Expression of an Efficient Selection Marker Out of a Duplicated Site in the ITRs of a Modified Vaccinia Virus Ankara (MVA)

Abstract

Background/Objectives: Poxviruses are large DNA viruses that replicate in the host cytoplasm without a nuclear phase. As vaccine vectors, they can package and express large recombinant cassettes from different positions of their genomic core region. We present a comparison between wildtype modified vaccinia Ankara (MVA) and isolate CR19, which has significantly expanded inverted terminal repeats (ITRs). With this expansion, a site in wildtype MVA, called deletion site (DS) IV, has been duplicated at both ends of the genome and now occupies an almost central position in the newly formed ITRs. Methods: We inserted various reporter genes into this site and found that the ITRs can be used for transgene expression. However, ITRs are genomic structures that can rapidly adapt to selective pressure through transient duplication and contraction. To test the potential utility of insertions into viral telomers, we inserted a factor from the cellular innate immune system that interferes with viral replication as an example of a difficult transgene. Results: A site almost in the centre of the ITRs can be used for transgene expression, and both sides are mirrored into identical copies. The example of a challenging transgene, tetherin, proved to be surprisingly efficient in selecting candidate vectors against the large background of parental viruses. Conclusions: Insertion of transgenes into ITRs automatically doubles the gene doses. The functionalisation of viruses with tetherin may accelerate the identification and generation of recombinant vectors for personalised medicine and pandemic preparedness.

Keywords: MVA; innate immune system; inverted terminal repeat; tetherin; vaccinia.

Figure 1

(a) Genomic organisation of WT MVA and strain CR19. Deletion sites I to VI are marked with Roman numerals and the inverted terminal repeats (ITRs) with grey arrow boxes. The ITRs have expanded in size in MVA-CR19 by a recombination event that has caused loss of DS I at the left side and duplication of DS IV (up-pointing red arrows) within the expanded ITRs at both sides of the genome. The distance from DS IV to the apex is 11 kb and to the core of the genome is 16 kb. (b) Possible genotypes and phenotypes for recombination of different markers into DS IV of WT MVA and MVA-CR19. Only the positions of DS I and DS IV are indicated with down-pointing arrows and roman numerals. The two ITRs are drawn as arrow boxes. DS IV is localised outside of the right ITR of WT and within both ITRs of MVA-CR19. DS I is localised outside of the left ITR of WT and is not present anymore in MVA-CR19. Grey arrow boxes are for non-modified ITRs. The letters in the filled arrow boxes of MVA-CR19 stand for the insertion of EGFP (G) or mCherry (R) into DS IV within the ITR. The bp numbers refer to the sizes of diagnostic PCR products obtained with the shown configuration using primers listed in Table S1.

Figure 2

Relative composition of focus populations isolated after simultaneous recombination of MVA-CR19 (a) and WT virus (b) with shuttle plasmids for EGFP and mCherry reporters. The numbers in the columns give absolute focus numbers. Shown here is the development of one green (g1), red (r1) and six independently picked yellow (y1–y6) foci. Red and green foci could be purified, and yellow foci were always dispersed into a mixture of red and green foci in addition to yellow foci.

Figure 3

Phenotype distribution of foci after recombination of WT (a) and CR19 viruses (b) that already carry mCherry in DS IV with a shuttle plasmid that codes for EGFP. Yellow foci were picked into pools (py) at passages 1 and 2. The foci were not pooled at passages 3–5 (individually named y1–y5). For WT, all yellow pools and yellow clones were separated into green, red, and yellow components independent of passage level. For CR19, a clone was identified in P3 (y1) that had no apparent red components and a very high frequency of yellow. That clone was focus-picked for a further 2 passages (P4 and P5, P4* is an independent passage of the same clone).

