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Applied and Environmental Microbiology, June 2005, p. 2894-2901, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2894-2901.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

Gregory D. Wiens^1* and Jennifer Owen²

USDA-ARS, National Center for Cool and Cold Water Aquaculture, 11861 Leetown Rd., Kearneysville, West Virginia 25430,¹ Department of Molecular Microbiology and Immunology, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd., Portland, Oregon 97201²

Received 2 November 2004/ Accepted 21 December 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Renibacterium salmoninarum is an important cause of disease in wild and cultured salmon, trout, and char (reviewed in references 12, 16, and 40). Fish are often chronically infected with the pathogen, and in some locations, a large percentage of fish can be subclinically infected (11, 26, 28). Accurate diagnosis of infected fish is important for the limitation of horizontal and vertical transmission of this pathogen (10). A number of immunological and molecular assays have been developed to identify Renibacterium-infected fish (reviewed in reference 29). The most widely used technique is the enzyme-linked immunosorbent assay (ELISA), which allows rapid screening of large sample numbers using semiautomated procedures (29). Several ELISAs have been developed to measure p57, a unique cell surface and secreted R. salmoninarum protein (17, 41).

p57 is an immunodominant antigen for rabbits and salmon (2, 13), and paradoxically, this protein also has immunosuppressive activities (5, 15, 20, 34, 36). p57 binds to salmonid leukocytes (42) and mammalian red blood cells (8). The binding site on p57 for these cells remains uncharacterized. We previously described three monoclonal antibodies (MAbs) that inhibit the agglutinating activity of p57 and determined that they bind to a recombinant amino-terminal fragment of p57 encoding amino acids 32 through 243 (39). Here, we use transposon mutagenesis and peptide mapping to identify neutralizing MAb epitopes to better understand bioactivity and develop improved detection assays. In addition, we used 15-mer synthetic peptides, spanning the entire p57 protein, to map the recognition of salmon anti-p57 and goat anti-Renibacterium salmoninarum antisera. Epitope localization is an initial step toward the development of second-generation immunoassays with higher sensitivity and precision.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monoclonal antibodies.
MAbs 4D3, 4H8, 4C11, 1A1, and 4D10 have been previously described (41, 42). Anti-salmonid immunoglobulin MAb 1-14 has been previously described (9). Hybridoma cells were grown in either tissue culture medium or in BALB/c mouse ascites fluid. MAbs were purified using protein A-Sepharose (Bio-Rad) and stored at –20°C in 50% glycerol. MAbs were biotinylated as previously described (41).

Polyclonal antisera.
Phosphatase-labeled affinity-purified goat antibody to Renibacterium salmoninarum cells (catalog no. 05-96-91, lot VC126) was obtained from Kirkegaard and Perry (Gaithersburg, MD). Polyclonal salmon anti-p57 was generated in spring chinook salmon obtained from Dworshak National Fish Hatchery. These fish were offspring of brood stock with undetectable or low levels of R. salmoninarum infection. Fish were immunized by intraperitoneal injection with 50 µg of recombinant p57 (rp57) protein (31) in a total volume of 100 µl of Freund's complete adjuvant. Serum from control and immunized fish were collected after 12 weeks and stored at –70°C.

Transposon mutagenesis.
Epitope mapping was achieved with an in vitro transposon mutagenesis system (EPICENTRE, Madison, WI). Briefly, 19 codon (57 nucleotide) insertions were introduced into a previously described expression plasmid (pGW) containing amino acids 32 to 243 of p57 (39). Insertions were introduced using a Tn5 in vitro transposition system (19). The EZ::TN (NotI/KAN-3)-modified Tn5 transposon (0.05 pmol) was incubated with pGW plasmid DNA (0.05 pmol) and transposase for 2 h, and the reaction mixture was transformed into TransforMax EC100 Escherichia coli cells by electroporation according to the manufacturer's instructions (EPICENTRE, Madison, WI). Kanamycin-resistant E. coli colonies were directly screened by colony PCR (35) for the presence of a transposon insertion within the p57 gene fragment. PCRs were carried out with T7 promoter and terminator primers (Novagen primers 69348-3 and 69337-3) flanking the p57 gene. PCR-positive colonies were inoculated into 3-ml overnight cultures, and plasmid DNA was isolated using a Qiaprep spin mini-prep kit (QIAGEN, Valencia, CA). The kanamycin resistance gene was removed by digestion with the NotI restriction enzyme, and linear plasmid DNA was gel purified, religated, and transformed into E. coli BL21(DE3) cells (Novagen). Plasmid DNA was isolated from overnight cultures and sequenced using T7 promoter or terminator primers. Nucleotide sequencing was performed using an ABI Prism terminator cycle-sequencing kit and AmpliTaq DNA polymerase according to the manufacturer's directions (Applied Biosystems). A 377 PRISM automated DNA sequencer (PE Applied Biosystems, Foster City, CA) was used for sequence determination at the Nucleic Acid Core Facility of the Department of Molecular Microbiology and Immunology at Oregon Health and Science University, Portland, OR.

