Immunology Division, Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, Republic of South Africa1
Department of Genetics, University of Pretoria, Pretoria, Republic of South Africa2
Author for correspondence: Dion du Plessis. Fax +27 12 5299431. e-mail dion{at}moon.ovi.ac.za
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Abstract |
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Introduction |
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Sequence comparisons show that, of the four major structural proteins, VP2 has the greatest variability between serotypes (Iwata et al., 1992 ; Williams et al., 1998
). Moreover, together with VP5, it is one of two proteins recognized by virus-neutralizing antibodies (Burrage et al., 1993
; Vreede & Huismans, 1994
; Martínez-Torrecuadrada & Casal, 1995
; Martínez-Torrecuadrada et al., 1999
). Expression in E. coli of restriction enzyme fragments has broadly defined a neutralization domain located between amino acid residues 253 and 413 on VP2 of AHSV serotype 4 (Martínez-Torrecuadrada & Casal, 1995
). While immunogenic regions capable of eliciting neutralizing antibodies have been mapped to a resolution of 34 residues with this serotype, very little is known regarding the sites on VP2 of any serotype recognized by the antibodies in an immune serum. Such information is likely to be of paramount importance in developing new immunodiagnostic approaches. For example, epidemiological studies and disease surveillance often require a knowledge of which serotype occurs in a particular area. Even if no virus can be isolated, this can be done by plaque-reduction assays with antiserum from infected or carrier animals (Barnard, 1993
). For rapid serotyping, however, an ELISA that can distinguish antibodies to the different serotypes would have distinct advantages. Accordingly, it would be extremely useful to find out whether VP2 has any highly antigenic regions that can be mimicked by linear peptides and, at the same time, correlate with regions of significant inter-serotype sequence variability. A knowledge of where antibodies bind to VP2 could also contribute towards further characterization of neutralization domains and, in the absence of X-ray diffraction data, can give some indication of how the polypeptide is folded by identifying accessible amino acids. Computer programs can sometimes predict epitopes (Van Regenmortel & de Marcillac, 1988
), but to locate the antigenic regions by experimental means probably remains a more reliable approach.
Filamentous phage display (Smith, 1985 ) offers a relatively direct and highly selective approach to the fine-mapping of epitopes on viral and other proteins. A collection of random fusion peptides encoded either by degenerate oligonucleotides (Scott & Smith, 1990
; Devlin et al., 1990
; Cwirla et al., 1990
; Kay et al., 1993
) or fragments of a target gene (Wang et al., 1995
; Petersen et al., 1995
) is expressed as part of the attachment protein pIII (Smith, 1985
; Parmley & Smith, 1988
) or the major capsid protein pVIII (Felici et al., 1991
; Ilyichev et al., 1992
). The foreign peptides are accessible on the phage surface. Consequently, phages displaying antigenic sequences can be selected specifically from a library by binding to immobilized antibodies. The peptide is identified by sequencing the phage DNA. With the orbiviruses, phage display techniques have so far been used to map antigenic determinants on VP7 (Du Plessis et al., 1994
), VP5 (Wang et al., 1995
) and NS1 (Du Plessis et al., 1995
) of BTV.
To locate epitopes on VP2 of AHSV, a filamentous phage library (Lib-VP2) displaying short peptides derived from fragments of the VP2 gene of AHSV serotype 3 (AHSV-3) was constructed and screened with polyclonal antisera and a neutralizing monoclonal antibody (MAb). By using this approach, several continuous epitopes located in the N-terminal half of the polypeptide could be identified. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. In addition, by using a MAb and a random 17-mer epitope library (Bonnycastle et al., 1996 ), residues possibly involved in the formation of a discontinuous neutralizing epitope could be identified. Finally, a comparison of the antigenic regions identified by phage display with corresponding regions on three other serotypes revealed at least two regions with the potential to discriminate serologically between AHSV serotypes.
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Methods |
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Antigens and antibodies.
VP2 of AHSV-3 (Vreede & Huismans, 1994 ) truncated by 305 C-terminal residues (VP2-tr) was constructed by subcloning into the pFastBacHTc vector and expressed by recombinant baculoviruses in Spodoptera frugiperda (Sf9) cells (Life Technologies). AHSV-3-specific neutralizing chicken IgY was obtained by PEG precipitation (Polson et al., 1985
) from the yolk of an egg laid by a Leghorn hen immunized with density-gradient-purified AHSV (Huismans et al., 1987
). Protein-specific antibodies were isolated from electroblotted VP2-tr (Du Plessis et al., 1995
) and their specificity for VP2 was reconfirmed by immunoblotting. AHSV-specific IgY was immunoaffinity-purified on whole virus immobilized on a 4% cross-linked beaded agarose support according to the manufacturers instructions (Pierce). Anti-AHSV equine antibodies were purified by ammonium sulphate precipitation and DEAE ion-exchange chromatography (Clark & Adams, 1977
) from antiserum (a gift of C. Vroon, Onderstepoort Veterinary Institute) obtained from a horse infected with cell culture-attenuated AHSV-3. The neutralizing MAb 2F2 (Van Wyngaardt et al., 1992
) was purified from ascitic fluid as above.
