Multi-Antigen Elephant Endotheliotropic Herpesvirus (EEHV) mRNA Vaccine Induces Humoral and Cell-Mediated Responses in Mice

<b>Background/Objectives:</b> Elephant endotheliotropic herpesvirus (EEHV) causes lethal hemorrhagic disease (HD) in Asian and African elephants in human care and the wild. It is the leading cause of death for young Asian elephants in North American and European zoos despite sensitive diagnostic tests and improved treatments. Thus, there is a critical need to develop an effective vaccine to prevent severe illness and reduce mortality from EEHV-HD. We generated a multi-antigenic EEHV mRNA vaccine to address this need that encodes the EEHV1A-subtype glycoproteins gB, gH, gL, and gO. These conserved proteins are the entry machinery for several herpesviruses in the betaherpesvirus subfamily and elicit humoral and cellular immunity in naturally infected elephants. <b>Methods:</b> Outbred CD-1 mice were vaccinated with two doses of an mRNA vaccine comprising modified EEHV1A gB, gH, gL, and gO mRNAs encapsulated into lipid nanoparticles. Humoral and T-cell immunity was assessed three weeks after the first dose or three weeks after the booster dose using luciferase immunoprecipitation system assays and flow cytometry, respectively. <b>Results:</b> The CD-1 mice vaccinated once had detectable antibody titers against gB, gH, and gL that increased significantly three weeks after a booster dose. Activated CD4⁺ and CD8⁺ T cells secreting cytokines associated with a T_H1 response were induced against all four glycoproteins. No adverse effects were observed following one or two doses of the vaccine. <b>Conclusions:</b> We found that gB, gH, gL, and gO as a multivalent vaccine stimulated robust humoral and cell-mediated immunity. This is a critical step for moving this candidate EEHV1A mRNA vaccine into clinical trials in Asian elephants.