Research Department, Pasteur Mérieux Connaught, 69290 Marcy lEtoile, France1
UMR 103 CNRS-bioMérieux, ENS, 69007 Lyon, France2
Lindsley F. Kimball Research Institute, NY 10021, USA3
Author for correspondence: Veronique Barban. Fax +33 437373189. e-mail vbarban{at}fr.pmc-vacc.com
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Abstract |
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Introduction |
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The main target for HCV diagnosis is the structural nucleocapsid protein. This protein is well conserved among different HCV genotypes (Bukh et al., 1994 ) and anti-capsid antibodies are present in the majority of patients with chronic infection (Harada et al., 1991
; Manzini et al., 1993
). As an example, the recombinant C223 protein, spanning amino acids (aa) 2120 of the nucleocapsid protein, is the major component of the commercially available second-generation anti-HCV tests (Chemello et al., 1993
; Hosein et al., 1991
).
The capsid gene is located at the 5' end of the genome. A large unique polyprotein precursor (Grakoui et al., 1993 a , b
, c
; Manabe et al., 1994
; Tanji et al., 1994
) is directly translated from the viral RNA (Choo et al., 1989
) and post-translationally processed by both host and viral proteases (Hijikata et al., 1993
; Manabe et al., 1994
). Structural proteins, including capsid (C) and two envelope proteins (E1 and E2), are generated by sequential cleavages in the first one-third of the polyprotein, while non-structural proteins (NS2, NS3, NS4 and NS5) are generated from the remaining two-thirds.
Several studies, based on scanning of the protein with synthetic peptides, have indicated that the HCV capsid protein contains multiple linear, highly immunogenic epitopes (Akatsuka et al., 1993 ; Cerino et al., 1993
; Chiba et al., 1991
; Ferroni et al., 1993
; Goeser et al., 1994
; Moradpour et al., 1996
; Nasoff et al., 1991
; Pujol et al., 1996
; Sallberg et al., 1992
, 1994
; Yoshikawa et al., 1992
). Conversely, very little is known about naturally occurring nucleocapsid conformational epitopes, mainly because only a few antibodies have been recovered from HCV-infected individuals (Akatsuka et al., 1993
; Cerino et al., 1993
; Siemoneit et al., 1994b
).
In recent years, the phage display system, originally developed by Parmley & Smith (1988) for peptide library presentation and identification of epitopes, has been widely applied to express antibody fragments on the phage surface (for a review see Winter et al., 1994
). This process may be considered as a powerful alternative to the hybridoma technology of Kohler & Milstein (1975)
. Human antibody libraries can be generated by random combinatorial linkage of diverse repertoires of VH and VL genes from lymphocytes (Barbas et al., 1991
; Burton & Barbas, 1994
; Clackson et al., 1991
; Hoogenboom et al., 1992
; Huse et al., 1989
; Lerner et al., 1992
; Marks et al., 1992
; McCafferty et al., 1990
; Waterhouse et al., 1993
). Antibody fragments are displayed on the surface of phage fd fused to a minor coat protein (pIII), and specific antibodies are obtained by sequential rounds of selection (biopanning) on variously presented antigens (Griffiths et al., 1994
; Williamson et al., 1993
).
In this paper, we present the construction of a combinatorial human antibody library from an HCV-positive non-symptomatic patient, and the isolation and characterization of specific recombinant Fabs directed against core antigen. By screening a synthetic peptide library, we also show that one of these Fabs defines a new human B cell epitope within the immunodominant N-terminal region of the HCV nucleocapsid.
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Methods |
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Human sera.