Fragmented-gene libraries.
Libraries displaying AHSV peptides were constructed according to methods described by Wang et al. (1995) and Du Plessis & Jordaan (1996)
. In essence, the double-stranded replicative form (RF) of the display vector fUSE2 DNA and pBS-VP2 plasmid DNA were prepared by alkaline lysis and CsClethidium bromide gradient centrifugation (Sambrook et al., 1989
). Vector DNA was digested with BglII, dephosphorylated and recovered after agarose gel electrophoresis (Glassmilk, BIO 101). Random fragmentation of target DNA encoding VP2 (pBS-VP2) with DNase I was used to produce fragments of between 50 and 300 bp. Library 1 utilized the VP2 gene excised from the vector as target, while the entire plasmid was used for Library 2. For the linker-ligated PCR library (Library 3), approximately 200 ng blunt-ended fragments was incubated in ligation buffer in the presence of 1 U T4 DNA ligase (Boehringer) before adding 250 pmol phosphorylated BamHI linkers. The linker-ligated DNA was then amplified by PCR (Nagesha et al., 1996
).
Modified DNA fragments of 100300 bp were separated electrophoretically on 5% polyacrylamide gels. Recovered fragments were ligated into approximately 400 ng linear dephosphorylated fUSE2 DNA. One-fifth of a 10 µl ligation was electroporated into electrocompetent MC1061 cells. Transformed clones were selected on LB agar containing 40 µg/ml tetracycline (Tet). Resulting libraries were harvested by scraping the colonies into Tris-buffered saline (TBS), removing cells by centrifugation and precipitating phages twice with 0·15 vols 16·7% PEG, 3·3 M NaCl (PEG/NaCl). Each library was titrated as transducing units (TU) (Smith & Scott, 1993 ). Colony PCR (Wang et al., 1995
) was used to screen for bacterial clones that contained fragments of DNA inserted at the BglII site of the vector by using primers III-5 (5' GGTTGGTGCCTTCGTAGT 3') and III-3 (5' CCATGTACCGTAACACTG 3'). The three different libraries were pooled, resulting in a combined library designated Lib-VP2.
Affinity selection by panning.
Panning of the phage libraries was done in ELISA plate wells by using antibody preparations adsorbed directly to the plastic surface (Wang et al., 1995 ). Any phage-binding antibodies were blocked by adding 50 µl UV-killed f1 phage particles suspended in 1% BSA in TBS containing 0·05% Tween 20 prior to panning. After washing, approximately 4x1010 TU of the phage library suspended in 200 µl of the same buffer was used in the first round of selection.
Sequencing of selected inserts.
Single-stranded DNA of phage clones selected by panning was prepared by standard methods (Sambrook et al., 1989 ). Cycle sequencing with dye-labelled terminators was with the primer 5' CCCTCATAGTTAGCGTAACG 3' and the Big Dye Ready reaction mix (PE Applied Biosystems). PCR products were analysed with an ABI PRISM 310 Genetic Analyser. DNA from the XCX15 library was sequenced manually (Sequenase kit) by using the f88.4 sequencing primers (Bonnycastle et al., 1996
).
ELISA.
Phages were multiplied in E. coli K91 in LB broth containing 40 µg/ml Tet and isolated from the culture supernatant by precipitation with 0·15 vols PEG/NaCl. Phages were diluted to 20 µg protein/ml in TBS and 50 µl aliquots were used to coat microtitre plate wells (Corning, Easywash) overnight at 4 °C. ELISAs were done in triplicate as described previously (Du Plessis et al., 1995 ). In some experiments, antibodies were cross-absorbed by incubating at least 5x1010 phage particles per µg antibody at 37 °C for 60 min in PBS containing 0·5% Tween 20 and 1% (w/v) non-fat milk powder prior to ELISA.
