Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments

Abstract

Edward Jenner and his contemporaries believed that his variolae vaccinae originated in horses and molecular analyses show that modern vaccinia virus (VACV) strains share common ancestry with horsepox virus (HPXV). Given concerns relating to the toxicity of modern VACV vaccines, we asked whether an HPXV-based vaccine might provide a superior alternative. Since HPXV may be extinct and the only specimen of HPXV that has been identified is unavailable for investigation, we explored whether HPXV could be obtained by large-scale gene synthesis. Ten large (10-30 kb) fragments of DNA were synthesized based on the HPXV sequence along with two 157 nt VACV terminal sequences, and were recombined into a live synthetic chimeric HPXV (scHPXV) in cells infected with Shope fibroma virus (SFV). Sequencing of the 212 kbp scHPXV confirmed it encoded a faithful copy of the input DNA. We believe this is the first complete synthesis of a poxvirus using synthetic biology approaches. This scHPXV produced smaller plaques, produced less extracellular virus and exhibited less virulence in mice than VACV, but still provided vaccine protection against a lethal VACV challenge. Collectively, these findings support further development of scHPXV as a novel replication-proficient smallpox vaccine.

Fig 1. Virus design strategy.

(a) Cloned synthetic DNA fragments used to assemble HPXV. Nine different clones were synthesized spanning all but the first and last 40 bp in GenBank entry DQ792504, each overlapping the adjacent fragment by ~1 kbp. All of the AarI and BsaI restriction sites were eliminated from fragments 1A to 7, inclusive, using silent mutations and the same strategy was used to add AvaI and StuI sites in Frag_2. To facilitate virus recovery a gene encoding a YFP-gpt fusion protein was inserted into Frag_3, at the site of the HPXV thymidine kinase locus. An additional HPXV095 fragment spans the thymidine kinase locus and was subsequently used to delete and replace the YFP-gpt marker using homologous recombination. (b) Synthetic hairpin telomeres. Because the HPXV genome was not sequenced to the ends, we substituted two hairpin sequences based upon those reported for VACV strain WR (green coloured nucleotides). These are called “fast” and “slow” forms based upon their electrophoretic properties. The nucleotides coloured in black come from the HPXV genome sequence and provide an element essential for telomere resolution.

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.

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.

Fig 4. scHPXV YFP-gpt::095 and scHPXV growth properties in culture.

a. Plaque size measurements. BSC-40 cells were infected with the indicated viruses and cultured for three days. The dishes were stained and the plaque areas measured using a scanned digital image. Approximately 8 plaques were measured per virus treatment from three independent infections (closed circles). A similar independent experiment comparing scHPXV YFP-gpt::095 and scHPXV to VACV WR was also completed as described above (open circles). DPP15 is a VACV clone isolated from Dryvax vaccine and CPXV is cowpox virus (strain Brighton Red). scHPXV YFP-gpt::095 and scHPXV form plaques that are significantly smaller than any of the other two viruses tested. Statistically significant differences are noted (****P² 0.0001). b. Plaque morphology seen at three days post-infection on BSC-40 cells. The cells were fixed and stained with crystal violet. c. Multi-step growth kinetics measured using human (HEL, HeLa) or monkey (BSC-40, Vero) cells. The cells were infected at a multiplicity of infection of 0.01, the virus harvested at the indicated times, and the virus titrated on BSC-40 cells in triplicate. The error bars indicate the SEM.

Fig 5. Virulence and vaccine properties of scHPXV.

a. Virulence studies. Immune competent BALB/c mice (5 per group) were inoculated intranasally with the indicated viruses. scHPXV was tested using doses ranging from 10⁵−10⁷ pfu per mouse. Some weight loss due to illness was seen in animals inoculated with 5×10³ pfu VACV strain WR (¢), or 1×10⁷ pfu of VACV strain DPP15 (Δ), but no illness was detected in any of the animals infected with HPXV clones. b. VACV challenge studies. Four weeks after exposure to the indicated agent as shown in panel a (or mock treatment with buffered saline), the mice were exposed to a lethal dose of VACV strain WR (1×10⁶ pfu) by the same route (day 0 in b is day 28 in graph a). The animals were monitored for signs of disease and euthanized if the weight loss exceeded 25% of the initial body weight. The scHPXV strain provided good protection at the two highest pre-challenge doses (1×10⁶ and 1×10⁷ pfu). c. Disease course in HPXV vaccinated mice. The mice were inspected to detect four signs of disease (ruffled fur, hunched posture, difficulty breathing, and reduced mobility) and a clinical score calculated from the sum of the individual scores averaged across all five (or surviving) mice per cohort. Little or no signs of illness were detected in mice first vaccinated with 1×10⁶ or 1×10⁷ pfu scHPXV d. Kaplan-Meyer analysis of survivorship. All of the mock-vaccinated (i.e. saline-treated) mice succumbed to VACV strain WR challenge, whereas treatment of animals vaccinated with either VACV strain WR, VACV strain DPP15, or all doses of scHPXV (10⁵−10⁷ pfu per mouse) protected 100% of the animals.