Figure 4

Insertion at DS IV has increased in size for a consistently yellow focus. Recombination may have occurred by a chance integration of different inserts at the distal termini. Intermediate bands that may be caused by transitory states are weakly visible in P3 y1 and are marked with asterisks (*). The foci shown in the right panel are foci from P4* (that were derived from P3 y1) in Figure 3. RED and GFP for reference PCRs with shuttle plasmids as a template, NTC for non-template control, and MVA represents a wild-type virus without insertion in DS IV. The size marker in this and all other agarose gels is the 1 kb Plus DNA Ladder (ThermoFisher).

Figure 5

Quantification of foci that were obtained in a replacement of an EGFP expression cassette in DS IV of MVA-CR19 against mCherry expression cassette: (a) Foci with a predominantly monofluorescent red phenotype were obtained after 2 passages. A pure yellow phenotype was not recovered and a high frequency of distinct red or green foci was observed for all 14 isolated yellow P3 foci. (b) Confirmation that recombination towards large dual-expressing insert is a rare event. Focus y01^• was also compared to a pure yellow focus in a comet assay, see Figure S5. PCR with r01 is a positive control for PCR on known viral DNA with the mixed isolates in Figure S4 and the partially consistent yellow isolate y10 in Figure S3 (here denoted as y10*). RED and GFP are reference controls as described above. Expected amplicons are 1099 bp for DS IV containing mCherry, 1231 bp for DS IV containing EGFP, approx. 2000 bp for a combination of both reporter genes, and 220 bp for an empty (WT) DS IV (see also Supplementary Table S1).

Figure 6

The addition of doxycycline has clear effects on the replication of tetherin-inducible viruses even if added 24 h post-infection. (a) Infection was performed with an MOI of 0.01 in adherent cultures. The tested virus is MVA-CR19 with tetherin in DS IV under the control of the HYBdx promoter. Co-expression of mCherry is used to quantify replication. Both, the number (left panel) and size of foci (right panel) are reduced compared to the uninhibited control, symbolised by (−). (b) The appearance of infected cultures that are quantified in (a) after doxycycline-induced de-blocking of tetherin expression.

Figure 7

Infectious titers of tetherin-containing viruses in suspension cultures. WT and MVA-CR19 viruses replicate to high titers if the expression of tetherin is suppressed (open symbols) and to 1000-fold lower titers if the expression is deblocked. MVA-CR19 contains two copies of tetherin and replicates to lower titers also if tetherin is under the control of the strong M52dx promoter. This experiment was performed twice with similar results.

Figure 8

The tetherin expression cassette is replaced within one passage. Successful replacement of the tetherin-containing insertion in DS IV through the dual-expression cassette in the shuttle plasmid for the same DS IV results in a loss of tetherin expression and gain of EGFP expression. Faithful homologous recombination furthermore maintains the constitutive expression of mCherry: (a) Infection and transfection for recombination without application of the semisolid medium. Only if tetherin expression is blocked virus can replicate for successful recombination. (b) A lysate of the infected/transfected cultures was transferred to a fresh cell monolayer in the presence or absence of doxycycline. A semisolid medium was applied for quantification. No parental viruses appear to be present if tetherin expression is allowed. (c) A lysate of the doxycycline-treated well of passage 2 was used to infect a fresh cell monolayer. All observed viruses appear to be desired recombinants, independent of further induction with doxycycline.

Figure 9

PCR over DS IV confirms the successful replacement of the tetherin marker with the dual expression cassette within a single passage. The upper panel shows the PCR reaction. The amplicons for tetherin/mCherry and EGFP/mCherry cassettes are similar in size (1839 bp and 2016 bp, respectively, marked by the bullets on the right side). Restriction enzyme digestion was employed to confirm the identity of the amplicons, NdeI within the tetherin gene (resulting in 986 bp and 853 bp, expected position indicated with the t) and BsrGI within EGFP (leaving 1025 bp and 991 bp, indicated with g). Left two lanes to show PCR on the shuttle plasmids as a positive control (PC). NTC for non-template control. An empty DS IV would give a signal of 220 bp.