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Clinical and Diagnostic Laboratory Immunology, July 2004, p. 658-664, Vol. 11, No. 4
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.4.658-664.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of Immunodominant Linear B-Cell Epitopes on the Carboxy Terminus of the Rinderpest Virus Nucleocapsid Protein

Kang-Seuk Choi,1* Jin-Ju Nah,1 Young-Joon Ko,1 Shien-Young Kang,2 Kyoung-Jin Yoon,3 and Yi-Seok Joo1

Foreign Animal Disease Research Division, National Veterinary Research and Quarantine Service, Anyang, Kyounggi 430-824,1 Research Institute of Veterinary Medicine/College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, South Korea,2 Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa 500113

Received 13 January 2004/ Returned for modification 9 March 2004/ Accepted 6 April 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The nucleocapsid (N) protein of rinderpest virus (RPV) is one of the most abundant and immunogenic viral proteins expressed during natural or experimental infection. To identify immunogenic epitopes on the N protein, different forms of RPV N protein, including the full-length protein (N1-525), an amino-terminal construct (N1-179), and a carboxy-terminal construct (N414-496), were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The antigenicity of each recombinant protein was evaluated by Western immunoblotting. All recombinants were recognized by hyperimmune RPV bovine antisera, indicating that immunoreactive epitopes may be present at both ends of the N protein. However, GST-N414-496 was much more antigenic than GST-N1-179 when tested with sera from vaccinated cattle, suggesting that an immunodominant or highly immunogenic epitope(s) may be located at the carboxy terminus of the N protein. Epitope mapping with overlapping peptides representing different regions of the carboxy terminus (amino acids 415 to 524) revealed three nonoverlapping antigenic sites in regions containing the residues 440VPQVRKETRASSR452 (site 1), 479PEADTDPL486 (site 2), and 520DKDLL524 (site 3). Among these, antigenic site 2 showed the strongest reactivity with hyperimmune anti-RPV bovine sera in a peptide enzyme-linked immunosorbent assay but did not react with hyperimmune caprine sera raised against peste-des-petits-ruminants virus, which is antigenically closely related to RPV. Identification of an immunodominant linear antigenic site at the carboxy terminus of the N protein may provide an antigen basis for designing diagnostics specific for RPV.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rinderpest is an acute and highly contagious viral disease causing high morbidity and mortality in cattle and wild bovine species. The disease has accounted for significant economic losses to the livestock industry in Africa, Europe, the Middle East, the Near East, and South Asia (27, 30). Rinderpest is now largely eradicated, but it is still endemic in some areas in East Africa and Asia (20, 27, 29).

Rinderpest virus (RPV), the causative agent of rinderpest, is an enveloped RNA virus belonging to the Morbillivirus genus in the family Paramyxoviridae. The other members of the genus include peste-des-petits-ruminants virus (PPRV), measles virus (MV), canine distemper virus, phocine distemper virus, and dolphin morbillivirus (1, 11). RPV, despite having a single serotype, can be grouped into three distinct lineages (i.e., Asian, African I, and African II) on the basis of partial sequence analysis of the fusion (F) protein gene (5, 27).

The genomes of morbilliviruses, including RPV, contain six structural protein genes, encoding F protein, hemagglutinin (H) protein, nucleocapsid (N) protein, matrix (M) protein, polymerase (L) protein, and phosphoprotein (P), and two nonstructural protein genes (encoding C and V) (5, 15, 18). Among the structural proteins, N protein has been used as a diagnostic antigen for various purposes. The N protein has been used as an antigen to detect antibodies specific for RPV (22), PPRV (24), and canine distemper virus (37), as well as antibodies that are cross-reactive to morbilliviruses (9, 32), since it is highly immunogenic in infected animals despite its internal location (14, 34, 35). In case of RPV, N protein was expressed in a baculovirus and used in an indirect enzyme-linked immunosorbent assay (ELISA) for differentiation of natural infection from vaccination with recombinant RPV H or F protein (18, 19). The N protein or its corresponding gene also have been used as a target for virus detection in clinical specimens, since it is the most abundant and highly conserved viral protein among the morbilliviruses (10, 17, 23).

