A bivalent VP2 based vaccine approach reveals co expression driven interference between VP2 antigens impairing BTV neutralizing antibody induction

Bluetongue virus (BTV) is an important arboviral pathogen of ruminants that causes a significant impact on the primary sector. More than 29 different BTV serotypes exist, hindering control and prevention of Bluetongue (BT) disease. Conventional vaccines possess safety and efficacy limitation and do not permit to confer multiserotype protection and differentiate between vaccinated and infected animals (DIVA strategy). Here, we developed recombinant Modified Vaccinia Ankara (rMVA) viral vectors individually expressing or co-expressing the neutralizing antibody (nAb) target proteins VP2 of BTV serotypes 4 and 8. We evaluated their immunogenicity and protective efficacy in IFNAR(-/-) mice and sheep. Immunization of IFNAR(-/-) mice with the rMVA co-expressing of BTV-4 VP2 and BTV-8 VP2 led to abolished nAb induction while eliciting non neutralizing antibodies against both VP2 proteins, suggesting a previously unrecognized interference between VP2 antigens. Combined immunization of IFNAR(-/-) mice with rMVAs expressing either BTV-4 VP2 or BTV-8 VP2 elicited a strong neutralizing immune response and complete protection against lethal challenge with BTV-4 or BTV-8. In sheep, combinatorial prime-boost immunization with these monovalent rMVAs induced nAbs against both homologous BTV serotypes. Immunized sheep were fully protected against BTV-4, as no fever or viral replication were detected after challenge. Similarly, immunized sheep were also protected against BTV-8 although detectable but mild RNAemia and slight increase in rectal temperatures were observed after challenge. Overall, we identified a relevant constraint for multivalent VP2 based vaccine design and present a DIVA vaccination strategy that offers bivalent protection against BTV in a natural host of the disease.