Sublingual Immunization with E2-CD154 Protein and the STING agonist c-di-AMP Confers Protection against Classical Swine Fever Virus in Pigs

Sara Puente-Marin¹Talía Sardina-González²Liani Coronado¹Cristina Riquelme¹Saray Heredia¹Adriana Munoz Aguilera¹Yusmel Sordo-Puga²Danny Pérez-Pérez²Alina Rodríguez-Mallon²Mario Pablo Estrada Garcia²Carlos A. Duarte²Maria Pilar Rodriguez Molto²Llilianne Ganges^1*

• ¹Institute of Agrifood Research and Technology (IRTA), Barcelona, Spain
• ²Centro de Ingenieria Genetica y Biotecnologia, Havana, Cuba

Subunit vaccines represent a safer alternative to live attenuated formulations. However, they often require potent adjuvants and delivery systems to elicit robust immunity, particularly against highly contagious diseases such as Classical Swine Fever (CSF). In this study, we investigated the immunogenicity and protective efficacy of a novel mucosal subunit vaccine comprising the chimeric E2-CD154 protein, co-administered with the mucosal adjuvant c-di-AMP, in domestic pigs. Optimal dosing and immunization schedules for sublingual immunization were determined, followed by a challenge experiment using a highly virulent CSF virus (CSFV) strain. Our results showed that sublingual co-administration of E2-CD154 and the STING agonist c-di-AMP conferred robust clinical protection, effectively prevented viral replication, and restricted the dissemination of infectious virus. This combination induced strong systemic IgG and IgA responses and neutralizing antibodies against multiple CSFV strains, achieving outcomes comparable to the commercial Porvac® vaccine, administered intramuscularly. Importantly, virus isolation from tonsils confirmed the absence of infectious virus in pigs immunized with E2-CD154 and c-di-AMP, unlike those receiving E2-CD154 or the adjuvant alone. Moreover, immunized animals exhibited minimal IFN-α serum levels post-challenge, indicating reduced innate activation and viral replication. These findings This is a provisional file, not the final typeset article provide evidence, in a large mammalian host such as the pig, that c-di-AMP functions as an adjuvant for a recombinant E2-CD154 protein delivered sublingually, enhancing immune responses consistent with protection against viral replication. Together, these results offer insights into the development of non-replicating, DIVA-compatible platforms against CSFV and support the rational design of next-generation subunit vaccines targeting viral pathogens relevant to both veterinary and human medicine.