Immunoblotting.
The wild type and 45 transposon insertion mutants were grown to mid-logarithmic phase in LB medium. Proteins were induced with 1 mM isopropyl-ß-D-thiogalactopyranoside for 3 hours. The bacterial cells were harvested by microcentrifugation and resuspended in 20 µl sodium dodecyl sulfate (SDS) sample buffer (50 mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol) per 1 mg of wet cell pellet. DNase I (Novagen, Madison, WI) was added to degrade E. coli genomic DNA. Proteins were separated on a 12% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad) overnight. Blots were probed with 1 µg ml^–1 MAb 4D3 or 4H8 or 2 µg ml^–1 MAb 4C11 for 1.5 h at room temperature. Blots were washed three times in phosphate-buffered saline containing 0.01% azide and 0.1% Tween 20 followed by three washes in PBSA (phosphate-buffered saline containing 0.01% azide). MAb binding was detected with a 1:1,000 dilution of alkaline phosphatase-coupled goat anti-mouse kappa antiserum (Southern Biotechnology Associates, Birmingham AL). After further washing, the blots were developed using nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Gibco BRL, Rockville, Maryland). Binding was scored positive (+) if a strong band was identified in three experiments. Binding was scored as weak (±) if the staining was faint or inconsistent or scored as negative (–) if there was an absence of binding.

Peptides.
Custom overlapping, biotinylated peptides were obtained from Mimotopes (San Diego, CA). Sixty-nine pentadecapeptides spanning the entire p57 amino acid sequence were synthesized by the multipin synthesis method (37) and amidated at the carboxy terminus. Peptides overlapped by 7 amino acids, with the exception of the last two carboxy-terminal peptides, which overlapped by 8 amino acids. Each peptide was synthesized with a four-residue spacer (SGSG) and biotinylated at the amino terminus. Freeze-dried peptides were dissolved in 200 µl 100% dimethyl sulfoxide to an approximate concentration of 3.5 mM and stored at –70°C. Aliquots of a 1:100 dilution were prepared in sodium phosphate buffer containing 0.1% bovine serum albumin (BSA) and 0.1% sodium azide, stored at –20°C, and diluted prior to use.

ELISA.
Direct binding assays were used to measure MAb and polyclonal antibody recognition of peptides. Half-area microtiter plates (Fisher, Pittsburgh, PA) were coated with 5 µg ml^–1 streptavidin (Sigma, St. Louis, MO) overnight at 37°C. Plates were washed three times with a sodium phosphate buffer (pH 7.4) containing (0.1%) sodium azide (PBSA) and 0.05% Tween 20, followed by three washes with PBSA. Wells were blocked using PBSA containing 2% BSA for 1 h at room temperature. Peptides were added to the wells at a 1:1,000 dilution in 2% BSA-PBSA and incubated at room temperature for 1.5 h. After washing, 1 µg ml^–1 of MAb 4D3 or 4H8 or 2 µg ml^–1 MAb 4C11 diluted in 2% BSA-PBSA was added to wells and mixtures were incubated for 1 h at room temperature. After washing, a 1:1,000 dilution of secondary antibody, alkaline phosphatase-coupled goat anti-mouse kappa (Southern Biotechnology Associates, Birmingham, AL) was applied and incubated for 1 h. Substrate p-nitrophenyl phosphate (Sigma, St. Louis, MO) in diethanolamine buffer (pH 9.8) was added to detect the antibody conjugate, and the optical density (OD) of the wells was determined after 0.5 to 2 h. For direct binding assays using polyclonal antisera, a 1:1,000 dilution of chinook salmon or phosphatase-labeled goat antibody to Renibacterium salmoninarum cells (Kirkegaard and Perry, Gaithersburg, MD), diluted in 2% BSA-PBSA, was incubated with the streptavidin-bound peptide either overnight at 10°C (fish antibodies) or 1.5 h for goat antibodies. Salmon antibodies were detected with a 1:1,000 dilution of MAb 1-14 (mouse anti-fish immunoglobulin) applied for 1 h at room temperature. MAb 1-14 was detected with alkaline phosphatase-labeled affinity-purified goat anti-mouse kappa.

