scHPXV design strategy

Given that the toxicity of modern VACV vaccines limits their use, and molecular analyses show that modern VACV vaccine strains share common ancestry with HPXV, we asked whether a HPXV-based vaccine might provide a safer alternative. Using the HPXV (strain WNR-76) sequence [4] we divided the 212,633 bp genome into 10-overlapping DNAs sharing ~1 kbp of flanking homology (Fig 1). Each fragment was synthesized and cloned into a bacterial vector. To facilitate the assembly of these clones we deleted all of the AarI or BsaI sites in fragments 1–7 using silent mutations, but did not mutate the BsaI sites in the two inverted terminal repeat (ITR) fragments, as these regions contain elements required for genome replication and resolution. In addition we used silent substitutions to introduce AvaI and StuI sites into the HPXV044 gene and replaced HPXV095 in Frag_3 with sequences encoding yellow fluorescent protein fused to guanine phosphoribosyltransferase protein (YFP-gpt) [21]. HPXV095 encodes the HPXV homolog of the non-essential VACV J2R gene.

As a proof of principle, co-transfection of HPXV Frag_3 along with VACV genomic DNA into SFV-infected BGMK cells led to the recovery of a hybrid virus (VACV/HPXV+Frag_3), validating the selection strategy (Fig 2). The addition of other HPXV synthetic DNA fragments along with genomic DNA from this hybrid virus, however, did not result in complete incorporation of the HPXV fragment lacking BsaI sites into the genomes of these hybrid virus clones (Fig 2a). Instead, these viruses exhibited a “patchy” pattern of DNA integration, similar to what is detected in virus-by-virus crosses (Fig 2b) [25]. This patchy pattern is easily detected because of the sequence polymorphisms that differentiate HPXV from VACV. These sequence differences are illustrated by an alignment of the two viruses (Fig 2c).

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larger image TIFF original image Download: Fig 2. Characterization of VACV-HPXV hybrid viruses. a. A PCR-based screening approach was used to identify hybrid and reactivated viruses. PCR primers were designed to target both HPXV and VACV (S2 Table) and used to amplify DNA segments spanning the BsaI sites that were disrupted in the synthetic HPXV clones. Following PCR amplification, the products were digested with BsaI to differentiate VACV sequences (which cut) from HPXV (which do not cut). The VACV/HPXV hybrids exhibit a mix of BsaI sensitive and resistant sites whereas a reactivated scHPXV YFP-gpt::095 clone is fully BsaI resistant. b. Sequence mapping of HPXV inserts in VACV strain WR. Virus genomes were sequenced using an Illumina platform, assembled, and LAGAN [26] and “Base-by-Base” [27] software were used to align and generate the maps shown. Places where VACV sequences (white) have been replaced by HPXV sequences are colour coded according to the difference. The first hybrid virus (“VACV/HPXV + fragment 3”) was obtained by co-transfecting VACV DNA plus HPXV Frag_3 (Fig 1) into SFV-infected cells. The green-tagged insertion encodes the YFP-gpt selection marker. Clones 1–3 were obtained by purifying the DNA from this first hybrid genome and transfecting it again, along with HPXV fragments 2, 4, 5, and 7, into SFV-infected cells. c. Genomic sequence comparison of scHPXV YFP-gpt::095 to VACV WR. https://doi.org/10.1371/journal.pone.0188453.g002

The HPXV ITRs presented more challenges, because the available HPXV genome was not sequenced through the hairpin ends. The HPXV sequence stops at a feature needed for concatemer resolution [28, 29], which in VACV is located ~60 nt from the end of the genome and it is identical to the putative resolution site in the HPXV sequence. Given the similarities between the two viruses, we tested whether VACV hairpins could substitute for the missing HPXV ends. The fast [F] and slow [S] VACV hairpins (Fig 1b) were synthesized and these two 157 nt oligonucleotides were ligated onto the left and right HPXV ITR fragments using standard molecular biology techniques. This produced four combinations of ITR-hairpin species with joints that copied the desired chimera and with no adventitious sequences (S1 Fig). We pooled these DNAs in reactivation reactions.

We then transfected an equimolar mix of all ten HPXV fragments into SFV-infected cells to test whether these synthetic HPXV fragments could be recombined into replication-competent viruses. Five days later, the virus mix was harvested and re-plated on BSC-40 cells, where Orthopoxviruses will form plaques. Although this process is inefficient compared to transfection of complete poxvirus genomes into cells where we and others can get up to 106 pfu/μg [18, 19], we obtained from 0-to-4 fluorescent plaques on the BSC-40 cells, in each of six replicate experiments, or ~30 pfu/μg of transfected DNA. Judging by pulse field gel electrophoresis these viruses appeared to be chimeras that encoded synthetic HPXV sequences bounded by HPXV/VACV hairpin ends (Fig 3a). We sequenced five viruses from two reactivation reactions and all faithfully replicated the sequence of the input clones. We confirmed that this method doesn't introduce SFV DNA into the reactivated viruses [25] and the only mutations detected were those we had incorporated by design, for example novel AvaI and StuI sites in HPXV044 (S2 Fig). Our Illumina sequencing also detected reads extending completely through the VACV-derived hairpin telomeres. The 1:1 distribution of F- and S-reads in each of the five reactivated viruses suggests that both ends are required to produce a virus (Fig 3b and S3 Table). However, because of the way that these sequences are produced by fragmentation of a pool of viruses bearing inverted terminal repeats, it is not possible to establish how they are oriented or distributed with respect to any particular individual genome.

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larger image TIFF original image Download: Fig 3. Sequencing and restriction endonuclease mapping of reactivated scHPXV YFP-gpt::095 clones. a. Pulsed field gel electrophoretic analysis. Three independent HPXV clones plus a VACV (strain WR) control virus were purified and then left either untreated, or digested with BsaI or HindIII. The larger size of HPXV relative to VACV is apparent in the uncut samples, and the absence of nearly all of the BsaI sites in the HPXV clones is also apparent. The selective manner in which only the BsaI sites were modified by gene synthesis is illustrated by the retention of all the HindIII restriction sites in scHPXV YFP-gpt::095. The HindIII digest also demonstrates the similarities between HPXV and VACV as illustrated by the related digestion patterns. The faint DNA bands marked with asterisks (*) are from monkey cell mitochondria, which co-purify with poxvirus particles. b. Sequence reads associated with the hairpin telomeres. The viruses were sequenced using Illumina technology and the genomes assembled using CLC genomics software. A subset of the longest sequencing reads, extending beyond the known end of the HPXV sequence, are shown aligned against a poly·dC template. (This method captures sequences extending beyond the point where the reference sequence ends.) These reads span the entire length of the unfolded hairpins as was provided using synthetic oligonucleotides. Because of the inverted terminal duplications, all of the reads “pile up” together. Both F- and S-forms of the VACV hairpin are detected and the ratio of F- to S-reads in this region was 1.03±0.01 (SEM) in eight different virus-sequencing reactions. https://doi.org/10.1371/journal.pone.0188453.g003

We subsequently synthesized a 1,349 bp DNA encoding the HPXV095 locus along with flanking sequences (Fig 1a) and transfected it into cells infected with one of the reactivated HPXV. Three non-fluorescent recombinant viruses were recovered and purified, and PCR and sequencing were used to confirm that we had restored the wild-type HPXV095 gene. This “synthetic and chimeric” virus was labeled scHPXV to differentiate it from the reactivated thymidine kinase deficient parent virus (scHPXV YFP-gpt::095) and the native virus (HPXV strain WNR-76).