Strategies to obtain multiple recombinant modified vaccinia Ankara vectors. Applications to influenza vaccines

•Evolution of rMVA “Red-to-Green gene swapping” construction strategy.•Construction rMVA influenza vaccines containing internal proteins NP, M1 and PB1.•In vivo assays of double and triple rMVA influenza vaccines.•Optimal immunogenicity and protection against lethal challenge (A/California/7/09). As a vaccination vector, MVA has been widely investigated both in animal models and humans. The construction of recombinant MVA (rMVA) relies on homologous recombination between an acceptor virus and a donor plasmid in infected/transfected permissive cells. Our construction strategy “Red-to-Green gene swapping” – based on the exchange of two fluorescent markers within the flanking regions of MVA deletion ΔIII, coupled to fluorescence activated cell sorting – is here extended to a second insertion site, within the flanking regions of MVA deletion ΔVI. Exploiting this strategy, both double and triple rMVA were constructed, expressing as transgenes the influenza A proteins HA, NP, M1, and PB1. Upon validation of the harbored transgenes co-expression, double and triple recombinants rMVA(ΔIII)-NP-P2A-M1 and rMVA(ΔIII)-NP-P2A-M1-(ΔVI)-PB1 were assayed for in vivo immunogenicity and protection against lethal challenge. In vivo responses were identical to those obtained with the reported combinations of single recombinants, supporting the feasibility and reliability of the present improvement and the extension of Red-to-Green gene swapping to insertion sites other than ΔIII.