Inhibition ELISAs were performed to determine whether synthetic peptides competed with MAb 4H8, 4D3, and 4C11 binding to recombinant p57 or extracellular protein. In these assays, 5 µg ml^–1 extracellular protein from isolate ATCC 33209 or recombinant p57 was coated onto ELISA plate wells overnight at 37°C. For single-point assays, peptides (17.5 µM) were preincubated with 1 or 2 µg ml^–1 MAb for 1.5 h prior to transfer to ELISA wells. For multipoint assays, dilutions of peptides were used. Peptide-antibody mixtures were incubated on the plate for 1 h. Plate-bound antibody was detected with alkaline phosphatase-labeled, affinity-purified goat anti-mouse kappa antisera.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mapping neutralizing epitopes using transposon mutagenesis.
The locations of the 4D3, 4H8, and 4C11 epitopes were mapped using an in vitro transposition epitope-disruption strategy (Fig. 1). In this approach, the epitope location was inferred from transposon insertions that disrupt MAb binding. A bank of transposon mutants was created and a total of 733 Kan^r colonies were screened for plasmids that contained a single transposition in the p57 coding region. A total of 45 clones (6% of the total screened) contained a transposon inserted between the XhoI to EcoRI restriction site boundaries. This was lower than the 10% expected insertion frequency calculated from the ratio of the insert to the total plasmid size (636-bp target in a 6,129-bp plasmid). The reason for the lower efficiency is unclear. After Kan^r gene excision and religation, mutants were sequenced using forward and reverse primers to determine the precise location and reading frame of the 57-nucleotide (nt) mutation (supplemental material is available at http://ncccwa.ars.usda.gov/New%20Public%20Information.htm). Forty-five mutants were sequenced, and 38 contained unique mutations (Table 1). There were five pairs of mutants containing the same insertion site and reading frame (plasmids pGW 8 and 9, 12 and 13, 21 and 22, 24 and 25, and 33 and 34). One insertion was obtained three times (plasmids pGW 14, 15, and 16). All 45 mutant proteins were overexpressed in E. coli, and the binding of MAbs 4D3, 4C11, and 4H8 to each mutant was determined by immunoblotting. Each mutant was successfully expressed as determined by Coomassie blue protein staining (data not shown), and at least one MAb bound to each mutant. Thus, lack of MAb binding to some mutants indicated epitope disruption or occlusion resulting from the 19-amino-acid insertion. MAb 4D3, 4C11, and 4H8 binding to wild-type p57NH2 was robust, and examples of binding to several mutant p57 proteins are shown in Fig. 2. For example, p57NH2.Y⁷⁸ was not recognized by MAbs 4H8 and 4C11, while 4D3 binding was not altered by the 19-amino-acid insertion between Tyr⁷⁸ and Ser⁷⁹. The binding profile of each antibody is listed in Table 1. In total, MAb 4H8 failed to bind 16% (7/45) of the transposon mutants. The seven mutants exhibiting disrupted 4H8 binding contained insertions clustering into a single region between amino acids 51 and 112. In contrast, the binding of MAb 4C11 was disrupted by insertions spanning a larger region of the protein. MAb 4C11 failed to bind 87% (39/45) of the mutants. The epitope-disrupting insertion sites clustered into two regions: one located between amino acid 78 and 95 and the second between amino acids 138 and 210. MAb 4D3 failed to bind 64% (29/45) of the mutants, and the locations of these insertions spanned one continuous region between amino acids 158 and 234. This region corresponds to the A1 repeat (Fig. 3) of p57 (7, 39). In summary, the binding profiles suggest that the 4H8 and 4D3 epitopes are distinct from one another, while the 4C11 epitope partially overlaps with both the 4H8 and 4D3 epitopes.

