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

Abstract

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.

Identification of Vaccinia Virus Inhibitors and Cellular Functions Necessary for Efficient Viral Replication by Screening Bioactives and FDA-Approved Drugs

“…A comparison between the genomes of VACV-WR and ACAM2000 showed the ACAM2000 has two major deletions in the inverted terminal repeats (ITR, C12-C18 at the left ITR and the corresponding duplicates at the right ITR), as well as mutations throughout the genome [ 77 , 80 ]. Among those deleted, C12 has been shown to be important for enabling modified vaccinia virus Ankara (MVA) replication in human cells [ 81 , 82 ]. Some of those genes lost in ACAM2000 might regulate innate immune responses in cells, which complicates the role of STAT3 in VACV replication.…”

Zinc-finger antiviral protein (ZAP) is a restriction factor for replication of modified vaccinia virus Ankara (MVA) in human cells

“…from inactivation of the genes encoding the C12 and C16 proteins [7,8]. Several lines of evidence implicated C16 as the ZAP antagonist.…”

“…MVA 47.1+GFP was made by inserting the enhanced GFP ORF directed by a synthetic early/ late promoter [41] into MVA between the F12 and F13 ORFs. Deletion of C16, and insertion of C12 and/or C16 were described previously [7,8].…”

Stability of the HSV-2 US-6 Gene in the del II, del III, CP77, and I8R-G1L Sites in Modified Vaccinia Virus Ankara After Serial Passage of Recombinant Vectors in Cells

“…The sum total of the deletions from CVA (GenBank #: AM501482) that resulted in MVA (GenBank accession # AY603355.1 for Acambis 3000 MVA) was > 25 kilobase pairs, representing >13% of the entire virus genome [3], and includes genes encoding immune evasion and virulence factors. MVA is capable of infecting mammalian cells and expressing proteins but it is impaired in morphogenesis of infectious virions [5] due in part to the loss of the C12L gene encoding serine protease inhibitor-1 [6] and truncation of the C16L/B22R host-range gene [7]. MVA has been shown to be safe in humans, and a strain of MVA (MVA-BN) has been approved by regulatory authorities in Canada, the European Union [8], and the United States, as a prophylactic smallpox (and monkeypox in the USA) vaccine [9].…”