Presentation of Recombinant Proteins in Modified Vaccinia Virus Ankara Extracellular Enveloped Virions

Modified Vaccinia Virus Ankara is a highly attenuated vaccinia
virus strain developed during the smallpox eradication campaign.
Nowadays recombinant attenuated poxviruses gain importance as live
carrier vaccines against different infectious diseases and in
cancer therapy. The aim of this work was to develop recombinant
viral vectors, for presentation of a foreign antigen on the surface
of extracellular enveloped particles (EEV). First, it was tested
whether significant amounts of this viral form are produced by MVA
in comparison to replication competent and widely used vaccinia
virus strains. Using a number of independent approaches it could be
shown that MVA infection in primary chicken embryo fibroblasts
results in the production of enveloped viruses, but strikingly most
of these were not released into the culture medium but remained
attached to the plasma membrane. The results also showed that the
replication competent vaccinia virus IHD-J is more efficient in
trans-Golgi-network-wrapping and in releasing enveloped virions
into the extracellular medium, while the WR strain is less
efficient than MVA. Two different strategies were followed to
target the recombinant protein to the surface of extracellular
enveloped viruses. Since it was shown that non-vaccinia virus
proteins can be incorporated to some extent into the outer
membrane, a native model type II membrane protein was used. To
increase the chance of foreign protein incorporation a fusion
protein was used which consisted of the transmembrane domain of a
protein known to be specific for the outer membrane of
extracellular eveloped virus and the extracellular domain of the
foreign antigen which was used in its native form. The data show
that both proteins were incorporated into the extracellular
enveloped virions produced in MVA infected chicken embryo
fibroblasts, albeit with low efficiency. the ransmembrane domain of
the EEV pecific protein was not sufficient to target the foreign
protein specifically to the outer envelope.