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FIG. 1. In vitro transposition epitope-disruption strategy. An in vitro transposition reaction was carried out with a modified Tn5 transposon and the pGW plasmid containing a 636-nt EcoRI-to-XhoI msa fragment (step 1). This fragment encodes amino acids 32 to 243 of p57. After transformation, kanamycin-resistant colonies containing a transposon inserted within the p57 fragment were identified by direct colony PCR (step 2). The kanamycin resistance gene was removed from each mutant by NotI restriction enzyme digestion, religated, and transformed into E. coli BL21(DE3) cells (step 3). Insertion sites and insertion reading frames were identified by DNA sequencing. All three reading frames of the 57-nt transposon insertion lack stop codons, and thus, three different mutant sequences can be obtained depending on which reading frame the transposon inserts. The three 19-amino-acid mutant sequences are LSLVHILRPQDVYKRQXXX for reading frame 1, XVSCTHLAAARCVQETXXX for reading frame 2, and XCLLYTSCGRKMCTRDXXX for reading frame 3. The three X's represent amino acids introduced by the 9-bp insertion site duplication (19). Each mutant protein was overexpressed, and MAb binding was determined (step 4).

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TABLE 1. Transposon insertion location and MAb recognition of p57NH2 wild-type and mutant proteins

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FIG. 2. MAbs 4D3, 4H8, and 4C11 display differential recognition of mutant p57 proteins. A representative of five different binding profiles is shown. Lanes: 1, p57NH2.Y78; 2, p57NH2.K107; 3, p57NH2.A139; 4, p57NH2.Q181; 5, p57NH2.Y233. No mutant was disrupted for both 4D3 and 4H8 binding. The binding to wild-type (WT) p57NH2 is shown in the last column, and the WT migrates faster than the mutant proteins (not shown). Data are representative of the results from three separate experiments and are a composite of several immunoblots.

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FIG. 3. The amino acid sequence of p57 with repeated regions underlined. Shown below are the sequences of 69 overlapping synthetic peptides spanning the entire length of p57. The peptide constructs include biotin-SGSG-peptide-amide, with SGSG as the linker designed by the manufacturer. The repeat regions in p57 consist of two A repeats (A1 and A2) and five B repeats (B1 to B5) (7, 39). The invariant amino acids in the repeats are shown in boldface type. Also in boldface type are the leader sequence (amino acids 1 to 26) and amino acid Ala139, which when changed to Glu, disrupts MAb 4C11 binding (43).

Epitope mapping using synthetic peptides.
To determine whether epitopes could be further mapped, biotinylated 15-mer peptides were synthesized (Fig. 3). A 7-amino-acid overlap was chosen to reduce the possibility of missing epitopes residing between peptides. Both direct binding and competition assays were used to measure peptide-MAb interaction. In the direct binding ELISA, the biotinylated peptides were anchored to the ELISA plate via streptavidin. Surprisingly, the neutralizing MAbs 4H8, 4C11, and 4D3 did not bind peptides located in the regions defined by transposon mutagenesis (Fig. 4). To examine the functionality of the peptides, additional monoclonal antibodies were assayed for binding. Nonneutralizing MAbs 4D10 and 1A1 both bound strongly to peptide 44 and weakly to peptides 45 and 46 (Fig. 4 and data not shown), demonstrating that the peptides could be used to map the recognition sites of some MAbs. We also examined the binding of MAb 3H1 to peptides in the direct binding assay, as this antibody is used in several diagnostic assays. However, similar to the neutralizing MAbs, 3H1 did not bind any of the peptides (data not shown). To rule out the possibility of biotin-avidin interaction occluding epitopes, a competitive solution-phase assay was used to measure the ability of peptides to inhibit MAb binding to rp57. None of the peptides, at the highest concentrations tested (17.5 µM), inhibited the binding of MAbs 4H8, 4C11, 4D3, or 3H1 to p57 (data not shown). In contrast, peptide 44 inhibited 4D10 and 1A1 binding to rp57 in a dose-dependent manner (Fig. 5A and data not shown). The binding of these MAbs to peptide 44 appears to be of relatively high affinity, as the concentration of peptide that inhibited 50% binding was 20 nM. In summary, the 15-mer peptides were not useful for further localization of the neutralizing MAb epitopes on p57; however, the nonneutralizing 4D10 and 1A1 MAb epitopes were localized predominantly within the 15-mer sequence of peptide 44.