Although the N protein is known to play an important role in humoral immunity against morbillivirus, many questions related to the locations and antigenicities of antigenic sites, especially immunodominant epitopes, that would be very useful for diagnostic applications remain unclear. Buckland et al. (4) have identified two antigenic sites (amino acids [aa] 457 to 476 and 519 to 523) on the carboxy terminus and one antigenic site (aa 122 to 150) on the amino terminus of the MV N protein by using N deletion mutants and monoclonal antibodies, but they did not investigate the degrees of immunogenicity of these epitopes. The carboxy-terminal region of the morbillivirus N protein protrudes from nucleocapsid core (16, 25, 26), so it has the potential to contain immunodominant antigenic sites, as found in N protein of Sendai virus, one of the morbilliviruses (13).

In a previous study conducted by our laboratory (8), antigenic sites in the amino terminus of the N protein of RPV were mapped and identified by using anti-N monoclonal antibodies and N deletion mutants. However, results obtained by use of hyperimmune RPV antiserum indicated that antigenic sites might exist in the carboxy terminus and that they might be more continuous (i.e., be linear epitopes) than ones in the amino terminus. In this study, linear antigenic sites in the carboxy terminus of the N protein of RPV were identified by using synthetic peptides and hyperimmune RPV antisera. The relative degree of antigenicity of each site was evaluated by using sera from RPV-infected cattle.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Virus. The LATC strain of RPV (6), the seed virus for the production of rinderpest vaccine for emergency use in Korea, was used for the generation of recombinant N proteins. The LATC strain belongs to Asian lineage. The virus was propagated in Vero cells prepared in roller bottle cultures, concentrated, and semipurified as previously described (8).

Preparation of full-length N and truncated mutants. The pGEX recombinants carrying the full-length open reading frame and truncations of the RPV N protein gene were constructed by previously described procedures (8), using the following three sets of PCR primers with XhoI and NotI sites (underlined): NP-F1 (5'-CTCGAGTTCTTTGAAATGGCTTCT-3') and NP-R1 (5'-GCGGCCGCTCATTTAGCTGAGGAG-3') for pGEX-N1-525, NP-F1 and NP-R179 (5'-GCGGCCGCAACAGCGACAGGATGCTGA-3') for pGEX-N1-179, and NP-F414 (5'-CTCGAGCAGACCCAGGTTTCATTTCT-3') and NP-R496 (5'-GCGGCCGCTCTGCTGACTTCTGCT-3') for pGEX-N414-496. Three pGEX constructs containing full-length protein (pGEX-N1-525), the amino-terminal construct (pGEX-N1-179), or the carboxy-terminal construct (pGEX-N414-496) were transformed into Escherichia coli strain BL21 (Amersham Biosciences) and were expressed by using isopropyl-ß-D-thiogalactopyranoside (IPTG) at a final concentration of 0.2 mM. The resulting fusion proteins glutathione S-transferase (GST)-N1-525, GST-N1-179, and GST-N414-496 were extracted from the transformed cells as previously described (8).

Antisera. Hyperimmune antisera raised against virus strains representing three lineages of RPV (Asian lineage and African lineages I and II) and four lineages of PPRV (lineages I, II, III, and IV) were used in this study. Two bovine antisera against RPV African lineages I ({alpha}RPV-I) and II ({alpha}RPV-II) were kindly supplied by H. M. Wamwayi (Kari NRVC, Muguga, Kenya). One bovine antiserum against RPV Asian lineage ({alpha}RPV-Asian), stored in our laboratory, was used. One bovine antiserum against RPV RBOK (vaccine) was supplied by G. Libeau (CIRAD-EMVT, Montpellier, France). Four goat antisera against PPRV lineages I ({alpha}PPRV-I), II ({alpha}PPRV-II), III ({alpha}PPRV-III), and IV ({alpha}PPRV-IV) were kindly supplied by E. Couacy-Hymann (LANADA/LCPA, Bingerville, Ivory Coast). All hyperimmune sera had neutralizing antibody titers of ≥1:256 against the respective virus. In addition to hyperimmune goat sera, anti-PPRV bovine sera were obtained by the courtesy of E. Couacy-Hymann in Africa. These sera were already confirmed positive by cross neutralization tests for PRV and PPRV (data not shown).

