Design of Multi‐Epitope <scp>DIVA</scp> ‐Compatible Vaccine Candidate Against Canine Parvovirus 2

Canine parvovirus type 2 (CPV-2) is a highly contagious and often fatal canine pathogen, with antigenic variants (CPV-2a, 2b, 2c), maternally derived antibody (MDA) interference, and lack of Differentiating Infected from Vaccinated Animals (DIVA) capability limiting current vaccines. Using an immunoinformatics-based reverse vaccinology approach, we identified conserved B-cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes from CPV-2 structural (VP1, VP2) and nonstructural (NS1, NS2) proteins. These were screened for antigenicity, immunogenicity, safety, and stability. The screening identified three B-cell, two CTL, and five HTL epitopes. Out of these, two representative epitopes were utilized for in vitro evaluation. The peripheral blood mononuclear cells (PBMCs) were collected from 10 dogs 28 days after vaccination with a live-attenuated whole-virus CPV vaccine and pooled. This pool was expected to contain memory cytotoxic T cells recognizing a broad viral epitope repertoire. On antigenic stimulation, PCPV2 induced significant proliferation of canine PBMCs, with the highest response at 33.33 μg/mL, alongside ~15.67-fold upregulation of IFN-γ mRNA, indicating a memory T-cell response. A multi-epitope vaccine was designed by integrating validated epitopes with the TLR-4 agonist RS09 and a Capripoxvirus-derived DIVA marker, linked by AAY/EAAAK spacers. The vaccine construct can be effectively delivered via a recombinant canine adenovirus-2 vector for efficient in vivo expression and cellular response. Structural modeling, molecular docking, immune simulations, molecular dynamic simulations, and codon optimization predicted strong receptor binding, stability, IgG/T-cell responses, and expression feasibility in Escherichia coli and canine cells. These findings support PCPV2 as a potent immunogen and demonstrate the promise of the designed construct for a potential adenovirus-vectored vaccine; however, in vivo validation will be essential to confirm efficacy, cross-variant protection, and DIVA performance.