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FIG. 4. MAb 4H8, 4C11, 4D3, and 4D10 binding to peptides derived from the entire p57 sequence. Peptide numbers along the x axis correspond to the peptide sequences listed in Fig. 3. Peptides 1 through 30 encompass the p57NH2 protein fragment, while the horizontal bar and adjacent peptide numbers represent the location of transposon insertion sites that disrupt MAb binding to p57NH2 as determined by immunoblotting results summarized in Table 1. The positive control (P) represents MAb binding to rp57, and the negative control (N) is MAb binding to streptavidin-coated wells. Data are average optical densities (O.D.) at 410 nm + standard deviations of the results from triplicate ELISA plates.

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FIG. 5. Peptide 44 preincubation with MAb 4D10 (A) but not 4C11 (B) inhibits binding to rp57-coated ELISA wells. Dilutions of peptide 44 or peptide 58 were preincubated with MAbs for 1.5 h prior to addition of the mixtures to p57-coated wells. Results are average optical densities (O.D.) + standard deviations of the results from triplicate wells.

The direct binding profile of MAb 4C11 (Fig. 4) was unusual, as there was consistent binding to peptide 58 (amino acids 456 to 470) located outside the region (amino acids 78 to 210) mapped by transposon mutagenesis (Table 1). In addition, we previously failed to detect binding of MAb 4C11 to a recombinant, carboxy-terminal p57 protein (amino acids 242 to 557) which includes the peptide 58 sequence (39). Peptide 58 is not "sticky," as no other MAb bound to peptide 58 in the direct ELISA (Fig. 4 and data not shown). To further investigate the binding of 4C11 to peptide 58, a competitive solution-phase assay was used. Peptide 58, at the highest concentrations tested (35 µm), did not inhibit the binding of 4C11 to rp57 (Fig. 5B), nor did it inhibit binding of MAb 4D10 to rp57 (Fig. 5A). In summary, these data indicate that if binding is occurring, it is a weak interaction, or that the peptide, when bound to streptavidin, may fortuitously mimic the native epitope.

Polyclonal antibody recognition of peptides spanning p57.
To identify sequence regions recognized by immunized fish, antiserum from p57-immunized and control fish were tested for binding to the panel of 69 peptides. Two of three fish sera (numbers 83 and 88) bound to select peptides (Fig. 6). The binding appeared to be specific, as sera from control fish failed to bind any of the peptides. Interestingly, three peptides, 41, 47, and 53, were recognized by both fish 83 and fish 88 antisera. These peptides correspond to the beginning of the B1, B2, and B3 repeats of p57 (Fig. 3). Peptides 47 and 53, located in the B2 and B3 repeats, share 13 of 15 of their amino acids. However, peptide 41 shares only limited homology with peptides 47 and 53. These data suggest that the B repeats may be generally immunogenic or cross-reactive. Minor reactivity was also observed to these same peptides by a commercial, affinity-purified goat polyclonal antisera generated against whole R. salmoninarum cells. Apart from recognition of these common peptides, a number of differences were observed between salmon and goat antibodies. In general, there was little chinook antibody binding to peptides within the A1 repeat (spanned by peptides 19 to 29) and A2 repeats (spanned by peptides 31 to 40). Minor reactivity of fish 83 was observed to peptides 29 and 30, which correspond to the end of the A1 repeat (peptide 29) or span the gap between the A1 and A2 repeats (peptide 30). The goat antisera elicited to whole R. salmoninarum exhibited minor reactivity to peptides from the A1 repeat and strong reactivity to A2 repeat peptides 31 and 33. In summary, fish and goat antiserum recognized peptides located within the B repeats, while the fish response to the A repeats was weaker than the goat antibody response.

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FIG. 6. Direct binding of chinook immune, control, or goat immune sera to p57 peptides as determined by ELISA. Positive control (P) represents binding to rp57, and negative control (N) represents binding to uncoated wells. Data are average optical densities (O.D.) + standard deviations of the results from three ELISA plates. Data are representative of results obtained from two or three experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Humoral recognition of Renibacterium salmoninarum surface proteins is poorly understood. In this study, we measured the binding of monoclonal and polyclonal anti-p57 antibodies to better characterize neutralizing epitopes on p57 and to suggest avenues for immunodiagnostic assay improvement. For these studies, we utilized two approaches: in vitro transposon mutagenesis and peptide binding assays. We report three main findings. First, using transposon mutagenesis we localized neutralizing murine antibody epitopes to the following regions: the 4H8 epitope was disrupted by insertions between amino acids 51 to 112; the 4C11 epitope was disrupted by insertions between amino acids 78 through 210; and the 4D3 epitope was disrupted by insertions between amino acids 158 through 234. Second, we identified that MAbs 1A1 and 4D10 recognized an epitope(s) localized within peptide 44 that spans amino acids 345 to 360 and contains part of the B1 repeat of p57. Our third finding was that both goat anti-R. salmoninarum antisera and chinook salmon anti-p57 antisera recognized peptides from the B1 to B3 repeats. To our knowledge, these results represent the most precise localization of neutralizing epitopes on p57 and the first mapping of polyclonal R. salmoninarum antiserum using synthetic peptides.

