Classical swine fever virus is a world-wide distributed highly-contagious disease affecting swine. The two main strategies for diseases control are prophylactic vaccination and non-vaccination stamping out policies. Marker vaccines are a promising strategy. Here we report the rational development of a doubly antigenic marker CSFV experimental live attenuated candidate strain vaccine (Flag/T4 virus). Flag/T virus (Flag/T4v) is based in the combination of two Brescia derived recombinant attenuated viruses: RB-C22 and T4. RB-C22v contains a 19mer insertion in the structural glycoprotein E1, while T4v posses mutated CSFV amino acid residues 830 to 834 in the structural glycoprotein E2, deleting the highly conserved epitope recognized by monoclonal antibody (mAb) WH303. Flag/T4 virus contains a positive foreign antigenic marker, due to the insertion of the highly antigenic epitope Flag in the 19mer insertion of E1, as well as a negative antigenic marker, the lack of reactivity with mAb WH303. Immunized with Flag/T4v induced a complete protection against the challenge with virulent strain Brescia both at 3 and 28 days post infection when nasally administered and since the second day post infection when intramuscularly administered. These results constitute an example of rational design of a CSFV antigenically marked LAV.