Anti-RPV sera were also obtained from 10 Holstein cows vaccinated with an attenuated RPV vaccine (LATC strain) at a rate of approximately 103.5 50% tissue culture infective doses per dose and used for the study. Sera designated 1 to 10 were collected from all vaccinated cattle 3 weeks after vaccination. All animals were kept in a biosafety level 3 containment animal research facility during the entire study period, in accordance with regulations of the Korean government.

Besides antisera raised in the natural host of morbillivirus, polyclonal antibodies ({alpha}N1-525) monospecific for recombinant full-length N polypeptide (N1-525) were produced in two guinea pigs and used for the study. The N1-525 was prepared from GST-recombinant N protein (GST-N1-525) by treating it with bovine factor Xa (Pharmacia Biotech) for 4 h at ambient temperature and then purifying the cleaved products through a single-step glutathione Sepharose 4B affinity column according to the procedure recommended by the manufacturer. The animals were injected subcutaneously twice with purified full-length N polypeptide (100 µg of protein per dose) mixed with Freund's adjuvant at an interval of 3 weeks. Sera were collected from two animals at 2 weeks after the last inoculation and pooled prior to use in this study. Goat anti-GST antibody was purchased from a commercial supplier (Amersham Biosciences) and used to identify fusion proteins expressed in the prokaryotic cells.

SDS-PAGE and Western immunoblotting. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) of protein materials (i.e., purified whole virus or recombinant GST fusion proteins) was performed with 12% vertical slab gels under denaturing conditions (21). Polypeptides separated on a polyacrylamide gel were subsequently electrotransferred to nitrocellulose membranes (36). Immunoblotting was performed by standard techniques with 1:100-diluted antisera, species-specific anti-immunoglobulin G (anti-IgG) conjugated with alkaline phosphatase (1:1,000), and a BCIP (5-bromo-4-chloro-3-indolylphosphate)-nitroblue tetrazolium solution (Kirkegaard-Perry Laboratories, Inc.) as the substrate.

GST capture ELISA. The immunoreactivity of whole or partial recombinant GST fusion N proteins was assessed by using hyperimmune ({alpha}RPV-Asian, {alpha}RPV-I, {alpha}RPV-II, {alpha}RPV-III, {alpha}RPV-IV, {alpha}PPRV-I, {alpha}PPRV-II, {alpha}PPRV-III, and {alpha}PPRV-IV), vaccination (no. 1 to 10), and N protein-specific ({alpha}N1-525) antisera. GST was used as a control protein. A 50-µl volume of one of the purified fusion proteins or GST control protein was added alternately to a row of wells in each of the ELISA plates (MaxiSorp; Nunc, Roskilde, Denmark) at a concentration of 1.0 µg/ml in 0.01 M phosphate-buffered saline (PBS) (pH 7.4 ± 0.2), and the plates were incubated for 1 h at 37°C. After GST proteins not bound to the plates were rinsed off, sera were diluted 1:50 in blocking buffer (0.01 M PBS containing 3% skim milk, 0.5% rabbit serum, 1% bacterial lysates, and 0.05% Tween 20) and 50 µl of each dilution was added to a pair of two wells (one with one of the recombinant fusion protein and the other with GST control protein). After 60 min of incubation at 37°C, the plates were washed three times with 0.002 M PBS containing 0.05% Tween 20 (PBST). After incubation for 60 min at 37°C, the plates were washed with PBST. Antigen-antibody reactions were visualized by adding 50 µl of optimally diluted anti-species IgG labeled with peroxidase (Kirkegaard-Perry Laboratories, Inc.) to each well, incubating for 1 h at 37°C, and then adding ortho-phenylenediamine substrate. After 10 min, the colorimetric reaction was stopped by adding 1.25 M sulfuric acid. The optical density (OD) of each well was measured at a wavelength of 492 nm. The net absorbance of each serum sample was corrected by subtracting the OD for the GST control from that for the recombinant fusion protein. Sera with net OD values of ≥0.2 were considered positive. The test was repeated three times.

