A Multi-Epitope Recombinant Vaccine Candidate Against Bovine Alphaherpesvirus 1 and 5 Elicits Robust Immune Responses in Mice and Rabbits

Background/Objectives: Varicellovirus bovinealpha1 and Varicellovirus bovinealpha5 (BoAHV-1 and BoAHV-5), respectively, are widely distributed pathogens that cause distinct clinical conditions in cattle including infectious bovine rhinotracheitis, infectious pustular vulvovaginitis/balanoposthitis, and meningoencephalitis. Due to the establishment of viral latency, controlling these infections is challenging, and vaccination remains the most effective strategy. In this study, vaccine candidates targeting both BoAHV-1 and BoAHV-5 were developed. Methods: A synthetic gene encoding immunodominant epitopes from the gB and gD proteins and tegument phosphoprotein of BoAHV-1 and BoAHV-5 was designed to produce a multi-epitope recombinant antigen, expressed both in a prokaryotic system (RecBoAHV) and by a modified vaccinia Ankara (MVA-BoAHV) viral vector. The binding affinity of MHC-I to bovine leukocyte antigens (BoLA) was predicted using the NetMHCpan tool (version 4.1). The immunogenicity of the vaccine candidates was evaluated in rabbit and mouse models, using prime-boost immunization protocols. Sera from bovines naturally infected with BoAHV-1 and/or BoAHV-5 were used to evaluate the chimeric protein antigenicity. Immune responses were assessed by indirect ELISA and Western blot. Results: The recombinant multi-epitope protein was effectively recognized by IgG and IgM antibodies in sera from cattle naturally infected with BoAHV-1 or BoAHV-5, confirming the antigenic specificity. Both RecBoAHV and MVA-RecBoAHV induced strong and specific humoral immune responses in rabbits following a homologous prime-boost regimen. In mice, both homologous and heterologous prime-boost protocols revealed robust immunogenicity, particularly after the second booster dose. Conclusions: These findings highlight the immunogenic potential of the RecBoAHV multi-epitope vaccine candidates for controlling BoAHV-1 and BoAHV-5 infections. Further characterization of these vaccine formulations is currently underway in bovine, the target specie.