Rabies Research and Diagnostic Group, Veterinary Laboratories Agency, Weybridge, Addlestone, Surrey KT15 3NB, UK1
Author for correspondence: Nicholas Johnson. Fax +44 1932 347046. e-mail n.johnson2{at}vla.defra.gsi.gov.uk
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Abstract |
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Introduction |
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Monoclonal antibodies (mAbs) have been instrumental in elucidating the antigenic sites on the RV glycoprotein and these can be classified into conformational sites and linear, non-conformational sites. Two major sites dominate the former. The first, termed antigenic site II, is formed from two sites between residues 34 and 42 and residues 198 and 200 (Prehaud et al., 1988 ). Conversely, a single region between residues 330 and 338 forms the second site, antigenic site III, with an arginine at position 333 being critical for virus neutralization (Seif et al., 1985
). This residue is also critical for neuroinvasion, possibly through its role in binding to a cellular receptor (Dietzschold et al., 1983
; Coulon et al., 1989
; Badrane et al., 2001
). There is also evidence that these antigenic sites are structurally conserved among all rhabdoviruses (Walker & Kongsuwan, 1999
), and the immunodominance of these sites is emphasized by the observation that 97% of mAbs recognized either site (Coulon et al., 1993
). Mapping the epitopes of neutralizing mAbs has also identified non-conformational sites (Bunschoten et al., 1989
; Dietzschold et al., 1990
) and a number of critical residues (Van der Heijden et al., 1993
; Ni et al., 1995
; Luo et al., 1997
). Alternative approaches to mapping antigenic sites have utilized cyanogen bromide (CNBr) cleavage fragments of purified glycoprotein (Dietzschold et al., 1982
) and short glycoprotein peptides expressed in yeast (Lafay et al., 1996
). The former study concluded that, in the rabbit, the principle regions recognized were internal sites between residues 103 and 330, whereas the latter mapped a panel of mAbs to a short region between residues 223 and 276. This contrast suggests that different species may respond to different sites on the RV glycoprotein.
In this study we have investigated the polyclonal antibody response to non-conformational epitopes in rabbit and dog recipients of a commercially available anti-rabies vaccine. Truncated fragments of the RV glycoprotein were fused to glutathione S-transferase (GST) and used to map the response to defined regions. This approach confirmed the immunodominance of an internal region of the glycoprotein observed with mAbs. It also identified a second region close to the N terminus of the protein towards which dogs in particular direct an antibody response.
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Methods |
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Fluorescent antibody virus neutralization assay (FAVN).
This assay was carried out following the protocol of Cliquet et al. (1998) . Briefly, sera were heat-inactivated by incubation at 56 °C for 30 min and then serially diluted in a 96-well plate. One hundred TCID50 of rabies Challenge Virus Standard (CVS-11) was added and the plate was incubated at 37 °C for 1 h. Finally, 50 µl baby hamster kidney (BHK) cells at 4x105 cells/ml were added in Dulbeccos modified Eagles medium with penicillin (100 U/ml), streptomycin (100 µg/ml), mycostatin (25 U/ml). The plate was incubated for 48 h at 37 °C. The supernatant was then discarded and the adherent cells fixed with acetone (80% in H2O) for 20 min. The plate was allowed to air-dry and then stained with an anti-rabiesFITC conjugate (Centocor) at a dilution of 1:50. Fluorescence was measured at each dilution of sera and titres were calculated using the SpearmanKarber method. Results are presented as the number of International Units by comparison with an Office International des Epizooties (OIE) positive and negative standard for rabies immunoglobulin.
Construction of truncated glycoprotein fragments.
DNA fragments encoding the four regions were amplified using PCR. The target for the amplification was RNA (1 µg/µl) extracted from BHK cells infected with RV (Pasteur Virus strain) (Heaton et al., 1997 ). Briefly, RNA was reverse-transcribed using the protocol of Heaton et al. (1997)
using the upstream primer and then amplified using the combinations of primers detailed in Table 1
. The product for each reaction was immediately cloned into plasmid pCR2.1 using the TA cloning system (Invitrogen) and transformed into competent E. coli TOP10F cells (Invitrogen) following the manufacturers instructions. Plasmids were then extracted using the Wizard plasmid preparation kit (Promega) and digested with BamHI and SalI restriction endonucleases. The digested glycoprotein fragments were purified and cloned into the pGEX-4T-3 plasmid (Pharmacia), and then transformed into competent E. coli BL21 cells (Pharmacia).
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Immunoblotting.
