Genetic stability of recombinant MVA-BN

MVA-BN (modified vaccinia Ankara-Bavarian Nordic) is a highly attenuated vaccinia virus, which serves for development of highly immunogenic recombinant vaccines against infectious diseases and cancer. For generation of recombinant vaccines, insertion of two or more genes at one integration site is preferable, because it is less time consuming and labour intensive. The conventional approach of controlling the two inserts by two different promoters may result in different expression levels of the transgenes. In generating a recombinant MVA-BN (recMVA-BN) vaccine expressing HIV genes, we inserted a tat gene and the fusion gene gag-pol, each under the control of the cowpox ATI promoter into one intergenic region (IGR). The IGRs are sites we specifically developed to stably integrate genes of interest. After 20 passages of the recombinant virus under selective conditions, PCR analysis of IGR showed no remaining contamination with empty vector for recMVA-BN. Further PCR analysis results demonstrated that no homologous recombination between the two ATI sequences had occurred. Moreover, sequencing of the inserted genes and surrounding region demonstrated sequence stability, and RT-PCR confirmed transcription of the inserted genes. The results of our investigation clearly show that a stable insertion of multiple genes each carrying the same promoter at one IGR site is possible and that these insertions show neither detectable genetic instability nor alterations in the transcription of the inserted genes even after many passages.