Systemic and Mucosal Immune Responses From Serotype-Switched Viral-Vectored Ebola Vaccines.

Ebola virus is a highly virulent pathogen causing severe hemorrhagic fever with 25%-90% mortality. The 2014 outbreak infected over 28,000 people, causing 11,325 deaths. Although there is one FDA-approved vaccine available for limited use, additional vaccines are needed. We developed adenoviral-vectored vaccines expressing the ebolavirus glycoprotein (GP), the sole viral surface protein and primary immunogen. Vaccine constructs spanned three species and five human adenovirus subtypes, including low-seroprevalent serotypes to minimize pre-existing immunity. We compared single-dose and prime-boost regimens, intramuscular versus needle-free intranasal delivery, and serotype-switching strategies. Immunogenicity was assessed by quantifying GP-specific antibodies and T cell responses in BALB/c mice. Species C vectors (Ad5 and Ad6) elicited strong antibody responses and promoted rapid class switching to IgG compared to species B and D vectors. Serotype-switched regimens further enhanced antibody levels via intramuscular or intranasal routes and generated robust T cell responses when delivered intramuscularly. Intranasal vaccination induced detectable IgA but no measurable T cell responses, indicating route-dependent immune compartmentalization. Sera partially inhibited viral entry in a dose-dependent manner, with the greatest reduction observed in the Ad5/Ad6 regimen. Collectively, adenoviral species selection, serotype switching, and route of administration significantly influence GP-specific immunity and warrant further evaluation in protective challenge models.