective antibodies to HCV by classical neutralization assays. In our study, we used an alternative blocking assay for predicting the potentially protective ability of antibodies to HCV. The ability of cAbs to inhibit HCV binding to human target cells in vitro was measured by native and IgG-depleted HCV plasmas. At low concentrations (0·8 µg ml1), cAbs blocked HCV binding to Molt-4 cells substantially. This function, associated with a high capacity to capture HCV, suggests that cAbs to HVR1 possess a strong potential to neutralize HCV.
One of several strategies for immunotherapy or prophylaxis of HCV infection is to prevent the binding of infectious virus to target cells. Neutralizing mAbs with a broad cross-reactivity to HCV are suitable for that purpose (Mondelli et al., 2003). The blocking and broadly cross-reactive cAbs to HVR1 presented here might be of substantial help in preventing nosocomial infection and transplanted liver reinfection by HCV.
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ACKNOWLEDGEMENTS |
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Received 21 January 2005;
accepted 17 February 2005.
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