Serum 1 was obtained from a 35-year-old female blood donor woman who had been anti-HCV positive for at least 5 years but had normal ALT levels and no clinical symptoms of disease. Viral genome was transiently detected in blood by RTPCR (Simmonds et al., 1993 ), and corresponded to HCV genotype 1a (Bukh et al., 1992
). Serological studies with the Innolia AbIII diagnostic kit (Innogenetics) showed strong reactivity against all HCV antigens except NS5, suggesting that HCV infection is immunologically regulated in this patient (Table 1
). Serum 2 was obtained from a chronic carrier, successfully treated with IFN-
for 18 months. Six months after the end of the treatment, this patient exhibited normal ALT and was RTPCR negative. Serum 2 exhibits a clear reactivity against HCV antigens in the RIBA-2 assay (Chiron), with the Innolia AbIII diagnostic kit and in ELISA against core antigen. Serum 3 was obtained from a patient who developed acute hepatitis, confirmed by RTPCR, after an accidental needle stick injury. Seroconversion (RIBA-2 assay) was observed and the patient was treated with IFN for 3 months. At the end of the treatment the patient exhibited normal ALT and was RTPCR negative, suggesting that a chronic infection was not successfully established. Serum 3 is positive in RIBA-2, but reacts only partially in Innolia AbIII against non-structural proteins NS3 and NS4. No reactivity against core antigens has been observed, either in Innolia AbIII or by ELISA.
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Panning of libraries.
For panning of the Fab library, immunotubes (Maxisorp, Nunc) were coated with C1119 recombinant antigen at a concentration of 1 mg/ml, for 1 h at 37 °C, blocked in 5% non-fat milk in PBS, and incubated with 5x1011 phages for 2 h at 37 °C. Several washes in PBS0·05% Tween 20 (PBS-T) were performed as described in Engberg et al. (1995) , and specific phages were eluted with 500 µl 100 mM triethanolamine for 15 min. The pH was decreased to neutrality by adding 100 µl 1 M TrisHCl pH 7 and eluted phages were amplified in E. coli XL-1 Blue strain (Stratagene). After concentration and titration, 1011 phages were used for another round of panning. Four successive rounds were performed in this way. Panning of the peptide library (pHD7, New England Biolabs) was performed as recommended by the manufacturer. Washes, elution and amplification of specific phages were done as described above (this section). At the third and fourth round, the eluted phages were titrated and 12 individual plaques were picked and amplified in E. coli. Phage DNA minipreparations were performed using standard M13 procedures and DNA was sequenced on an automated sequencer (LI-COR), according to the manufacturers instructions.
Anti-phage ELISA.
Antigen solutions were coated onto an enzyme immunoassay microtitration plate at 1 or 2 mg/ml, depending on the antigen, and the plate was incubated for 16 h at 4 °C. Goat anti-human Fab IgGs (Sigma), C1119 and C148 recombinant antigens were diluted to 1 µg/ml in PBS; core-derived peptides were diluted to 5 µg/ml in PBS. Wells were then blocked for 1 h in 5% non-fat milk in PBS, 1091010 phages in PBS+1% non-fat milk added and the mixture incubated for 2 h at 37 °C. After extensive washing with PBS-T, 100 µl anti-fd bacteriophage biotin conjugate (Sigma) diluted to 1:2000 was added to each well for an additional 1 h of incubation at 37 °C. After another round of extensive washes in PBS-T, plates were incubated for 10 min with streptavidin biotinylated horseradish peroxidase complex (Amersham). The reaction was then developed for 45 min with o-phenylenediamine as substrate and stopped with 50 µl 1 M sulfuric acid before reading on an automated system equipped with a 492 nm filter (Multiscan Titertek, ICN Flow). Results are expressed as the ratio of relative absorbance on specific antigen to relative absorbance on anti-human Fab, after deduction of background absorbance.
Competition experiments.
For inhibition of binding experiments, a phage dilution which results in approximately 40% reduction of the absorbance value after twofold serial dilutions on antigen-coated plates was mixed with serial tenfold dilutions of peptides in PBS. For competition experiments, the phage dilution which results in approximately 80% reduction of the absorbance value in the same conditions was mixed with twofold serial dilutions of human antisera in PBS. These mixtures were tested in a standard anti-fd ELISA. Values are expressed as a percentage of the relative A490 absorbance obtained with the same quantity of phages incubated without competing peptides or sera.
