SPI-1 is a missing host-range factor required for replication of the attenuated modified vaccinia Ankara (MVA) vaccine vector in human cells

Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements.
Author summary Poxvirus vectors have outstanding properties for development of vaccines against a myriad of infectious agents due to their ability to retain long segments of foreign DNA and high-level gene expression. Safety concerns led to a preference for attenuated poxviruses that lost the ability to produce infectious progeny in human cells. The most widely used poxvirus vector is modified vaccinia virus Ankara (MVA), which exhibits an extreme host-range restriction in most mammalian cells. MVA was attenuated by passaging more than 500 times in chicken embryo fibroblasts during which large deletions and numerous additional genetic changes occurred. Despite ongoing clinical testing of MVA-vectored vaccines, the basis for its host-range restriction remained unknown. Here we show that re-introduction of the SPI-1 gene into MVA or host cells increased virus spread by more than 100-fold in a human diploid cell line, providing an important insight into the mechanism responsible for the host-range restriction. This information could help design improved vectors and develop non-avian cell lines for propagation of candidate MVA vaccines.