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.
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