Construction and purification of soluble Fabs.
Phagemid DNA was digested with SpeI and NheI (1040 U/µl, Boehringer Mannheim) to excise gene III, gel-purified, and cohesive ends ligated to allow expression of a His-tailed VH gene. This construction lead to the insertion of two extra amino acids (a tyrosine and a serine) between the His tail and the N terminus of the VH region. A 2 l fermenter of TGM-16 broth containing carbenicillin (50 µg/ml) and MgCl2 (20 mM) was inoculated with a preculture of the appropriate clone. Secretion of soluble rFabs (sFabs) was induced, when the culture reached an OD600 of 0·6, with 1 mM IPTG for 8 h at 37 °C. Cells were collected by centrifugation (Beckman J-6B; 3000 g, 45 min, 4 °C) and the supernatant fraction was removed. The cell pellet was resuspended in 1/20 of the original volume of PBS and submitted to four rounds of freezing (liquid nitrogen) and thawing (56 °C). After centrifugation (Beckman J-21B; 10000 g, 30 min, 4 °C), the supernatant was recovered and dialysed overnight against 50 mM TrisHCl, 500 mM NaCl, pH 8·0. The filtered (0·45 µm) supernatant was loaded on a 5 ml HiTrap chelating column. (Pharmacia), washed with dialysis buffer and eluted with 50 mM TrisHCl, 500 mM NaCl, 500 mM imidazole, pH 8·0 (1 ml/min). Eluted fractions were pooled, dialysed against PBS overnight at 4 °C, concentrated (Centricon 3, Millipore), and then analysed by SDSPAGE (Phast system, Pharmacia) according to the manufacturers instructions, and by Western blot using a light chain-specific anti-human IgG (Fab) (data not shown). Purity was around 80%. Culture yields were in the order of 5 mg/l for sFabC3, and 0·8 mg/litre for sFabC14. This difference is probably due to toxicity of sFabC14 when over-produced in bacteria. Indeed, we observed a clear inhibition of bacterial growth after induction with IPTG. The sFab concentration was determined in a sandwich ELISA by comparison with known concentrations of purified human IgG (Fab) fragment, and compared to the total protein concentration of the sample.
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Results |
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Panning of the surface library against C1119 antigen
The phage surface expression library was panned against a core-derived recombinant antigen (C1119, aa 1119) coated on immunotubes. We used a truncated form, deleted from the C-terminal hydrophobic region, and purified after solubilization in 1 M guanidium.HCl, in order to break core recombinant antigennucleic acid interactions. This truncated antigen is very similar to C22 (aa 2120), which was a component of the first generation diagnostic kits. Recombinant phages were analysed by anti-fd ELISA. As it is difficult to control Fab surface expression on phage [it is commonly considered that only 110% of phages actually carry an Fab molecule at their surface, due to proteolysis (Hoess et al., 1994 )], the anti-fd ELISA assay was standardized by incubating the phage minipreparations on anti-human Fab (light chain-specific)-coated plates, and expressing the results as the ratio of specific absorbance of HCV antigen to anti-human Fab.
