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

Shujie Wang,Haijuan He,Wenjiao Xue,Chunsheng Wang,Xuehui Cai,Yanzhu Zhu,Xiao Ren,Hongfeng Wang,Gang Wang

doi:10.3390/vaccines9020135

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.

Highlights

Infectious canine hepatitis (ICH) is an acute septic infectious disease, and its etiological agent is canine adenovirus type 1 (CAdV-1)
Immunoprecipitation, Western blot and protein spectral analysis indicated that both neutralizing monoclonal antibodies (mAbs) recognized the hexon protein (Hex) of Canine adenovirus (CAdV)-2
CAdV type 2 (CAdV-2) can cause infectious tracheobronchitis (ITB) and pneumonia in dogs, known as kennel cough, fever, serous to mucinous rhinorrhea, tonsillitis and laryngotracheitis in dogs, which has a high prevalence in canine populations [4,5]

Summary

Introduction

Infectious canine hepatitis (ICH) is an acute septic infectious disease, and its etiological agent is canine adenovirus type 1 (CAdV-1). CadVs have non-enveloped capsids with pseudo-icosahedral symmetry, linear doublestranded DNA of about 30 kb and are included in the genus Mastadenovirus of the family Adenoviridae [6]. The major coat protein of CAdV is hexon (Hex), which plays a major role in viral tropism and neutralization [8]. The CAdV capsid structure is relatively well conserved, and is primarily composed of homo-trimers of Hex; 240 trimers form the icosahedron’s 20 facets [9,10]. Apart from these, the capsid is stabilized by other Hex-associated proteins [13]. It is estimated that most of the adenoviral neutralizing antibodies are generated against Hex [14]

Methods

Results

Conclusion