Analysis of neutralizing determinants: MAb 4D3, 4H8, and 4C11 recognition of assembled epitopes.
Knowledge of neutralizing epitopes is important for understanding pathogenic mechanisms and for vaccine design. Antibody epitopes can be classified into two types: linear (or continuous) and assembled (or discontinuous) (3, 32). Antibodies that bind reduced and SDS-denatured proteins often recognize linear epitopes. The murine MAbs 4H8, 4C11, and 4H3 recognize SDS-denatured and reduced p57 and also neutralize the agglutinating activity of the native protein (39, 42). Thus, it was expected that the binding could be mapped to short linear sequences; however, transposon mutagenesis identified large regions that, when mutated, disrupt MAb binding. The regions, disrupted by insertion, spanned 61 amino acids for the 4H8 antibody, 76 amino acids for the 4D3 antibody, and 132 amino acids for the 4C11 antibody. This contrasts with the expected size of linear peptide epitopes which are usually 8 to 12 amino acids but can comprise up to 15 amino acids (32). Two possible explanations for these data are (i) the 19-amino-acid insertions either block or modify the linear epitope(s) or (ii) the MAbs may recognize assembled epitopes that are disrupted due to changes in secondary protein structure or folding. In support of the later possibility, the failure of the neutralizing MAbs to bind 15-mer overlapping peptides suggests that these antibodies do not recognize linear epitopes but rather bind assembled epitopes. If these antibodies bind assembled epitopes, ß-strand structure may be involved, as this is the predominant secondary structure predicted for the amino-terminal region (data not shown). To fully characterize assembled, neutralizing epitopes, MAb cocrystallization with p57 is required (25).

These mapping results have implications for understanding p57 binding to eukaryotic cells. Previously, we found that MAbs 4H8, 4C11, and 4D3 inhibited the binding of p57 to eukaryotic cells differently. MAbs 4H8 and 4C11 inhibited p57 binding to fish leukocytes and rabbit erythrocytes, while 4D3 inhibited binding to only rabbit erythrocytes and not to fish leukocytes (42). Here, we identify that 4D3 binds to the p57 A1 repeat. This repeat is 81 amino acids long and contains a transcription factor immunoglobulin-like domain which is found in the extracellular domain of members of the plexin protein family of adhesion-repulsion molecules (1, 4). These results indirectly implicate the A1 repeat in the erythrocyte agglutinating activity of p57. Regions outside the A repeat also appear to be involved in leukocyte binding, as the 4H8 epitope was localized outside this region. At present, we do not know whether antibody neutralization occurs by direct or indirect blocking of the leukocyte-binding site. While less frequent, neutralization can occur by antibodies that bind outside the receptor binding site, as has been documented for the anti-influenza hemagglutinin monoclonal antibody HC45 (14). We attempted to model the p57NH2 protein in an effort to understand the spatial proximity of these regions; however, these efforts were unsuccessful due to low homology to other proteins of known structure. Examination of the binding activity of the transposon mutants may provide a further avenue for analysis of the eukaryotic cell binding domain(s) on p57.