Twelve out of eighteen (70%) isolated Fab-carrying phages (rFabCs) from panning 4 were specific for C1119 recombinant antigen in this ELISA and were further analysed for their ability to recognize C1119-derived polypeptides. Only two different clones were identified, on the basis of sequence analysis: a major one, rFabC14 (11/12 clones), and a minor one, rFabC3 (1/12). These two clones recognize C148 (aa 148) recombinant antigen, but not C3881 (aa 3881) (Fig. 1). More precisely, phage rFabC3 recognizes C220 (aa 221) peptide, while no significant reactivity against either C220 or C2245 (aa 2245) peptides was observed with rFabC14. To test the hypothesis that rFabC14 could recognize a linear epitope overlapping C220 and C2245, reactivity of this recombinant was assayed on peptide C1824 (aa 1824), which is known to contain an immunodominant epitope (Jolivet-Reynaud et al., 1998
). No significant reactivity was observed (A470=0·040 with 1011 phages; not shown). This may indicate that rFabC14 is directed against a conformation-dependent epitope present on C148 but not present on the derived peptides. We cannot exclude, however, that coating of these uncoupled peptides to plastic drastically affects their conformation, as suggested by Chan et al. (1996)
from similar observations, leading to an absence of reactivity.
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Competition with human sera
To confirm that the recombinant phages, rFabC3 and rFabC14, present anti-HCV-specific Fabs on their surface, they were tested for their ability to compete with human anti-HCV-positive sera for recognition of anti-C148 recombinant antigen. The initial serum from the patient whose antibody repertoire was cloned (serum 1) as well as two other anti-HCV-positive sera (sera 2 and 3) was independently tested. All three exhibit a clear reactivity against HCV antigens; serum 1 and 2 also react against core epitopes, while serum 3 does not. Fig. 2 shows that serum 1 and serum 2, but not a pool of 10 sera from non-immune donors, are able to block the binding of the two Fab-carrying phages, rFabC3 and rFabC14, in a concentration-dependent manner. There is a correlation between the titres in ELISA against C148 and the ability to block the two rFabCs: serum 1 has an ELISA titre of 1:600, and complete inhibition is reached at a serum dilution of 1:16, while serum 2, with an ELISA titre of 1:200, completely inhibits rFabCs binding at a dilution of 1:4. Using serum 3 as a source of competing antibodies, we observed an incomplete, but significant, inhibition effect (around 20% at dilution 1:2) of the binding of rFabC14 but not of rFabC3. This last result was unexpected considering that serum from this patient does not recognize capsid polypeptides in direct detection assays. It may indicate that serum 3 contains low, but significant, titres of antibodies with the same specificity as rFabC14.
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The identified peptide sequences were chemically synthesized. The C terminus was blocked in order to mimic the peptide link with pIII protein and to avoid modifications of the ionic charge of the peptides by introducing an extra negative charge. As shown in Fig. 5, binding to C148 recombinant antigen of soluble Fabs, sFabC3 and sFabC14, can be completely inhibited by their respective synthetic peptides, but not by C1824 peptide. 50% inhibition of sFabC3 was observed with concentration of QLITKPL and QRKTKRN peptides between 105 and 106 M-1. However, the natural peptide QRKTKRN was unable to inhibit more than 50% of sFabC3 binding, suggesting that this peptide do not contain the entire epitope sequence recognized by sFabC3. A similar concentration was necessary to reach 50% inhibition of sFabC14 using HAFPHLH peptide, while only 107108 M-1 was necessary using SAPSSKN peptide, indicating that this sequence has a greater affinity for sFabC14 than the former. No inhibition of sFabC3 or sFab C14 was observed using a non-related peptide sequence with similar isoelectric point (not shown).
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Discussion |
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Only two different recombinant phages were selected at the end of the panning, one (rFabC14) being represented 10 times more than the other (rFabC3). This low number can be attributed to the panning conditions, especially washes, which were done in order to select high-affinity binders. These phages carry human antibody specificities naturally induced during human infection, as demonstrated by inhibition of binding by human anti-HCV-positive sera, and we observed a correlation between anti-core serum titres and the level of inhibition. Such a correlation has been observed previously in a similar study with a recombinant scFv selected against a peptide scanning aa 6688 on core protein. (Chan et al., 1996 ). However, despite high serum titres of anti-peptide antibodies (1:12800), complete inhibition of scFv binding was never observed (76%), probably reflecting the fact that antigen presentation by small peptides is not optimal.
