Immunoinformatics design and experimental expression of a multi-epitope vaccine simultaneously targeting AAV2 and HAdV-F41 against acute hepatitis of unknown etiology.

The recent global outbreak of acute hepatitis of unknown etiology (AHUE) in children has raised significant health concerns due to the severity of infections, some of which require liver transplants and can lead to fatalities. Emerging evidence suggests that AHUE is caused by a co-infection involving AAV2 and HAdV-F41. Through immunoinformatics, we identified optimal T-cell and B-cell epitopes from AAV2's VP1 and AAP, as well as from HAdV-F41's long fiber, short fiber, and hexon proteins. To enhance specific immune responses, we incorporated the pan DR-binding epitope (PADRE) and Mycobacterium tuberculosis resuscitation-promoting factor RpfE as adjuvants, linking these elements with appropriate linkers to create a multi-epitope vaccine (MEV). The MEV gene was codon-optimized, cloned into the pET-15b vector, expressed in bacterial hosts, and purified using affinity chromatography. The resulting candidate vaccine, MEV-3, demonstrated high antigenicity, non-allergenicity, and non-toxicity, with a low instability index and favorable molecular characteristics. Molecular dynamics simulations confirmed the vaccine's stable binding to immune receptors, and prokaryotic expression yielded stable and pure MEV-3. Computational immune analysis further predicted a strong immune response induced by this vaccine. In conclusion, we developed an MEV that simultaneously targets AAV2 and HAdV-F41, potentially offering effective prevention and treatment for AHUE.