Protein separations were carried out by SDSPAGE and transferred to nitrocellulose membranes (Bio-Rad) in transfer buffer (0·025 M TrisHCl, pH 8·8, 0·192 mM glycine, 0·1%, w/v, SDS, 25%, v/v, methanol). Membranes were blocked with 5% non-fat milk in PBS (pH 7·2) with 0·1% Tween 20 (PBS-T). Probing with polyclonal sera was carried out at a dilution of 1:2000 in 1% non-fat milk in PBS-T for 1 h at 37 °C. Each membrane was washed three times with PBS-T and then incubated with swine anti-rabbit horseradish peroxidase (Dako) or rabbit anti-dogperoxidase (Sigma) conjugates as appropriate, both at a dilution of 1:2000 for 1 h at 37 °C. Membranes were washed five times and developed using enhanced chemiluminescence (Amersham Pharmacia). Protein sizes were estimated by comparison with pre-stained broad-range markers (Bio-Rad).
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Results |
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Discussion |
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These observations compare favourably with two previous studies that identified antigenic sites on the RV glycoprotein. Dietzschold et al. (1982) generated shortened fragments of the ERA strain with CNBr. Immunoprecipitation of such radiolabelled CNBr fragments with hyperimmune rabbit sera identified two regions of the glycoprotein that formed antigenic sites. The second of these partially overlaps the RvG5-6 fragment over the sequence 293332. These two regions, and a non-precipitatable peptide from the N-terminal 50 residues, were capable of inducing neutralizing antibodies. The second study by Lafay et al. (1996)
using peptide fragments of the CVS strain expressed in yeast identified a region between residues 223 and 276 that bound six of twelve non-conformationally dependent mAbs. The site is located within the core of the RvG5-6 peptide. The remaining mAbs bound to sites flanking this region. Prediction studies on this region suggest that there are multiple sites that could form antigenic determinants, although this is not matched by the limited number of predicted surface-exposed sites (N. Johnson, unpublished observations). In addition, three separate studies (van der Heijden et al., 1993
; Ni et al., 1995
; Luo et al., 1997
) have characterized neutralizing mAbs that bind to a region between residues 248 and 268. It is possible that within polyclonal dog sera, the total binding of neutralizing antibodies to the RvG5-6 fragment could be due to binding of antibodies to this site. However, a more detailed analysis is required to assess the number of epitopes within this truncated region.
As with both previous studies (Dietzschold et al., 1982 ; Lafay et al., 1996
) that mapped the RV glycoprotein, the C-terminal fragment between residues 327 and 443 does not appear to contain any antigenic determinants. One possible explanation for this could be that this region is protected from immune surveillance by N-terminal domains of the glycoprotein. However, in the absence of a full secondary structure, this remains speculative, despite being a consistent finding between research groups.
Depletion studies using insoluble RvG5-6 caused a limited reduction in the neutralizing titre of both rabbit and dog sera. Insoluble material from sonicated E. coli alone caused no reduction in titre (data not shown) and this step could be used to remove non-specific binding without any reduction in RV glycoprotein detection. The remaining components that constitute the neutralizing titre within a polyclonal serum are antibodies that bind to linear epitopes at the N-terminal region, which could be induced following immunization with a peptide covering this site by Dietzschold et al. (1983) . Also, antibodies that bind conformational sites such as the previously described antigenic sites II and III (Coulon et al., 1993
) will make up the neutralizing component of immune sera.
It should be noted that the approach described above only identifies non-conformational antigenic regions. The relative importance of each component is impossible to compare within polyclonal sera, although the experience with neutralizing mAbs suggests that conformational sites are dominant (Lafay et al., 1996 ). However, the absence of studies in carnivores means that this may not be the case in animals such as the dog. Approaches that develop non-mouse mAbs could be used to address this issue (Champion et al., 2000
).
The importance of investigating the antibody response in vaccine recipients lies in the information it provides in identifying alternative approaches to vaccine design. The identification of short linear epitopes allows their replication in the form of peptides, which can then be used as vaccines (Dietzschold et al., 1990 ). However, the ability of RV to accommodate point mutations that allow escape from neutralization, and ironically the method used to identify many epitopes, suggests that this approach may be limited. Using a larger region of the glycoprotein as the antigen, containing more than one neutralizing epitope, may overcome this problem. The major observation of this study suggests that some epitopes recognized by mAbs are probably also recognized by vaccinated dogs. This should allow the design of a large fragment of the glycoprotein, which is capable of inducing a neutralizing polyclonal response following immunization, that could protect against challenge with a divergent range of classical RV isolates.
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Acknowledgments |
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References |
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Received 24 April 2002;
accepted 10 July 2002.