We found antibodies with the same specificity as FabC14 in serum 3. The serum of this patient does not contain any detectable antibody against major capsid epitopes, probably because the infection was not successfully established (possibly due to IFN treatment). Consequently, we deduced that FabC14-like antibodies could have appeared very early after infection. The absence of reactivity against core in Innolia assay is expected as FabC14 does not recognize any peptide in this assay, and we believe that the ELISA assay is not adapted for detection of low-abundant antibodies of these type.
Epitope mapping of the two recombinant antibodies
rFabC3 recognizes a linear epitope contained in the first 21 aa of the capsid molecule, as deduced from anti-C220 ELISA. Using overlapping peptides, several authors have described a linear epitope in this region, both in the murine and in the human system (Nasoff et al., 1991 ; Okamoto et al., 1992
; Pujol et al., 1996
; Jolivet-Reynaud et al., 1998
) The sequence RKTKRNTN (aa 916) has been identified as a major antigenic site in humans and in chimpanzees (Sallberg et al., 1992
; Wang et al., 1996). The sequence QLITKPL that we isolated from the peptide library exhibits significant homology with the region QRKTKRN within aa 814. Both sequences are able to compete-out sFabC3 from recombinant antigen with similar concentrations, but competition is only partial with QRKTKRN peptide. We concluded from this experiment that the region between aa 814 is part of the epitope mimicked by QLITKPL sequence. However, sFabC3 has a low affinity for both peptides: we calculated that a 100- to 1000-fold molar excess of peptide is needed to reach maximum inhibition. We suggest that either neither of these two sequences contains the entire FabC3 epitope, or FabC3 is a low-affinity antibody. By further screening of the peptide library, higher-affinity sequences could be isolated which would help to discriminate between these two hypotheses.
Of the two peptide sequences we have identified as ligands for sFabC14, SAPSSKN is the one which exhibits the highest affinity for sFabC14: complete inhibition of binding of the soluble Fab occurs in conditions of molar excess of peptide ranging from 1 to 10. However, this sequence shares no homology with the primary HCV core sequence, and we suggest that it could represent a mimotope of a conformation-dependent epitope. The N-terminal region of the capsid contains multiple overlapping epitopes, and studies in the murine system as well as in humans suggest that there is at least one major conformational epitope residing within the first 82 aa of the HCV core protein (Cerino et al., 1993 ; Goeser et al., 1994
; Moradpour et al., 1996
; Nasoff et al., 1991
). More precisely, a conformational epitope has been previously identified by several teams in the region spanned by aa 2045 (Cerino & Mondelli, 1991
; Nasoff et al., 1991
; Pujol et al., 1996
; Siemoneit et al., 1994a
). However, there are no data about a conformational epitope being present in the region spanned within aa 221. Consequently, the epitope recognized by sFabC14 may represent a new human conformational epitope on HCV core protein.
Recombinant FabC14 does not recognize C220 peptide in ELISA, but it can be competed out from C148 recombinant antigen with low doses of the same peptide (107108 M-1). This suggests that the two recombinant antibodies recognize either two different epitopes, or two regions, a structured and a non-structured one, which are parts of the same epitope. Only structural analysis will allow us to discriminate between these two possibilities, and further studies will include localization of SAPSSKN, HAFPHFL and QLITKPL peptides on the three-dimensional model of HCV core region 148, which has been recently deduced from NMR studies (Penin et al., 1997 ).
Conclusions
The library reported in this paper represents an important tool for isolating specific antibodies against different HCV viral antigens, such as anti-envelope antibodies that are present at low titres in naturally infected individuals. The fact that HCV infection was naturally down-regulated for at least 5 years in the patient we chose encourages us in our effort to isolate anti-HCV neutralizing antibodies, which are of crucial interest for serotherapy or vaccine development.
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Acknowledgments |
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Footnotes |
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References |
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Received 19 August 1999;
accepted 28 October 1999.