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Results |
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PCR amplification was used to determine the size and frequency of the VP2-derived inserts. With Library 1, 51·5% of the colonies had inserts, with a mean size of 130 bp. Assuming that only 1/18 of the c.f.u. in a primary library can produce an infective phage particle displaying an authentic sequence (Parmley & Smith, 1988 ; Wang et al., 1995
), it required 2012 c.f.u. with inserts to obtain a 99% probability of representing the entire VP2 gene (Clarke & Carbon, 1976
). Similarly, Library 2, with its mean insert size of 73 bp and inserts in 78% of the clones, required 7226 c.f.u. with inserts. The actual number of c.f.u. was 1·3x104 for Library 1 and 3·2x105 for Library 2. Each was therefore likely to be sufficiently large to represent VP2 in its entirety. In addition, DNA from several Library 3 clones was sequenced, both before and after affinity selection, but phages displaying anything other than a single contiguous VP2 sequence were rare and did not identify any novel epitopes after panning (not shown). The three precursor libraries were pooled by mixing 1·12x1010 TU from Library 1, 2·8x109 TU from Library 2 and 1·9x1010 TU from Library 3, resulting in the composite display library, Lib-VP2.
Selection with antibodies immunoaffinity-purified on electroblotted VP2-tr
IgY antibodies directed against purified AHSV were raised in a chicken. To enrich for those most likely to recognize continuous epitopes, recombinant viral protein was separated by SDSPAGE and transferred to a PVDF membrane for use as an immunoaffinity matrix. Since the 305 C-terminal residues of VP2 were not antigenic in immunoblotting (L. Bentley, unpublished), it was possible to use a baculovirus-expressed truncated version of VP2 (VP2-tr) in this step. Lib-VP2 was panned with eluted antibodies at a concentration of 10 µg/ml for two rounds and at 1 µg/ml for a third. A total of 25 phage clones with VP2-derived inserts in the correct orientation and reading frame were selected (Fig. 1). Comparison with the authentic VP2 sequence indicated that most encoded fusion peptides clustering in three regions located between amino acid residues 323 and 529 (Fig. 1b
d
). Seven peptides overlapped in the region encompassing residues 324 and 365. These all contained the sequence IRRA (Fig. 1b
). Similarly, peptides displayed by the clones in the other two clusters overlapped residues HKAEVKKFL (Fig. 1c
) or QGTRTAAIVET (Fig. 1d
). In addition, two single clones, one displaying residues nearer the N terminus (228282) and the other towards the C terminus, were also selected (Fig. 1a
, e
).
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Affinity selection with horse antibodies
AHSV does not normally replicate in chickens or mice. To permit epitopes mapped with IgY and the MAb to be compared with those recognized by a natural hosts immune system during infection, immunoglobulins from an infected horse were used to pan Lib-VP2. An IgG concentration of 20 µg/ml was used for the first two rounds and 2 µg/ml for the final round. Sequencing revealed a peptide representing amino acids 228282 (Fig. 4a; peptide 3). It was identical to peptide 2 (Fig. 1a
), which was selected with IgY released from the electroblotted VP2-tr. A further 11 clones, eight of which were identical siblings, displayed peptides that included residues 393432, with a minimal overlap (398432) shifted relative to overlaps identified by chicken antibodies (Figs 4b
and 5
). A further potentially antigenic region near the C terminus (Fig. 4c
, peptide 14) was also identified.
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Discussion |
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When Lib-VP2 was panned with either chicken IgY affinity-purified on denatured protein or with antibodies eluted from whole virus, the selected peptide clusters corresponded closely, identifying three major antigenic regions between residues 324 and 543 (Figs 1 and 2
). Antigenicity in these regions was not eliminated by reduction, SDS denaturation and binding to a membrane. Operationally, these sequences therefore represent continuous epitopes. Unlike the electroblot-purified antibodies, however, the virus-specific preparation eluted from intact virus did not select any peptides corresponding to amino acids 228282 (Fig. 1a
, peptide 2). This sequence, although only selected once by the electroblot-purified antibodies, bound the anti-AHSV IgY in ELISA (Fig. 6
). Hence, it may represent a cryptotope. The original AHSV used as immunogen could conceivably have contained some free or possibly even proteolytically degraded VP2 that gave rise to antibodies that bound epitopes that are normally hidden. The horse immunoglobulins also selected a single phage clone displaying this sequence (peptide 3, Fig. 4a
). This was not surprising, since cryptotopes could be revealed to the horse immune system during uncoating or before assembly.