Preparation of synthetic peptides. Twelve overlapping synthetic peptides covering the carboxy terminus (aa 414 to 524) were used in this study. Their amino acid sequences were based on those of the N protein of the RPV LATC strain (Table 1). Nonoverlapping peptide P1 was used as an unrelated control peptide. All synthetic peptides were produced by a commercial manufacturer (Peptron Co., Daejeon, Korea) by the 9-fluorenylmethoxy carbonyl solid-phase method on amide resin. Peptides synthesized on the resin were cleaved from the resin with reagent K (950 µl of trifluoroacetic acid, 25 µl of tri-isopropyl silane, and 25 µl of water), precipitated with a mixture of ether and hexanes at a ratio of 2:1, and purified by C18 reverse-phase high-performance liquid chromatography. Final peptide products were adjusted to a concentration of 5 mg/ml in distilled water.


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  		TABLE 1. Synthetic peptides used for identifying antigenic sites

 
Peptide ELISA. Fifty microliters of each synthetic peptide (10 µg/ml) and control peptide (P1) in 0.01 M PBS were added alternately to each row of the wells of MaxiSorp ELISA plates and incubated overnight at 37°C to coat the plates. After unbound synthetic peptide was washed off with PBST, 50 µl of each serum sample diluted 1:10 in 0.01 M PBS containing 1x Genosys blocking buffer (Sigma Genosys), 5% skim milk, and 0.05% Tween 20 (Genosys blocking buffer) was added to a pair of wells (one with an RPV peptide and the other with P1). Each serum sample was run in three replicates. After 1 h of incubation at 37°C, plates were washed three times with PBST. To visualize specific antigen (peptide)-antibody reactions, 50 µl of peroxidase-labeled anti-species IgG (Kirkegaard-Perry Laboratories, Inc.) diluted 1:2,000 in the Genosys blocking buffer was added to each well, and the plates were incubated for 1 h at 37°C. After that, ortho-phenylenediamine was added to each well and incubated for 10 min at room temperature. The colorimetric reaction was stopped by adding 1.25 M sulfuric acid, and the OD of each sample was measured at a wavelength of 492 nm. The T/C value (test peptide OD/control peptide OD) for each sample was then calculated as (mean OD of sample for an RPV peptide)/(mean OD of sample for control peptide + 3 standard deviations). Samples with T/C values of ≥2 were considered positive for antibody against the specific RPV peptide. The test was repeated three times.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and characterization of the GST fusion proteins. GST-N1-525, GST-N1-179, GST-N414-496, and GST polypeptides were expressed with apparent molecular masses of 87, 48, 37, and 28 kDa, respectively, which correlated with the predicted molecular mass of each product (Fig. 1A). The presence of recombinant GST fusion protein and GST itself was confirmed by Western immunoblotting with anti-GST antibody (Fig. 1B) and anti-N polypeptide antibody (Fig. 1C). Their expression was optimized by inducing the cells with 0.2 mM IPTG for 6 h at room temperature. After purification on a glutathione-Sepharose 4B affinity column, the recombinant GST fusion N proteins were recovered at concentrations of approximately 4, 3.8, and 4 mg per liter of culture, respectively. GST itself was recovered at a concentration of 4 mg per liter of culture after affinity column chromatography. GST and two truncated forms of the N protein (GST-N1-179 and GST-N414-496) were successfully expressed in soluble form, while the full-length N protein (GST-N1-525) was insoluble under nondenaturing conditions after mechanical lysis of the bacteria and incubation with 5% Triton X-100 (data not shown). Insoluble protein GST-N1-525 was successfully extracted from bacterial cells after treatment with lysozyme (0.2 mg/ml) and sodium sarkosyl (0.5%).



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  		FIG. 1. SDS-PAGE and Western immunoblot analyses of GST fusion proteins containing the full length (GST-N1-525), the amino terminus (GST-N1-179), and the carboxy-terminus (GST-N414-496) of the N protein of RPV from E. coli cells transformed with recombinant pGEX-derived expression vectors. Ab, antibody.

