Rabies Research and Diagnostic Group (WHO Collaborating Centre), Veterinary Laboratories Agency-Weybridge, Woodham Lane, New Haw, Surrey, KT15 3NB, UK
Correspondence
N. Johnson
n.johnson2{at}vla.defra.gsi.gov.uk
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ABSTRACT |
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MAIN TEXT |
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In order to identify linear epitope sites on the glycoprotein of the rabies Pasteur virus (PV), we have used phage display (Smith & Scott, 1993), a technique that has successfully identified linear epitopes on a number of viral glycoproteins (Grabowska et al., 1999
). Two mAbs that recognized the virus glycoprotein, MnAb1 and MnAb2 (a gift from Merial, France), were studied by Western blot. The PhD12 peptide library (New England Biolabs) was used to characterize the mAb-binding site and was performed by following the manufacturer's protocols. Escherichia coli strain ER2738 was used for amplification of eluted phage, which provided input phage for the next round of biopanning. After four rounds of biopanning for each mAb, ten clones were amplified and the region encoding the peptide insert was sequenced. Synthetic peptides were manufactured (Alta Bioscience) in a linear form or as a multiple antigenic peptide (MAP) and purified to >95 %. Peptide inoculations were carried out in rabbits by using 2 mg MAP ml1, injected subcutaneously with Freund's complete adjuvant, with subsequent inoculations given with Freund's incomplete adjuvant. Groups of five mice were inoculated with saline, monomeric peptide or MAP. The peptides were all diluted to 2 mg ml1 in saline and the inocula were combined with equal quantities of MPL+TDM adjuvant (Sigma). The mice were inoculated and given boosters on days 21 and 42. Each mouse was tail-bled prior to the first inoculation and then on days 14 and 28. On day 59, they were killed humanely and a blood sample was retained. For rabbits, mice and rabies-vaccinated dogs, blood samples were tested by ELISA for reactivity to the peptide as follows. Each sample was tested in triplicate against the peptide at a concentration of 10 µg ml1 in PBS or against a PBS-only background control in a Maxisorp plate (Nunc). The coated plate was incubated at 37 °C for 2 h. Plate contents were discarded, each well was filled with blocking buffer (PBS, 1 % Tween 20, 1 % non-fat milk) and the plate was incubated at 37 °C for 1 h. During the blocking incubation, samples were pre-diluted 1 : 250 in antibody diluent (PBS, 1 % Tween 80, 2 % fetal calf serum) and incubated at room temperature for 30 min. Blocking was followed by six washes with 1 % PBST (PBS, 1 % Tween 20; 1 min per wash, with agitation). On addition to the coated plate, samples were incubated for 1 h at 37 °C and then washed as above. Protein Ahorseradish peroxidase (Sigma), diluted 1 : 6000 in antibody diluent, was incubated with the plate for 1 h at 37 °C, again followed by six washes with 1 % PBST. 3',3',5',5'-Tetramethylbenzidine (Sigma) substrate was incubated with the plate for 25 min at room temperature in the dark. The reaction was stopped with 1 M sulphuric acid and A450 was measured. The fluorescent antibody virus neutralization (FAVN) test was used for the detection of rabies vaccine-specific antibodies in companion animal sera, as described by Cliquet et al. (1998)
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Consensus sequences were obtained for both mAbs MnAb1 and MnAb2, panned against the peptide library [Fig. 1a (i and ii)]. For both target molecules, the amino acids tryptophan, aspartic acid and isoleucine were common to all sequences. In addition, four phage sequences for MnAb1 were identical to the consensus sequence of MnAb2, indicating that both antibodies probably bound to the same epitope site. Alignment of the consensus sequences with the mature PV glycoprotein (lacking the signal peptide) suggested that the proposed binding site was in a region near to the N terminus between aa 14 and 19 [Fig. 1a
(iii)]. Further homology was observed at the terminal lysine for the consensus sequence for MnAb1 and at histidine and serine residues within the consensus sequence of MnAb2. In order to provide supporting evidence that the binding site of these antibodies was indeed the N-terminal domain, both were used to probe recombinant glutathione S transferase (GST) fragments of the RABV glycoprotein (Johnson et al., 2002a
). Identification of the N terminus as the site of the epitope is supported by the immunoblotting of recombinant glycoprotein fusion proteins with mAb MnAb2 (Fig. 1b
). This antibody binds strongly to fragment RvG1-2, which encodes the first 128 aa of the mature glycoprotein, and supports the location of the epitope at the N-terminal region of the RABV glycoprotein. Confirmation that the region between aa 12 and 23 was the location of the epitope site was obtained by synthesis of a 12-mer peptide that was identical to this region. This peptide was detected by both mAbs by ELISA (data not shown). To establish immunogenicity of the epitope, a MAP of the epitope was synthesized and used to inoculate both rabbits and mice. Sera obtained from rabbits detected both the MAP and monomeric conformations of the peptide by ELISA (data not shown); both forms of the peptide were detectable after the first test bleed. Similar results were obtained following inoculation of mice with the MAP (Table 1
). An antibody response was detected after inoculation with the multimeric peptide, which was not observed following inoculation with either saline or the monomeric form of the peptide. However, when mouse serum raised against the MAP was tested by FAVN, it was shown to have no neutralizing activity against the challenge virus standard at neutral pH and reflected the failure of both original mAbs to neutralize virus. The prototype ELISA was modified for screening two panels of dog sera, one of which consisted of naïve animals with no vaccination history and the second of sera from recently vaccinated dogs with a detectable response, as measured by FAVN. The results from this screening (Fig. 1c
) suggest that the synthetic peptide mimic was recognized (A450>1) by sera from a subset of rabies-vaccinated dogs. This subset was characterized by elevated levels of neutralizing antibody (>10 IU ml1).
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Screening of post-rabies vaccination sera by using techniques such as ELISA is of increasing importance in the control of animal movements, when attempting to minimize the risk of rabies reintroduction. The aim of such screening is to identify those animals that have developed a measurable antibody response to vaccination, and are therefore protected against the disease, prior to movement of an animal into a new country. Screening assays use whole, inactivated virus (Cliquet et al., 2004) or recombinant viral proteins (Inoue et al., 2003
) as the antigenic target. Peptides offer a cost-effective alternative in the production of the appropriate antigenic component of such assays. From this study, we conclude that a single peptide mimic of this epitope when used alone is a poor discriminator of vaccination status of sera, compared with current methods. However, it is possible that a panel of peptides or larger peptide fragments may offer a viable alternative for future assay development.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 June 2004;
accepted 18 August 2004.
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