Identification of Two Novel Linear Neutralizing Epitopes within the Hexon Protein of Canine Adenovirus Using Monoclonal Antibodies

Abstract

Canine adenovirus (CAdV) has a high prevalence in canine populations. High affinity neutralizing antibodies against conserved epitopes can provide protective immunity against CAdV and protect against future outbreaks. In this study, we identified two CAdV-2-specific neutralizing monoclonal antibodies (mAbs), 2C1 and 7D7, which recognized two linear-dependent epitopes. MAb 2C1 potently neutralized CAdV-2 with a 50% neutralization titer (NT50) of 4096, and mAb 7D7 partially neutralized CAdV-2 with a 50% NT50 of 64. Immunoprecipitation, Western blot and protein spectral analysis indicated that both neutralizing mAbs recognized the hexon protein (Hex) of CAdV-2. Through a 12-mer random peptide phage display and synthetic peptides analysis, we finely mapped the neutralizing epitopes to two 10-amino acid (aa) peptides within the CAdV Hex: ⁶³⁴RIKQRETPAL⁶⁴³ located on the surface region; and ⁷³⁶PESYKDRMYS⁷⁴⁵ located in the inner region of the expected 3D structure of trimeric Hex. Importantly, the two epitopes are highly conserved among all CAdV isolates by sequence alignment analysis. Thus, these results provide insights into the interaction between virus and mAbs at the aa level and may have potential applications in the development of novel therapeutic or epitope-based vaccines, antibody therapeutics and a diagnostic method suitable for the rapid detection of all CAdVs.

Keywords: canine adenovirus; hexon protein; identification; monoclonal antibodies; novel epitopes.

Figure 1

Identification of mAbs reacting with CAdV-2 by indirect immunofluorescence and Western blot. MDCK cells were infected with CAdV-2 at a multiplicity of infection (MOI) = 10 for 24 h, and mAbs 2C1 and 7D7 were used to detect the virus (a) by indirect immunofluorescence assay (magnification = 200×); (b) Western blot (WB) after native PAGE; and (c) WB after SDS-PAGE.

Figure 2

MAbs 2C1 and 7D7 react with the CAdV-2 hexon. (a) SDS-PAGE of CAdV-2 proteins after immunoprecipitation with mAbs 2C1 and 7D7. Marker: PageRuler Prestained Protein Ladder. (b) Results of MALDI-TOF-MS analysis. Protein scores (n = 1074 for mAbs) that were greater than 26 were significant (p < 0.05). Protein scores were derived from ion scores as a non-probabilistic basis for ranking protein hits. (c) A truncated, recombinant peptide spanning the C-terminal 300 aa of the CAdV hexon protein was expressed in Escherichia coli. Coomassie-stained SDS-PAGE showing characteristic 40-kDa band; marker: PageRuler Prestained Protein Ladder. (d) Reactivity of the truncated hexon protein with mAbs 2C1 and 7D7 by Western blot analysis. (e) 3D structure of the expressed 300 aa from the truncated peptide (red spheres) predicted by the SWISS-MODEL online server.

Figure 3

Confirmation of the epitope recognized by mAbs 2C1 and 7D7. Selected positive phage clones reacted specifically with mAb 2C1 and 7D7 in phage ELISA. Eight and fourteen phage clones were selected after four rounds of biopanning for (a) 2C1 or (b) 7D7, respectively, and their binding was analyzed by phage ELISA (upper). Three independent assays were carried out. Lower: sequence comparison of random peptide inserts displayed on the positive phages to the CAdV-2 sequence.

Figure 4

Truncated peptides define the minimal linear epitope recognized by mAb 2C1 or 7D7. (a) ELISAs were performed using synthetic peptides (4–10 aa) with different deleted aa residues from the N and/or C termini of the peptide ⁶³⁴RIKQRETPAL⁶⁴³ to determine the minimal linear epitope sequence recognized by mAb 2C1. (b) ELISAs were performed using synthetic peptides (4–10 aa) with deleted different aa residues from the N and/or C termini of the peptide ⁷³⁶PESYKDRMYS⁷⁴⁵ to determine the minimal linear epitope sequence recognized by mAb 7D7.

Figure 5

Spatial structure and positions of the identified epitopes. (a) Relative locations of the identified epitopes are presented in spherical, cartoon and external surface depictions of monomer hexon from a 3D structure of CAdV-2 trimeric hexon that was predicted using the SWISS-MODEL online service. Epitopes recognized by mAbs 2C1 and 7D7 are shown in red and purple, respectively, separately, and from the front view and the bottom view. (b) Secondary structure features of the CAdV-2 hexon were predicted using PROTEAN software. The putative 2C1 and 7D7 epitopes are shown in the boxes.

Figure 6

Amino acid sequence alignments of the epitope regions from several different AdV hexon proteins. (a) Sequence comparison of the mAb epitopes of different CAdV isolates available in GenBank. The homologous sequences corresponding to the putative hexon epitopes for mAbs 2C1 and 7D7 are shown in red boxes. (b) Alignment of the CAdV mAb epitope sequences with 10 other AdV hexon proteins available in GenBank.