 
Antigenicity of GST-N fusion proteins. The antigenicities of three different forms of recombinant GST-N fusion proteins were assessed by Western immunoblot analysis and ELISA with three hyperimmune RPV antisera ({alpha}RPV-Asian, {alpha}RPV-I, and {alpha}RPV-II). All three forms of recombinant N protein reacted with the RPV-specific bovine hyperimmune antisera in Western immunoblotting (Fig. 2), suggesting that both the carboxy-terminal and the amino-terminal regions of the protein contained an antigenic determinant(s) recognized by antibodies raised against RPV in cattle. The reactivity pattern was similar to that with anti-N polypeptide guinea pig antiserum (Fig. 1C). However, based on band intensity, there was a difference between the amino-terminal form (GST-N1-179) and the carboxy-terminal form (GST-N414-496) in the degree of reactivity with the antisera in the immunoblotting. These hyperimmune anti-RPV bovine sera showed stronger reactivity with the GST-N1-525 and the GST-N414-496 than with the GST-N1-179, as shown in Fig. 2. All hyperimmune PPRV antisera reacted with the GST-N1-525 but not with the others (GST-N1-179 and GST-N414-496). In order to further characterize the antigenicity of GST-recombinant N fusion proteins, their reactivities with sera from cattle vaccinated against RPV, in which relatively low levels of RPV neutralizing antibody (1:8 to 1:16) were detected, were evaluated by Western immunoblotting. As shown in Fig. 3, eight sera reacted with both the native N (whole virus) and the full-length N (GST-N1-525) proteins. With the same set of antisera, the GST-N414-496 reacted with seven of the sera, whereas the GST-N1-179 reacted only with serum 1, suggesting that highly immunogenic epitopes were present within the carboxy terminus.



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  		FIG. 2. Reactivity of full-length, amino-terminal, and carboxy-terminal recombinant N proteins with hyperimmune RPV or PPRV sera in Western immunoblotting. All recombinants were expressed in the form of GST fusion proteins. Western immunoblotting was done with hyperimmune RPV bovine antisera (A and B), hyperimmune PPRV caprine antiserum (C) and N polypeptide guinea pig serum (D). The similar pattern observed in panels B and C was also observed with the other RPV antiserum ({alpha}RPV-II) and PPRV sera ({alpha}PPRV-II, {alpha}PPRV-III, and {alpha}PPRV-IV).

 


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  		FIG. 3. Western immunoblot analysis of immunoreactivity of whole virus and recombinant N polypeptides with sera from cattle vaccinated for RPV. (A) Whole virus; (B) full-length N; (C) amino terminus of N; (D) carboxy terminus of N. Lanes 1 through 10, sera from vaccinated cattle; lane 11, hyperimmune anti-RPV bovine serum ({alpha}RPV-Asian). Arrows represent positive precipitate lines as a result of a specific reaction.

 
Identification of immunodominant epitopes on the carboxy terminus. Immunodominant epitopes at the carboxy terminus of N protein were investigated by use of overlapping peptides covering the carboxy terminus (aa 415 to 524) of the N protein. Guinea pig polyclonal antibodies ({alpha}N1-525) recognized 4 peptides (R440, R470, R473, and R508), as illustrated in Fig. 4. Comparison of amino acid sequences of immunoreactive peptides revealed that the carboxy terminus of the N protein possessed at least three nonoverlapping antigenic sites. The first antigenic site (site 1) was considered to be present in residues 440VPQVRKETRASSR452 of immunoreactive peptide R440, since peptide R440 shared amino acid residues 453SDRYK457 with nonimmunoreactive peptide R453. The second antigenic site (site 2) appeared to be in residues 479PEADTDPL486, since these residues were included in immunoreactive peptides (R470 and R473) but not in nonimmunoreactive peptide R487. The last antigenic site (site 3) was considered to be in residues 520DKDLL524 of immunoreactive peptide R508, since R508 shared residues 508PTLGNDSLRTYN519 with nonimmunoreactive peptide R501.



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  		FIG. 4. Reactivities of synthetic peptides to hyperimmune RPV and N protein-specific antisera. Synthetic peptides were made to overlap amino acid residues (aa 414 to 524) in the carboxy terminus of RPV N protein. A peptide (P1) containing amino acid residues unrelated to RPV was used as a control. Bovine antisera specific for RPV ({alpha}RPV-Asian) and N polypeptide-specific guinea pig sera ({alpha}N1-525) were used in a peptide ELISA. Numbers following R indicate the corresponding position of the first amino acid residue of each peptide in the native N protein. The T/C value for each serum was calculated by dividing the mean OD of an RPV peptide by the mean OD of the control peptide plus 3 standard deviations. Samples with T/C values of ≥2 were considered positive for antibody against the specific RPV peptide. Error bars indicate standard deviations.

