A chiropteran factor of innate immunity can be recruited for rapid rescue and amplification of recombinant modified vaccinia Ankara (MVA)

Poxviruses are investigated as vectors for prophylactic and therapeutic vaccination. Transgenes are usually inserted via homologous recombination, and the desired vectors are isolated through sequential steps of plaque purification or limiting dilution. However, obtaining recombinants from the vast background of parental viruses is challenging for the ambitious timelines in personalised medicine and pandemic preparedness. We have developed a selection system based on tetherin from bats that eliminates parental viruses within the first passage after recombination. Tetherins are late-acting defensive factors that crosslink budding viruses to cellular membranes. Poxvirus morphogenesis is completed in the cytoplasm and only a subset of mature infectious particles acquires additional membranes from the trans-Golgi network. The outer of these membranes fuses with the plasma membrane for egress and is left behind. Tetherins have therefore not been suspected to interfere with release of poxviruses. We now describe that tetherin from the common vampire bat is as effective as tetherin from the Egyptian fruit bat and compare our results to conventional marker restoration based on a transiently deleted E3L gene. We demonstrate highly efficient simultaneous insertion of two transgenes into different positions of the genome of modified vaccinia Ankara (MVA) using the two tetherins. We furthermore show that recombination, rescue and amplification of vectors in suspension cultures in chemically-defined media is possible, a potential benefit for GMP campaigns. Observations with an EGFP fusion protein that is localised in the perinuclear space suggest that the bat tetherins may be active as a crosslinker during early steps of morphogenesis.