Implications of epitope mapping for improving immunodiagnostic assays.
Immunodiagnostic assays that measure p57 are important tools for the identification of subclinically infected fish (29). Detection of these fish is important for health monitoring and for culling progeny of infected adults to prevent vertical transmission (29). The ELISA is one of the most commonly used methods for bacterial kidney disease detection, as it can be semiautomated to run large numbers of samples. Since this assay detects a diffusible antigen (33, 36), a positive test can provide evidence for Renibacterium infection even if the bacteria reside outside the sample area (29). For example, localized infections can occur in the head and skin tissues, which are sites not usually assayed in screening programs (29). Several ELISAs have been described utilizing either polyclonal antibodies (21, 24, 27, 30), monoclonal antibodies (33), or a combination of both types of antibodies (23). Assays relying on polyclonal antisera are limited by reagent availability, lot consistency, and cross-reactivity (29). A potential limitation of the monoclonal ELISA is a lower reported sensitivity than that of several polyclonal ELISAs (24, 38). The epitope analysis described here has implications for incorporating additional MAbs to increase sensitivity. The principal antibodies utilized are the 4D3 and 3H1 MAbs (33). The inclusion of the 4H8 antibody might be one possible avenue for further investigation, as it recognizes a spatially separate epitope from 4D3. The inclusion of either the 1A1 or the 4D10 antibody, both of which recognize a linear epitope(s) between the B1 and B2 repeats, may also be used. Both of these antibodies are of relatively high affinity, as determined by competition experiments. The 4C11 antibody should be avoided, as we have identified antigenic variation in the epitope it recognizes. The 4C11 antibody does not recognize a Norwegian Renibacterium salmoninarum isolate, 684, that contains a single Ala¹³⁹Glu mutation in both copies of the msa genes (43). This mutation is present in a number of Scandinavian isolates (G. D. Wiens, unpublished data). Besides being sensitive to the Ala¹³⁹Glu mutation, the 4C11 antibody was the most sensitive to transposon mutagenesis, indicating that this epitope may be the easiest to disrupt by spontaneous mutations in p57. The ease of disruption may be related to an overall weaker binding of the 4C11 antibody to p57 or the greater dependence on protein conformation for binding. In summary, epitope mapping suggests the addition of the 4H8 and either the 1A1 or 4D10 antibodies as candidates for inclusion in the MAb ELISA.

Fish antibody recognition of p57 repeats.
The library of synthetic 15-mer peptides spanning the entire p57 protein allowed us to measure the mammalian and fish polyclonal antibody response to peptide epitopes. In general, the response of both the fish antisera and goat antisera was weak in comparison to the recognition of whole recombinant p57 (data not shown). In addition, one of three fish antisera did not bind any of the peptides yet had a high titer to recombinant p57 (data not shown). This is not surprising, as antipeptide responses are usually a minor component of the total antibody response to proteins (18). Nevertheless, ELISA binding profiles could be generated from two of three fish immunized with recombinant p57. It was of interest that the fish antibodies recognized peptides overlapping the p57 B repeats, while there was a lack of binding to peptides spanning the A1 and A2 repeat regions. Antibodies to Plasmodium falciparum circumsporozoite protein repeats are hypothesized to divert immune recognition away from single epitopes located outside the repeat region (6). It would be of interest to determine whether the polyclonal antisera specific for the p57 B repeats lacked neutralizing activity. Possibly, the B repeats may serve as a diversion from a response to the neutralizing regions in the amino terminus. It should be noted that only a limited number of antisera were studied and that antibody binding kinetics were not examined. Larger sample sizes and analysis of naturally infected fish are required to fully define the humoral immune response to p57. The peptides described here may be useful for studies comparing polyclonal antisera.

In summary, we have used two complementary techniques to map antibody binding regions on p57. The transposon mutagenesis technique is a rapid but indirect method for epitope mapping; 19-amino-acid insertions are created to identify locations that disrupt binding. This method has proven to be a very powerful strategy for dissecting protein structure-function relationships in other systems (reviewed in reference 22) and for epitope mapping described here. Transposon mutagenesis localized three neutralizing epitopes which could not be mapped with 15-mer overlapping synthetic peptides. However, the peptides were useful for identifying epitopes recognized by several nonneutralizing monoclonal antibodies as well as by fish and goat polyclonal antibodies.

 
This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education Service grant number 2002-35204-14256 to G.D.W.

We thank J. Winton and R. Pascho for helpful discussions and L. Rhodes and B. Wiens for critical review of the manuscript. We thank S. Alcorn for fish immunization and collection of antisera.

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

 
* Corresponding author. Mailing address: USDA-ARS, National Center for Cool and Cold Water Aquaculture, 11861 Leetown Rd., Kearneysville, WV 25430. Phone: (304) 724-8340. Fax: (304) 725-0351. E-mail: gwiens{at}ncccwa.ars.usda.gov.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, June 2005, p. 2894-2901, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2894-2901.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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