Apart from peptides 2 (Fig. 1a) and 3 (Fig. 4a
) and the one near the C terminus, the chicken and horse antibodies selected sequences that differed in their minimal overlapping residues. Nevertheless, with both the horse and the chicken antibodies, it is clear that significant antigenicity resides in the region encompassed by amino acids 393432. The horse antibodies bound a set of peptides that were located within the overall antigenic region stretching from residues 224 to 543, but some sequences identified as being highly antigenic with the chicken antibodies (e.g. the regions depicted in Figs 1b
and d
and 2a
and c
) were not identified at all. It is possible that, owing to intrinsic genetic differences, the equine immune system recognized a different set of epitopes. Horse antibodies have, for example, been reported not to be able to recognize dodecameric synthetic peptides reflecting VP5 sequences (Martínez-Torrecuadrada et al., 1999
) in PEPSCAN and to show epitope-boundary frame shifts and differences in immunodominance when compared with mouse and human antisera (Atassi et al., 1996
). It may also be significant that the chicken was immunized with non-replicating AHSV together with an adjuvant. The horse serum, on the other hand, was from a host animal infected with cell culture-derived inoculum. The immune responses to AHSV could therefore have involved differences in antigen presentation, resulting in different cytokine profiles, which in turn could affect the clonal expansion of B or T helper cells.
While most of the fusion peptides selected from Lib-VP2 were clearly antigenic, a sequence ARWVEWA (Fig. 1e) near to the N terminus was recognized by IgY that had been eluted from a truncated VP2 from which these residues were absent. Logically, therefore, it cannot be a true epitope. A very similar sequence nearer the middle of the polypeptide chain, namely WVDW (residues 428432), was selected with the horse immunoglobulins (Fig. 4b
). WVEWA is therefore a potential internal antigenic mimic (Fehrsen & Du Plessis, 1999
). Surprisingly, the MAb 2F2 also panned a peptide that included these residues. It is therefore not clear whether they represent an epitope, a mimotope or an adventitious binding sequence. WVEWA was, however, not highly antigenic in ELISA.
Two other clones were selected by the neutralizing MAb 2F2. The only correspondence with any of the other peptides was with peptide 12 (Fig. 3b), which overlapped the end of peptide 11 (Fig. 2c
) by ten residues. None produced any significant readings in ELISA. When the same MAb was used to pan the XCX15 random-epitope library, peptides that included the sequences LEKW (Table 1
, peptide X7) and MYPFTY (Table 1
, peptides X1, X2 and X3) were selected. Recognizably similar sequences occur near the C terminus (MYPVHY) and at the N terminus (LEKWRDL) of the MAb-selected peptide 1 (Fig. 3a
). These residues may therefore form part of a discontinuous epitope. The XCX15 peptide (sequence X7) that included the amino acids LEKW produced the stronger ELISA signal (Table 1
), indicating a greater contribution towards binding. On the phage, the peptide that includes LEKW is located between two cysteine residues and therefore has the potential to form a loop structure, which may assist it in mimicking the authentic epitope. No similarities with the other peptides selected by the MAb (Fig. 3
, peptide 12) were found, and it probably represents background binding.
ELISA was used to test the binding of representative phage-displayed peptides under conditions different from those under which they were selected. In general, the strongest ELISA signals correlated with antigenic regions characterized by clusters of panned peptides. With the chicken IgY, ELISA signals could be reduced by up to 90% with homologous phage preparations. Nonetheless, under similar conditions, the neutralizing activity of the chicken antibodies could not be reduced (not shown). With AHSV-4, bacterially expressed fragments of VP2 identified a domain extending from residues 253 to 413 that evoked neutralizing antibodies in mice but, like the phage-displayed fusion peptides, could not reduce serum neutralizing capacity (Martínez-Torrecuadrada & Casal, 1995 ). Neutralizing activity was localized further to residues 283379 and 379413 of the serotype 4 polypeptide. These regions fall within the overall antigenic region encompassing amino acids 324543 of AHSV-3 (Fig. 5
). None of the peptides identified by phage display, however, was shown to be involved directly in neutralization.
A comparison of the aligned amino acid sequences of four AHSV serotypes revealed that a tract of residues encompassing peptide 7, the sequence that bound strongest in ELISA, varied significantly between four different AHSV serotypes. This peptide, selected with chicken IgY, included a sequence also recognized by the horse antibodies. In the development of serotype-discriminatory immunoassays, the antigenicity of this region, combined with sequence variability, makes it a potentially worthwhile subject for further investigation. Another potentially useful region is included in peptide 11. The horse serum used for panning was, however, not a good binder in ELISA with any of the fusion phages that were tested. The bacteriophage fUSE2 displays at most five or six copies of the fusion peptide per phage (Specthrie et al., 1992 ). The use of synthetic peptides or other fusion proteins could, therefore, conceivably enable a higher epitope density to be achieved in ELISA, possibly resulting in more efficient assays. It may nevertheless be necessary to pan epitope libraries with more than one horse immune serum in order to locate regions recognized consistently by equine antibodies.
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Acknowledgments |
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References |
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Received 18 October 1999;
accepted 21 December 1999.