 
Antigenicity and specificity of each epitope on the carboxy terminus. Although all antigenic sites were recognized equally by the anti-N polypeptide antibody ({alpha}N1-525) in the peptide ELISA, peptides representing antigenic site 2 showed much stronger reactivity with the hyperimmune PRV-specific bovine antisera than with peptides representing the other two antigenic sites (Fig. 4). Similar observations were also made with sera from RPV-infected cattle (Table 2). Peptide R470 (antigenic site 2) was recognized by six of seven sera that reacted with GST-N414-496, whereas none and one of seven sera reacted with peptides R440 (antigenic site 1) and R508 (antigenic site 3), respectively. These results indicated that site 2 may be more antigenic or immunodominant than the other two sites when cattle are infected with RPV. Antigenic sites 2 and 3 reacted with all hyperimmune RPV sera, whereas site 1 reacted with {alpha}RPV-Asian only (Table 2). Antigenic sites 1 and 2 did not show reactivity with sera from goats and cattle infected with PPRV, while antigenic site 3 showed reactivity to some PPRV hyperimmune caprine sera.


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  		TABLE 2. Reactivities of hyperimmune, vaccination, and N protein-specific sera with various recombinant GST-N fusion proteins and synthetic peptides

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, Western immunoblot analyses of denatured GST-recombinant N fusion proteins with hyperimmune anti-RPV bovine sera ({alpha}RPV-Asian, {alpha}RPV-I, and {alpha}RPV-II) and anti-N polypeptide antibody ({alpha}N1-525) demonstrated that both ends of the N protein were antigenic. This observation is consistent with reports on MV N protein (4). Furthermore, both full-length N recombinant protein (GST-N1-525) and the GST fusion protein representing the carboxy terminus of the N protein (GST-N414-496) showed stronger reactivity with antisera in Western immunoblotting (Fig. 2) than did the fusion protein (GST-N1-179) containing the amino terminus of the N protein. This suggests the presence of highly immunogenic epitopes on the carboxy terminus of RPV N protein that are probably independent of conformation and/or are natively unfolded (26). This would not be a surprise, since the carboxy terminus is outside a normally folded protein (16, 25, 26). GST-N1-525 also detected anti-N antibodies in all of the vaccinated cattle by indirect ELISA (Table 2), suggesting that the alteration or masking of epitopes on the N protein by the GST fusion may be minimal with respect to the antigenicity. Nonetheless, on Western immunoblotting denatured antigens, including both whole virus (Fig. 3A) and GST-N1-525 (Fig. 3B), failed to detect anti-N antibodies in 2 of 10 vaccinated cattle which were positive in the indirect ELISA with GST-N1-525 (Table 2), indicating the presence of conformational epitopes which may be highly susceptible to denaturation and immunologically important for the native N protein.

In this study, overlapping peptides and polyclonal antibodies to both native and recombinant N proteins were used to map linear epitopes and assess the comparative antigenicities of identifiable epitopes. To our knowledge, this approach was the first attempt at defining epitopes on the N proteins of the morbilliviruses. Since amino-terminal epitopes of RPV N protein were identified in previous studies (7, 8), we attempted to determine the genomic locations of epitopes on the carboxy terminus of RPV N protein and obtained results similar to those for MV N protein (4). At least three antigenic sites (sites 1 to 3) were identified on the carboxy-terminal part of the N protein by using polyclonal anti-N polypeptide antiserum ({alpha}N1-525). It appeared that antigenic site 3, containing residues 520DKDLLS525 on the carboxy terminus, corresponded to site III (aa 519 to 523) on the MV N protein. Antigenic sites 1 (440VPQVRKETRASSR452) and 2 (479PEADTDPL486) were somewhat different from antigenic site II (aa 457 to 476) on the MV N protein. Our results from epitope mapping with overlapping peptides may raise an argument about the precise location and/or extent of residues of each of the epitopes identified in our study, which could be answered by applying site-directed mutagenesis, but this requires an infectious clone since RPV is a RNA virus. Furthermore, the overlapping peptide approach cannot demonstrate all conformational epitopes; hence, conformational epitopes on native PRV N protein may have not been identified in this study. Further work remains to be done to identify such epitopes and assess their antigenicities.

