Towards vaccine development against African swine fever virus in Eastern and Southern Africa
To enable control of African swine fever (ASF) in Eastern and Southern Africa, prototype live vaccine candidates were generated by targeted gene deletions from a Kenyan genotype IX ASF virus (ASFV). It was attempted to delete known nonessential genes involved in virulence (encoding TK, dUTPase, CD2v, 9GL), possibly essential genes (p12, pA104R, ribonucleotide reductase), and genes with widely unknown functions (pK145R). Isolation of the desired virus recombinants by plaque assays or limiting dilutions on a wild boar lung cell line (WSL-HP) was facilitated by substitutive reporter gene insertions encoding fluorescent proteins (GFP, DsRed), or the human membrane protein CD4. The latter protein permitted enrichment of recombinant virus particles by magnetic activated cell sorting (MACS). The isolated ASFV recombinants were characterized by PCR and sequencing of the mutated genome parts, and replication kinetics and virus spread in cell culture were investigated. Deletion of TK, CD2v, or pK145R had no detectable effect on in vitro growth of ASFV Kenya. Interestingly, virus mutants lacking the DNA binding protein pA104R which has been considered to be essential for DNA replication, also exhibited almost wild type-like growth properties. In contrast, ASFV mutants lacking ribonucleotide reductase or p12 could not be purified to homogeneity on WSL-HP cells, indicating these proteins are essential for virus replication in cell culture. Therefore, trans-complementing cells lines stably expressing ASFV p12 have been prepared which can now be used for mutant virus purification. If this approach is successful the resulting defective mutant ASFV Kenya-p12 might be suitable as a safe “disabled in second cycle” (DISC) live vaccine in swine. In a novel approach to improve reverse genetics of ASFV the CRISPR/Cas9 cell line WSL-gRp30 (Hübner et al., 2018a) was co-transfected with genomic DNA of ASFV-KenyaCD2vDsRed, sgRNA plasmids targeting K145R or 9GL, and GFP-expressing recombination plasmids for homology-directed repair. For booting up of the noninfectious virus genome the cells were infected with phylogenetically distant helper virus (genotype II ASFV Armenia, 84% identity) which was selectively inhibited on the used cell line. The desired double-fluorescent double-deletion mutants could be isolated after few plaque purification steps on selective WSL-gRp30 cells. Next generation sequence (NGS) analyses of reconstituted ASFV Kenya genomes showed that no unwanted recombination with the helper virus occurred, indicating that the method might be also suitable for booting of synthetic ASFV genomes cloned and mutagenized in E. coli or yeast. The modified CRISPR/Cas9 system of S. pyogenes might be also usable for generation of ASFV resistant pigs. To evaluate this alternative control measure WSL cell clones stably expressing Cas9 nuclease and single or multiple sgRNAs against essential ASFV proteins were prepared and tested for their susceptibility to infection. Strain specific sgRNAs targeting the p30 gene of ASFV Kenya or Armenia selectively inhibited the respective viruses, and a p12 gene-specific sgRNA abrogated replication of both genotypes almost completely. Interestingly, coexpression of four ASFV-specific sgRNAs did not enhance virus inhibition, but might help to reduce the frequency of escape mutants which were occasionally isolated from the single sgRNA-expressing cells, and exhibited silent base substitutions or in-frame deletions within the target genes. First attempts to express the in vitro tested CRISPR/Cas9 constructs in transgenic pigs are in progress. CRISPR/Cas9 supported rescue of a defective BAC clone of pseudorabies virus (PrV) vaccine strain Bartha (Hübner et al., 2018b) was used to develop putative vectored vaccines against ASFV. In the present study expression cassettes for the codon-optimized p12 and p54 genes of ASFV were successfully inserted into the PrV genome. The insertions did not significantly affect PrV recombination in cell culture, and the transgenes were expressed at similar levels as in ASFV-infected cells. It has to be tested whether coinfection with vector constructs for these and other immunogenic ASFV proteins is able to protect pigs against a lethal challenge. For characterization of the generated ASFV mutants and PrV vector constructs, monospecific antisera against several ASFV gene products (p11.5, p12, p54, pK145R, p285L) were prepared by immunization of rabbits with bacterial GST fusion proteins. The anti-p12 serum showed only weak and strain-specific reactions with the ASFV Kenya protein, but was nevertheless useful for identification of p12-expressing PrV recombinants and WSL cell lines. All other sera showed satisfying reactions in Western blot and mostly immunofluorescence analyses, and allowed i.a. precise localization of the pK145R and p285L proteins in ASFV-infected cells and virions (Hübner et al., 2019).
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