# A Highly Immunogenic and Cross-Reactive Multi-Epitope Vaccine Candidate Against Duck Hepatitis A Virus: Immunoinformatics Design and Preliminary Experimental Validation

## TL;DR

This paper presents a new multi-epitope vaccine candidate for duck hepatitis A virus that shows strong immune responses and cross-genotype reactivity.

## Contribution

A novel multi-epitope vaccine design for DHAV with demonstrated immunogenicity and cross-reactivity across genotypes.

## Key findings

- The vaccine candidate induced high-titer antibodies with potency up to 1:128,000 in immunized animals.
- Western blot analysis confirmed cross-reactivity of the vaccine with recombinant VP proteins across DHAV genotypes.
- Computational validation showed immune interaction potential and stability of the vaccine construct.

## Abstract

Duck viral hepatitis (DVH), a highly contagious disease, is caused primarily by duck hepatitis A virus (DHAV). The viral genotypes exhibit significant diversity, creating a challenge as monovalent vaccines fail to provide cross-genotype protection in ducklings. This study aimed to design a multi-epitope peptide vaccine targeting different genotypes of DHAV. Using immunoinformatics approaches, we systematically identified key antigenic determinants, including linear B-cell epitopes, cytotoxic T-cell epitopes (CTL), and helper T-cell epitopes (HTL). Based on these, a novel vaccine candidate was developed. The vaccine construct was subjected to rigorous computational validation: (1) Molecular docking with Toll-like receptors (TLRs) predicted immune interaction potential. (2) Molecular dynamics simulations assessed complex stability. (3) In silico cloning ensured prokaryotic expression feasibility. Then, we conducted preliminary experimental validation for the actual effect of the vaccine candidate, including recombinant protein expression in E. coli, enzyme-linked immunosorbent assay (ELISA) quantification of humoral responses, and Western blot analysis of cross-reactivity. ELISA results demonstrated that the vaccine candidate could induce high-titer antibodies in immunized animals, with potency reaching up to 1:128,000, and the immune serum showed strong reactivity with recombinant VP proteins. Western blot analysis using duck sera confirmed epitope conservancy across genotypes. Collectively, the multi-epitope vaccine candidate developed in this study represents a highly promising broad-spectrum strategy against DHAV. The robust humoral immunity it elicits, coupled with its demonstrated cross-reactivity, constitutes compelling proof-of-concept, laying a solid foundation for advancing to subsequent challenge trials and translational applications.