Notably, the degrees of immunoreactivity of various recombinant N proteins expressed in this study with sera from cattle experimentally infected with RPV were somewhat different from those with anti-N polypeptide guinea pig serum ({alpha}N1-525), as illustrated in Table 2 and Fig. 4. Both antigenic sites 1 and 3 were strongly recognized by {alpha}N1-525 but showed weak or no reactivity with hyperimmune anti-RPV bovine sera, which is probably due to weaker immunogenicity. In contrast, antigenic site 2 reacted strongly with both {alpha}N1-525 and these hyperimmune sera. The greater immunogenicity of antigenic site 2 was also demonstrated with sera from RPV-infected cattle (Table 2). Several factors might account for the difference in immunogenicity between recombinant N protein and RPV (i.e., native N protein). First, perhaps the antigenicities of sites 1 and 3 were reduced by conformational changes through formation of N-P complexes in infected cattle, since the carboxy terminus is intrinsically disordered and folds upon binding to the carboxy-terminal moiety of the phosphoprotein (3, 25, 26, 33). Second, the specificity of both species and the route of inoculation may also contribute to some degree of immunodominance. An alternative explanation, aside from actual differences in antigenicity, is that other protein regions may contribute to the antibody response. Antigenic site 2 may interact with other immune components, such as a helper T-cell epitope, influencing the repertoire specificity of antibody-producing cells (2, 31). In RPV, a hypervariable region (aa 452 to 501) of the N protein has been identified as a helper T-cell epitope (28). If this was the case in the antibody response to NP, differences in the response elicited by the site may affect properties of other sites, which remain to be further investigated.

Cross-reactivity between PRV and PPRV has been a diagnostic challenge, particularly for serological monitoring in areas of endemicity, which raises the need for a diagnostic antigen conserved among PRV strains but distinct from PPRV. In this regard, a promising feature of antigenic site 2 is that the site was demonstrated to be the most immunogenic among linear epitopes on the carboxy terminus of PRV N protein identified and was not recognized by any of the hyperimmune PPRV antisera in peptide ELISA, suggesting that site 2 is likely specific to RPV only (Table 2). In contrast, antigenic site 3 showed reactivity to all RPV hyperimmune sera and some of the PPRV hyperimmune sera (Table 2), perhaps due to the conserved property of this region among strains of RPV and PPRV (12, 18), while antigen site 1 was reactive only to {alpha}PRV-Asia. Thus, it is feasible to design diagnostic assays specific for RPV by using small polypeptides and synthetic peptides representing site 2 instead of full-length N polypeptides. Further work with extended RPV sera remains to be done to determine whether the antigenic site identified in this study is antigenically highly conserved among all RPV strains.


   ACKNOWLEDGMENTS
 
We thank Hoe-Wol Lee and Seung-Hyun Cho for their excellent technical and laboratory assistance. H. M. Wamwayi (Kari NRVC, Muguga, Kenya), E. Couacy-Hymann (LANADA/LCPA, Bingerville, Ivory Coast), and G. Libeau (CIRAD-EMVT, Montpellier, France) are also acknowledged for kindly providing hyperimmune and positive morbillivirus (RPV and PPRV) sera.

This project was supported in part by funding from the National Veterinary Research and Quarantine Service, Ministry of Agriculture and Forestry, Republic of Korea (NVRQS grant B-AD-16-1999-04).


   FOOTNOTES
 
* Corresponding author. Mailing address: Foreign Animal Disease Research Division, National Veterinary Research and Quarantine Service, 480 Anyang-6-dong, Anyang, Kyonggi 430-824, Republic of Korea. Phone: 82-31-467-1860. Fax: 82-31-449-5882. E-mail: choiks{at}nvrqs.go.kr. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Clinical and Diagnostic Laboratory Immunology, July 2004, p. 658-664, Vol. 11, No. 4
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.4.658-664.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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